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Network Publications

Network Topics

  1. DNA rearrangements, mechanisms of MAC development, non coding RNA and epigenetics
  1. Genome evolution, gene annotation, comparative genomics and molecular phylogeny
  1. Environmental adaptation: response to stress and effects on the genome
  1. Chromatin organisation
  1. Mating type determination
  1. Determination of cell polarity; role of structural inheritance; cilia as sensors
  1.  Symbiosis of ciliates

 

Topic 1

 

ParTIES: a toolbox for Paramecium interspersed DNA elimination studies.

Denby Wilkes C, Arnaiz O, Sperling L.

Bioinformatics. 2015 Nov 20. pii: btv691. [Epub ahead of print]

Abstract: http://www.ncbi.nlm.nih.gov/pubmed/?term=ParTIES%3A+a+toolbox+for+Paramecium+interspersed+DNA+elimination+studies

MOTIVATION: Developmental DNA elimination occurs in a wide variety of multicellular organisms, but ciliates are the only single-celled eukaryotes in which this phenomenon has been reported. Despite considerable interest in ciliates as models for DNA elimination, no standard methods for identification and characterization of the eliminated sequences are currently available.

RESULTS: We present the Paramecium Toolbox for Interspersed DNA Elimination Studies (ParTIES), designed for Paramecium species, that (i) identifies eliminated sequences, (ii) measures their presence in a sequencing sample and (iii) detects rare elimination polymorphisms.

AVAILABILITY AND IMPLEMENTATION: ParTIES is multi-threaded Perl software available at https://github.com/oarnaiz/ParTIES. ParTIES is distributed under the GNU General Public Licence v3.

CONTACT: linda.sperling@i2bc.paris-saclay.fr

SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

 

 

Experimental identification and analysis of macronuclear non-coding RNAs from the ciliateTetrahymena thermophila.  

Andersen KL, Nielsen H.

Nucleic Acids Res. 2012 Feb;40(3):1267-81. doi: 10.1093/nar/gkr792. Epub 2011 Oct 3

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3273799/

ABSTRACT

The ciliate Tetrahymena thermophila is an important eukaryotic model organism that has been used in pioneering studies of general phenomena, such as ribozymes, telomeres, chromatin structure and genome reorganization. Recent work has shown that Tetrahymena has many classes of small RNA molecules expressed during vegetative growth or sexual reorganization. In order to get an overview of medium-sized (40–500 nt) RNAs expressed from the Tetrahymena genome, we created a size-fractionated cDNA library from macronuclear RNA and analyzed 80 RNAs, most of which were previously unknown. The most abundant class was small nucleolar RNAs (snoRNAs), many of which are formed by an unusual maturation pathway. The modifications guided by the snoRNAs were analyzed bioinformatically and experimentally and many Tetrahymena-specific modifications were found, including several in an essential, but not conserved domain of ribosomal RNA. Of particular interest, we detected two methylations in the 5′-end of U6 small nuclear RNA (snRNA) that has an unusual structure in Tetrahymena. Further, we found a candidate for the first U8 outside metazoans, and an unusual U14 candidate. In addition, a number of candidates for new non-coding RNAs were characterized by expression analysis at different growth conditions.

 

RNA-guided DNA rearrangements in ciliates: is the best genome defence a good offence?  

Coyne RS, Lhuillier-Akakpo M, Duharcourt S.

Biol Cell. 2012 Jun;104(6):309-25. doi: 10.1111/boc.201100057. Epub 2012 Apr 18. Review

http://onlinelibrary.wiley.com/doi/10.1111/boc.201100057/full

Abstract

Genomes, like crazy patchwork quilts, are stitched together over evolutionary time from diverse elements, including some unwelcome invaders. To deal with parasitic mobile elements, most eukaryotes employ a genome self-defensive manoeuvre to recognise and silence such elements by homology-dependent interactions with RNA–protein complexes that alter chromatin. Ciliated protozoa employ more ‘offensive’ tactics by actually unstitching and reassembling their somatic genomes at every sexual generation to eliminate transposons and their remnants, using as patterns the maternal genomes that were rearranged in the previous cycle. Genetic and genomic studies of the distant relatives Paramecium and Tetrahymena have begun to reveal how such events are carried out with remarkable precision. Whole genome, non-coding transcripts from the maternal genome are compared with transcripts from the zygotic genome that are processed through an RNA interference (RNAi)-related process. Sequences found only in the latter are targeted for elimination by the resulting short ‘scanRNAs’ in many thousand DNA splicing reactions initiated by a domesticated transposase. The involvement of widely conserved mechanisms and protein factors clearly shows the relatedness of these phenomena to RNAi-mediated heterochromatic gene silencing. Such malleability of the genome on a generational time scale also has profound evolutionary implications, possibly including the epigenetic inheritance of acquired adaptive traits.

 

 

Epigenetics of ciliates.  

Chalker DL, Meyer E, Mochizuki K.

Cold Spring Harb Perspect Biol. 2013 Dec 1;5(12):a017764. doi: 10.1101/cshperspect.a017764. Review.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3839606/

Abstract

Research using ciliates revealed early examples of epigenetic phenomena and continues to provide novel findings. These protozoans maintain separate germline and somatic nuclei that carry transcriptionally silent and active genomes, respectively. Examining the differences in chromatin within distinct nuclei of Tetrahymena identified histone variants and established that transcriptional regulators act by modifying histones. Formation of somatic nuclei requires both transcriptional activation of silent chromatin and large-scale DNA elimination. This somatic genome remodeling is directed by homologous RNAs, acting with an RNA interference (RNAi)-related machinery. Furthermore, the content of the parental somatic genome provides a homologous template to guide this genome restructuring. The mechanisms regulating ciliate DNA rearrangements reveal the surprising power of homologous RNAs to remodel the genome and transmit information transgenerationally.

 

 

Analysis of Piwi-loaded small RNAs in Tetrahymena.  

Noto T, Kurth HM, Mochizuki K.

Methods Mol Biol. 2014;1093:209-24. doi: 10.1007/978-1-62703-694-8_17. Erratum in: Methods Mol Biol. 2014;1093:E1-2.

Abstract: http://www.ncbi.nlm.nih.gov/pubmed/?term=Analysis+of+Piwi-loaded+small+RNAs+in+Tetrahymena

Scan RNAs (scnRNAs) are developmentally regulated siRNAs of ~26-32 nucleotides in length that are involved in programmed DNA elimination in Tetrahymena. scnRNAs are loaded onto the Piwi-related protein Twi1p and 2'-O-methylated at their 3' termini. We describe two alternative strategies for analyzing the Twi1p-loaded scnRNAs: preparation of loaded scnRNAs by immuno-purification of the Twi1p-scnRNA complex and exclusion of non-methylated scnRNAs during cDNA library construction using periodate oxidation.

 

 

Transposon domestication versus mutualism in ciliate genome rearrangements. 

Vogt A, Goldman AD, Mochizuki K, Landweber LF.

PLoS Genet. 2013;9(8):e1003659. doi: 10.1371/journal.pgen.1003659. Epub 2013 Aug 1. Review.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3731211/

Abstract

Ciliated protists rearrange their genomes dramatically during nuclear development via chromosome fragmentation and DNA deletion to produce a trimmer and highly reorganized somatic genome. The deleted portion of the genome includes potentially active transposons or transposon-like sequences that reside in the germline. Three independent studies recently showed that transposase proteins of the DDE/DDD superfamily are indispensible for DNA processing in three distantly related ciliates. In the spirotrich Oxytricha trifallax, high copy-number germline-limited transposons mediate their own excision from the somatic genome but also contribute to programmed genome rearrangement through a remarkable transposon mutualism with the host. By contrast, the genomes of two oligohymenophorean ciliates, Tetrahymena thermophila and Paramecium tetraurelia, encode homologous PiggyBac-like transposases as single-copy genes in both their germline and somatic genomes. These domesticated transposases are essential for deletion of thousands of different internal sequences in these species. This review contrasts the events underlying somatic genome reduction in three different ciliates and considers their evolutionary origins and the relationships among their distinct mechanisms for genome remodeling.

 

 

Loading and pre-loading processes generate a distinct siRNA population in Tetrahymena.  

Mochizuki K, Kurth HM.

Biochem Biophys Res Commun. 2013 Jul 5;436(3):497-502. doi: 10.1016/j.bbrc.2013.05.133. Epub 2013 Jun 11.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3714595/

Abstract

The various properties of small RNAs, such as length, terminal nucleotide, thermodynamic asymmetry and duplex mismatches, can impact their sorting into different Argonaute proteins in diverse eukaryotes. The developmentally regulated 26- to 32-nt siRNAs (scnRNAs) are loaded to the Argonaute protein Twi1p and display a strong bias for uracil at the 5′ end. In this study, we used deep sequencing to analyze loaded and unloaded populations of scnRNAs. We show that the size of the scnRNA is determined during a pre-loading process, whereas their 5′ uracil bias is attributed to both pre-loading and loading processes. We also demonstrate that scnRNAs have a strong bias for adenine at the third base from the 3′ terminus, suggesting that most scnRNAs are direct Dicer products. Furthermore, we show that the thermodynamic asymmetry of the scnRNA duplex does not affect the guide and passenger strand decision. Finally, we show that scnRNAs frequently have templated uracil at the last base without a strong bias for adenine at the second base indicating non-sequential production of scnRNAs from substrates. These findings provide a biochemical basis for the varying attributes of scnRNAs, which should help improve our understanding of the production and turnover of scnRNAs in vivo.

 

 

Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena.   

Schoeberl UE, Kurth HM, Noto T, Mochizuki K.

Genes Dev. 2012 Aug 1;26(15):1729-42. doi: 10.1101/gad.196493.112.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3418590/

Abstract

The ciliated protozoan Tetrahymena undergoes extensive programmed DNA elimination when the germline micronucleus produces the new macronucleus during sexual reproduction. DNA elimination is epigenetically controlled by DNA sequences of the parental macronuclear genome, and this epigenetic regulation is mediated by small RNAs (scan RNAs [scnRNAs]) of 2830 nucleotides that are produced and function by an RNAi-related mechanism. Here, we examine scnRNA production and turnover by deep sequencing. scnRNAs are produced exclusively from the micronucleus and nonhomogeneously from a variety of chromosomal locations. scnRNAs are preferentially derived from the eliminated sequences, and this preference is mainly determined at the level of transcription. Despite this bias, a significant fraction of scnRNAs is also derived from the macronuclear-destined sequences, and these scnRNAs are degraded during the course of sexual reproduction. These results indicate that the pattern of DNA elimination in the new macronucleus is shaped by the biased transcription in the micronucleus and the selective degradation of scnRNAs in the parental macronucleus.

 

 

Developmentally programmed, RNA-directed genome rearrangement in Tetrahymena.   

Mochizuki K.

Dev Growth Differ. 2012 Jan;54(1):108-19. doi: 10.1111/j.1440-169X.2011.01305.x. Epub 2011 Nov 22. Review

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3746296/

Abstract

Developmentally programmed genome rearrangement has been observed in a variety of eukaryotes from vertebrates to worms to protists, and it provides an interesting exception to the general rule of the constancy of the genome. DNA elimination in the ciliated protozoan Tetrahymena is one of the most well-characterized programmed genome rearrangement events. DNA elimination in the newly formed macronucleus of Tetrahymena is epigenetically regulated by the DNA sequence of the parental macronucleus. Dicer-produced, Piwi-associated small RNAs mediate this epigenetic regulation, probably through a whole-genome comparison of the germline micronucleus to the somatic macronucleus. However, a correlation between small RNAs and programmed genome rearrangement could not be detected in the worm Ascaris suum. Therefore, different types of eukaryotes may have developed unique solutions to perform genome rearrangement.

 

 

Targeted Gene Disruption by Ectopic Induction of DNA Elimination in Tetrahymena.  

Hayashi A, Mochizuki K.

Genetics. 2015 Sep;201(1):55-64. doi: 10.1534/genetics.115.178525. Epub 2015 Jul 23.

Abstract: http://www.ncbi.nlm.nih.gov/pubmed/?term=Targeted+Gene+Disruption+by+Ectopic+Induction+of+DNA+Elimination+in+Tetrahymena.

Tetrahymena is a useful eukaryotic model for biochemistry and molecular cell biology studies. We previously demonstrated that targeted ectopic DNA elimination, also called co-Deletion (coDel), can be induced by the introduction of an internal eliminated sequence (IES)-target DNA chimeric construct. In this study, we demonstrate that coDel occurs at most of the loci tested and can be used for the production of somatic gene KO strains. We also showed that coDel at two loci can be simultaneously induced by a single transformation; thus, coDel can be used to disrupt multiple gene loci in a single cell. Therefore, coDel is a useful tool for functional genetics in Tetrahymena and further extends the usefulness of this model organism.

 

 

Small-RNA-Mediated Genome-wide trans-Recognition Network in Tetrahymena DNA Elimination. 

Noto T, Kataoka K, Suhren JH, Hayashi A, Woolcock KJ, Gorovsky MA, Mochizuki K.

Mol Cell. 2015 Jul 16;59(2):229-42. doi: 10.1016/j.molcel.2015.05.024. Epub 2015 Jun 18.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4518040/

Summary

Small RNAs are used to silence transposable elements (TEs) in many eukaryotes, which use diverse evolutionary solutions to identify TEs. In ciliated protozoans, small-RNA-mediated comparison of the germline and somatic genomes underlies identification of TE-related sequences, which are then eliminated from the soma. Here, we describe an additional mechanism of small-RNA-mediated identification of TE-related sequences in the ciliate Tetrahymena. We show that a limited set of internal eliminated sequences (IESs) containing potentially active TEs produces a class of small RNAs that recognize not only the IESs from which they are derived, but also other IESs in trans. This trans recognition triggers the expression of yet another class of small RNAs that identify other IESs. Therefore, TE-related sequences in Tetrahymenaare robustly targeted for elimination by a genome-wide trans-recognition network accompanied by a chain reaction of small RNA production.

 

 

Epigenetic regulation of serotype expression antagonizes transcriptome dynamics in Parameciumtetraurelia. 

Cheaib M, Dehghani Amirabad A, Nordström KJ, Schulz MH, Simon M.

DNA Res. 2015 Aug;22(4):293-305. doi: 10.1093/dnares/dsv014. Epub 2015 Jul 31.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4535620/

Summary

Small RNAs are used to silence transposable elements (TEs) in many eukaryotes, which use diverse evolutionary solutions to identify TEs. In ciliated protozoans, small-RNA-mediated comparison of the germline and somatic genomes underlies identification of TE-related sequences, which are then eliminated from the soma. Here, we describe an additional mechanism of small-RNA-mediated identification of TE-related sequences in the ciliate Tetrahymena. We show that a limited set of internal eliminated sequences (IESs) containing potentially active TEs produces a class of small RNAs that recognize not only the IESs from which they are derived, but also other IESs in trans. This trans recognition triggers the expression of yet another class of small RNAs that identify other IESs. Therefore, TE-related sequences in Tetrahymena are robustly targeted for elimination by a genome-wide trans-recognition network accompanied by a chain reaction of small RNA production.

 

 

Genomic characterization of variable surface antigens reveals a telomere position effect as a prerequisite for RNA interference-mediated silencing in Paramecium tetraurelia.  

Baranasic D, Oppermann T, Cheaib M, Cullum J, Schmidt H, Simon M.

MBio. 2014 Nov 11;5(6):e01328. doi: 10.1128/mBio.01328-14.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4235209/

ABSTRACT

Antigenic or phenotypic variation is a widespread phenomenon of expression of variable surface protein coats on eukaryotic microbes. To clarify the mechanism behind mutually exclusive gene expression, we characterized the genetic properties of the surface antigen multigene family in the ciliate Paramecium tetraurelia and the epigenetic factors controlling expression and silencing. Genome analysis indicated that the multigene family consists of intrachromosomal and subtelomeric genes; both classes apparently derive from different gene duplication events: whole-genome and intrachromosomal duplication. Expression analysis provides evidence for telomere position effects, because only subtelomeric genes follow mutually exclusive transcription. Microarray analysis of cultures deficient in Rdr3, an RNA-dependent RNA polymerase, in comparison to serotype-pure wild-type cultures, shows cotranscription of a subset of subtelomeric genes, indicating that the telomere position effect is due to a selective occurrence of Rdr3-mediated silencing in subtelomeric regions. We present a model of surface antigen evolution by intrachromosomal gene duplication involving the maintenance of positive selection of structurally relevant regions. Further analysis of chromosome heterogeneity shows that alternative telomere addition regions clearly affect transcription of closely related genes. Consequently, chromosome fragmentation appears to be of crucial importance for surface antigen expression and evolution. Our data suggest that RNAi-mediated control of this genetic network by trans-acting RNAs allows rapid epigenetic adaptation by phenotypic variation in combination with long-term genetic adaptation by Darwinian evolution of antigen genes.

 

 

Local effect of enhancer of zeste-like reveals cooperation of epigenetic and cis-acting determinants for zygotic genome rearrangements. 

Lhuillier-Akakpo M, Frapporti A, Denby Wilkes C, Matelot M, Vervoort M, Sperling L, Duharcourt S.

PLoS Genet. 2014 Sep 25;10(9):e1004665. doi: 10.1371/journal.pgen.1004665. eCollection 2014 Sep.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4177680/

Abstract

In the ciliate Paramecium tetraurelia, differentiation of the somatic nucleus from the zygotic nucleus is characterized by massive and reproducible deletion of transposable elements and of 45,000 short, dispersed, single-copy sequences. A specific class of small RNAs produced by the germline during meiosis, the scnRNAs, are involved in the epigenetic regulation of DNA deletion but the underlying mechanisms are poorly understood. Here, we show that trimethylation of histone H3 (H3K27me3 and H3K9me3) displays a dynamic nuclear localization that is altered when the endonuclease required for DNA elimination is depleted. We identified the putative histone methyltransferase Ezl1 necessary for H3K27me3 and H3K9me3 establishment and show that it is required for correct genome rearrangements. Genome-wide analyses show that scnRNA-mediated H3 trimethylation is necessary for the elimination of long, repeated germline DNA, while single copy sequences display differential sensitivity to depletion of proteins involved in the scnRNA pathway, Ezl1- a putative histone methyltransferase and Dcl5- a protein required for iesRNA biogenesis. Our study reveals cis-acting determinants, such as DNA length, also contribute to the definition of germline sequences to delete. We further show that precise excision of single copy DNA elements, as short as 26 bp, requires Ezl1, suggesting that development specific H3K27me3 and H3K9me3 ensure specific demarcation of very short germline sequences from the adjacent somatic sequences.

 

 

Genome-wide analysis of genetic and epigenetic control of programmed DNA deletion. 

Swart EC, Denby Wilkes C, Sandoval PY, Arambasic M, Sperling L, Nowacki M.

Nucleic Acids Res. 2014 Aug;42(14):8970-83. doi: 10.1093/nar/gku619. Epub 2014 Jul 12.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4132734/

ABSTRACT

During the development of the somatic genome from the Paramecium germline genome the bulk of the copies of 45 000 unique, internal eliminated sequences (IESs) are deleted. IES targeting is facilitated by two small RNA (sRNA) classes: scnRNAs, which relay epigenetic information from the parental nucleus to the developing nucleus, and iesRNAs, which are produced and used in the developing nucleus. Why only certain IESs require sRNAs for their removal has been enigmatic. By analyzing the silencing effects of three genes: PGM (responsible for DNA excision), DCL2/3 (scnRNA production) and DCL5 (iesRNA production), we identify key properties required for IES elimination. Based on these results, we propose that, depending on the exact combination of their lengths and end bases, some IESs are less efficiently recognized or excised and have a greater requirement for targeting by scnRNAs and iesRNAs. We suggest that the variation in IES retention following silencing of DCL2/3 is not primarily due to scnRNA density, which is comparatively uniform relative to IES retention, but rather the genetic properties of IESs. Taken together, our analyses demonstrate that in Paramecium the underlying genetic properties of developmentally deleted DNA sequences are essential in determining the sensitivity of these sequences to epigenetic control.

 

 

Primary and secondary siRNA synthesis triggered by RNAs from food bacteria in the ciliateParamecium tetraurelia. 

Carradec Q, Götz U, Arnaiz O, Pouch J, Simon M, Meyer E, Marker S.

Nucleic Acids Res. 2015 Feb 18;43(3):1818-33. doi: 10.1093/nar/gku1331. Epub 2015 Jan 15.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4330347/

ABSTRACT

In various organisms, an efficient RNAi response can be triggered by feeding cells with bacteria producing double-stranded RNA (dsRNA) against an endogenous gene. However, the detailed mechanisms and natural functions of this pathway are not well understood in most cases. Here, we studied siRNA biogenesis from exogenous RNA and its genetic overlap with endogenous RNAi in the ciliate Paramecium tetraurelia by high-throughput sequencing. Using wild-type and mutant strains deficient for dsRNA feeding we found that high levels of primary siRNAs of both strands are processed from the ingested dsRNA trigger by the Dicer Dcr1, the RNA-dependent RNA polymerases Rdr1 and Rdr2 and other factors. We further show that this induces the synthesis of secondary siRNAs spreading along the entire endogenous mRNA, demonstrating the occurrence of both 3′-to-5′ and 5′-to-3′ transitivity for the first time in the SAR clade of eukaryotes (Stramenopiles, Alveolates, Rhizaria). Secondary siRNAs depend on Rdr2 and show a strong antisense bias; they are produced at much lower levels than primary siRNAs and hardly contribute to RNAi efficiency. We further provide evidence that the Paramecium RNAi machinery also processes single-stranded RNAs from its bacterial food, broadening the possible natural functions of exogenously induced RNAi in this organism.

 

TFIIS-Dependent Non-coding Transcription Regulates Developmental Genome Rearrangements. 

Maliszewska-Olejniczak K, Gruchota J, Gromadka R, Denby Wilkes C, Arnaiz O, Mathy N, Duharcourt S, Bétermier M, Nowak JK.

PLoS Genet. 2015 Jul 15;11(7):e1005383. doi: 10.1371/journal.pgen.1005383. eCollection 2015 Jul.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4503560/

Abstract

Because of their nuclear dimorphism, ciliates provide a unique opportunity to study the role of non-coding RNAs (ncRNAs) in the communication between germline and somatic lineages. In these unicellular eukaryotes, a new somatic nucleus develops at each sexual cycle from a copy of the zygotic (germline) nucleus, while the old somatic nucleus degenerates. In the ciliate Paramecium tetraurelia, the genome is massively rearranged during this process through the reproducible elimination of repeated sequences and the precise excision of over 45,000 short, single-copy Internal Eliminated Sequences (IESs). Different types of ncRNAs resulting from genome-wide transcription were shown to be involved in the epigenetic regulation of genome rearrangements. To understand how ncRNAs are produced from the entire genome, we have focused on a homolog of the TFIIS elongation factor, which regulates RNA polymerase II transcriptional pausing. Six TFIIS-paralogs, representing four distinct families, can be found in P. tetraurelia genome. Using RNA interference, we showed that TFIIS4, which encodes a development-specific TFIIS protein, is essential for the formation of a functional somatic genome. Molecular analyses and high-throughput DNA sequencing upon TFIIS4 RNAi demonstrated that TFIIS4 is involved in all kinds of genome rearrangements, including excision of ~48% of IESs. Localization of a GFP-TFIIS4 fusion revealed that TFIIS4 appears specifically in the new somatic nucleus at an early developmental stage, before IES excision. RT-PCR experiments showed that TFIIS4 is necessary for the synthesis of IES-containing non-coding transcripts. We propose that these IES+ transcripts originate from the developing somatic nucleus and serve as pairing substrates for germline-specific short RNAs that target elimination of their homologous sequences. Our study, therefore, connects the onset of zygotic non coding transcription to the control of genome plasticity in Paramecium, and establishes for the first time a specific role of TFIIS in non-coding transcription in eukaryotes.

 

 

Programmed Rearrangement in Ciliates: Paramecium. 

Betermier M, Duharcourt S.

Microbiol Spectr. 2014 Dec;2(6). doi: 10.1128/microbiolspec.MDNA3-0035-2014.

http://www.asmscience.org/content/journal/microbiolspec/10.1128/microbiolspec.MDNA3-0035-2014#backarticlefulltext

Abstract

Programmed genome rearrangements in the ciliate Paramecium provide a nice illustration of the impact of transposons on genome evolution and plasticity. During the sexual cycle, development of the somatic macronucleus involves elimination of 30% of the germline genome, including repeated DNA (e.g., transposons) and 45,000 single-copy internal eliminated sequences (IES). IES excision is a precise cut-and-close process, in which double-stranded DNA cleavage at IES ends depends on PiggyMac, a domesticated piggyBac transposase. Genome-wide analysis has revealed that at least a fraction of IESs originate from Tc/mariner transposons unrelated to piggyBac. Moreover, genomic sequences with no transposon origin, such as gene promoters, can be excised reproducibly as IESs, indicating that genome rearrangements contribute to the control of gene expression. How the system has evolved to allow elimination of DNA sequences with no recognizable conserved motif has been the subject of extensive research during the past two decades. Increasing evidence has accumulated for the participation of noncoding RNAs in epigenetic control of elimination for a subset of IESs, and in trans-generational inheritance of alternative rearrangement patterns. This chapter summarizes our current knowledge of the structure of the germline and somatic genomes for the model species Paramecium tetraurelia, and describes the DNA cleavage and repair factors that constitute the IES excision machinery. We present an overview of the role of specialized RNA interference machineries and their associated noncoding RNAs in the control of DNA elimination. Finally, we discuss how RNA-dependent modification and/or remodeling of chromatin may guide PiggyMac to its cognate cleavage sites.

 

 

Transposon Invasion of the Paramecium Germline Genome Countered by a Domesticated PiggyBac Transposase and the NHEJ Pathway. 

Dubois E, Bischerour J, Marmignon A, Mathy N, Régnier V, Bétermier M.

Int J Evol Biol. 2012;2012:436196. doi: 10.1155/2012/436196. Epub 2012 Jul 22.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3408717/

Abstract

Sequences related to transposons constitute a large fraction of extant genomes, but insertions within coding sequences have generally not been tolerated during evolution. Thanks to their unique nuclear dimorphism and to their original mechanism of programmed DNA elimination from their somatic nucleus (macronucleus), ciliates are emerging model organisms for the study of the impact of transposable elements on genomes. The germline genome of the ciliate Paramecium, located in its micronucleus, contains thousands of short intervening sequences, the IESs, which interrupt 47% of genes. Recent data provided support to the hypothesis that an evolutionary link exists between Paramecium IESs and Tc1/mariner transposons. During development of the macronucleus, IESs are excised precisely thanks to the coordinated action of PiggyMac, a domesticated piggyBac transposase, and of the NHEJ double-strand break repair pathway. A PiggyMac homolog is also required for developmentally programmed DNA elimination in another ciliate, Tetrahymena. Here, we present an overview of the life cycle of these unicellular eukaryotes and of the developmentally programmed genome rearrangements that take place at each sexual cycle. We discuss how ancient domestication of a piggyBac transposase might have allowed Tc1/mariner elements to spread throughout the germline genome of Paramecium, without strong counterselection against insertion within genes.

 

 

The eukaryotic way to defend and edit genomes by sRNA-targeted DNA deletion.  

Swart EC, Nowacki M.

Ann N Y Acad Sci. 2015 Apr;1341:106-14. doi: 10.1111/nyas.12636. Epub 2015 Jan 7. Review.

http://onlinelibrary.wiley.com/doi/10.1111/nyas.12636/full

Abstract

While there is currently burgeoning interest in the application of the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) to genome editing, it is perhaps not widely appreciated that this is the second discovery of a small RNA (sRNA)–targeted DNA-deletion system. The first sRNA–targeted DNA-deletion system to be discovered, which we call IES/Ias (internal eliminated sequence/IES-associated genes) to contrast with CRISPR/Cas, is found in ciliates, and, like CRISPR/Cas, is thought to serve as a form of immune defense against invasive DNAs. The manner in which the ciliate IES/Ias system functions is distinct from that of the CRISPR/Cas system in archaea and bacteria, and arose independently through a synthesis of RNA interference–derived and DNA-specific molecular components. Despite the major differences between CRISPR/Cas and IES/Ias, both systems face similar conceptual challenges in targeting invasive DNAs. In this review, we focus on the discovery, effects, function, and evolutionary consequences of the IES/Ias system.

 

 

RNA-dependent genome processing during nuclear differentiation: the model systems of stichotrichous ciliates.  

Fuhrmann G, Swart E, Nowacki M, Lipps HJ.

Epigenomics. 2013 Apr;5(2):229-36. doi: 10.2217/epi.13.15.

Abstract: http://www.ncbi.nlm.nih.gov/pubmed/?term=RNA-dependent+genome+processing+during+nuclear+differentiation%3A+the+model+systems+of+stichotrichous+ciliates

We introduce ciliated protozoa, and more specifically the stichotrichous ciliates Oxytricha and Stylonychia, as biological model systems for the analysis of programmed DNA-reorganization processes during nuclear differentiation. These include DNA excision, DNA elimination, reordering of gene segments and specific gene amplification. We show that small nuclear RNAs specify DNA sequences to be excised or retained, but also discuss the need for a RNA template molecule derived from the parental nucleus for these processes. This RNA template guides reordering of gene segments to become functional genes and determines gene copy number in the differentiated nucleus. Since the template is derived from the parental macronucleus, gene reordering and DNA amplification are inherited in a non-Mendelian epigenetic manner.

Topic 2

 

The Oxytricha trifallax macronuclear genome: a complex eukaryotic genome with 16,000 tiny chromosomes.    

Swart EC, Bracht JR, Magrini V, Minx P, Chen X, Zhou Y, Khurana JS, Goldman AD, Nowacki M, Schotanus K, Jung S, Fulton RS, Ly A, McGrath S, Haub K, Wiggins JL, Storton D, Matese JC, Parsons L, Chang WJ, Bowen MS, Stover NA, Jones TA, Eddy SR, Herrick GA, Doak TG, Wilson RK, Mardis ER, Landweber LF.

PLoS Biol. 2013;11(1):e1001473. doi: 10.1371/journal.pbio.1001473. Epub 2013 Jan 29.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3558436/

Abstract

The macronuclear genome of the ciliate Oxytricha trifallax displays an extreme and unique eukaryotic genome architecture with extensive genomic variation. During sexual genome development, the expressed, somatic macronuclear genome is whittled down to the genic portion of a small fraction (5%) of its precursor silent germline micronuclear genome by a process of unscrambling and fragmentation. The tiny macronuclear nanochromosomes typically encode single, protein-coding genes (a small portion, 10%, encode 28 genes), have minimal noncoding regions, and are differentially amplified to an average of 2,000 copies. We report the high-quality genome assembly of 16,000 complete nanochromosomes (50 Mb haploid genome size) that vary from 469 bp to 66 kb long (mean 3.2 kb) and encode 18,500 genes. Alternative DNA fragmentation processes 10% of the nanochromosomes into multiple isoforms that usually encode complete genes. Nucleotide diversity in the macronucleus is very high (SNP heterozygosity is 4.0%), suggesting that Oxytricha trifallax may have one of the largest known effective population sizes of eukaryotes. Comparison to other ciliates with nonscrambled genomes and long macronuclear chromosomes (on the order of 100 kb) suggests several candidate proteins that could be involved in genome rearrangement, including domesticated MULE and IS1595-like DDE transposases. The assembly of the highly fragmented Oxytricha macronuclear genome is the first completed genome with such an unusual architecture. This genome sequence provides tantalizing glimpses into novel molecular biology and evolution. For example, Oxytricha maintains tens of millions of telomeres per cell and has also evolved an intriguing expansion of telomere end-binding proteins. In conjunction with the micronuclear genome in progress, the O. trifallax macronuclear genome will provide an invaluable resource for investigating programmed genome rearrangements, complementing studies of rearrangements arising during evolution and disease.

 

 

Copy number variations of 11 macronuclear chromosomes and their gene expression in Oxytricha trifallax.  

Xu K, Doak TG, Lipps HJ, Wang J, Swart EC, Chang WJ.

Gene. 2012 Aug 15;505(1):75-80. doi: 10.1016/j.gene.2012.05.045. Epub 2012 Jun 2.

http://www.sciencedirect.com/science/article/pii/S0378111912006518

Abstract

Ciliated protozoa are peculiar for their nuclear dimorphism, wherein two types of nuclei divide nuclear functions: a germline micronucleus (MIC) is transcriptionally inert during vegetative growth, but serves as the genetic blueprint for the somatic macronucleus (MAC), which is responsible for all transcripts supporting cell growth and reproduction. While all the advantages/disadvantages associated with nuclear dimorphism are not clear, an essential advantage seems to be the ability to produce a highly polyploid MAC, which then allows for the maintenance of extremely large single cells — many ciliate cells are larger than small metazoa. In some ciliate classes, chromosomes in the MAC are extensively fragmented to create extremely short chromosomes that often carry single genes, and these chromosomes may be present in different copy numbers, resulting in different ploidies. While using gene copy number to regulate gene expression is limited in most eukaryotic systems, the extensive fragmentation in some ciliate classes provides this opportunity to every MAC gene. However, it is still unclear if this mechanism is in fact used extensively in these ciliates. To address this, we have quantified copy numbers of 11 MAC chromosomes and their gene expression in Oxytricha trifallax (CI: Spirotrichea). We compared copy numbers between two subpopulations of O. trifallax, and copy numbers of 7 orthologous genes between O. trifallax and the closely related Stylonychia lemnae. We show that copy numbers of MAC chromosomes are variable, dynamic, and positively correlated to gene expression. These features might be conserved in all spirotrichs, and might exist in other classes of ciliates with heavily fragmented MAC chromosomes.

 

 

Characterization and taxonomic validity of the ciliate Oxytricha trifallax (Class Spirotrichea) based on multiple gene sequences: limitations in identifying genera solely by morphology.  

Zoller SD, Hammersmith RL, Swart EC, Higgins BP, Doak TG, Herrick G, Landweber LF.

Protist. 2012 Jul;163(4):643-57. doi: 10.1016/j.protis.2011.12.006. Epub 2012 Feb 9

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3433844/

Abstract

Oxytricha trifallax — an established model organism for studying genome rearrangements, chromosome structure, scrambled genes, RNA-mediated epigenetic inheritance, and other phenomena — has been the subject of a nomenclature controversy for several years. Originally isolated as a sibling species of O. fallax, O. trifallax was reclassified in 1999 as Sterkiella histriomuscorum, a previously identified species, based on morphological similarity. The proper identification of O. trifallax is crucial to resolve in order to prevent confusion in both the comparative genomics and the general scientific communities. We analyzed nine conserved nuclear gene sequences between the two given species and several related ciliates. Phylogenetic analyses suggest that O. trifallax and a bona fide S. histriomuscorum have accumulated significant evolutionary divergence from each other relative to other ciliates such that they should be unequivocally classified as separate species. We also describe the original isolation of O. trifallax, including its comparison to O. fallax, and we provide criteria to identify future isolates of O. trifallax.

 

 

Pdsg1 and Pdsg2, novel proteins involved in developmental genome remodelling in Paramecium.  

Arambasic M, Sandoval PY, Hoehener C, Singh A, Swart EC, Nowacki M.

PLoS One. 2014 Nov 14;9(11):e112899. doi: 10.1371/journal.pone.0112899. eCollection 2014. Erratum in: PLoS One. 2015;10(2):e0118384.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4232520/

Abstract

The epigenetic influence of maternal cells on the development of their progeny has long been studied in various eukaryotes. Multicellular organisms usually provide their zygotes not only with nutrients but also with functional elements required for proper development, such as coding and non-coding RNAs. These maternally deposited RNAs exhibit a variety of functions, from regulating gene expression to assuring genome integrity. In ciliates, such as Paramecium these RNAs participate in the programming of large-scale genome reorganization during development, distinguishing germline-limited DNA, which is excised, from somatic-destined DNA. Only a handful of proteins playing roles in this process have been identified so far, including typical RNAi-derived factors such as Dicer-like and Piwi proteins. Here we report and characterize two novel proteins, Pdsg1 and Pdsg2 (Paramecium protein involved in Development of the Somatic Genome 1 and 2), involved in Paramecium genome reorganization. We show that these proteins are necessary for the excision of germline-limited DNA during development and the survival of sexual progeny. Knockdown of PDSG1 and PDSG2 genes affects the populations of small RNAs known to be involved in the programming of DNA elimination (scanRNAs and iesRNAs) and chromatin modification patterns during development. Our results suggest an association between RNA-mediated trans-generational epigenetic signal and chromatin modifications in the process of Paramecium genome reorganization.

 

 

The draft assembly of the radically organized Stylonychia lemnae macronuclear genome. 

Aeschlimann SH, Jönsson F, Postberg J, Stover NA, Petera RL, Lipps HJ, Nowacki M, Swart EC.

Genome Biol Evol. 2014 Jun 20;6(7):1707-23. doi: 10.1093/gbe/evu139.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4122937/

Abstract

Stylonychia lemnae is a classical model single-celled eukaryote, and a quintessential ciliate typified by dimorphic nuclei: A small, germline micronucleus and a massive, vegetative macronucleus. The genome within Stylonychia’s macronucleus has a very unusual architecture, comprised variably and highly amplified “nanochromosomes,” each usually encoding a single gene with a minimal amount of surrounding noncoding DNA. As only a tiny fraction of the Stylonychia genes has been sequenced, and to promote research using this organism, we sequenced its macronuclear genome. We report the analysis of the 50.2-Mb draft S. lemnae macronuclear genome assembly, containing in excess of 16,000 complete nanochromosomes, assembled as less than 20,000 contigs. We found considerable conservation of fundamental genomic properties between S. lemnae and its close relative, Oxytricha trifallax, including nanochromosomal gene synteny, alternative fragmentation, and copy number. Protein domain searches in Stylonychia revealed two new telomere-binding protein homologs and the presence of linker histones. Among the diverse histone variants of S. lemnae and O. trifallax, we found divergent, coexpressed variants corresponding to four of the five core nucleosomal proteins (H1.2, H2A.6, H2B.4, and H3.7) suggesting that these ciliates may possess specialized nucleosomes involved in genome processing during nuclear differentiation. The assembly of the S. lemnae macronuclear genome demonstrates that largely complete, well-assembled highly fragmented genomes of similar size and complexity may be produced from one library and lane of Illumina HiSeq 2000 shotgun sequencing. The provision of the S. lemnae macronuclear genome sets the stage for future detailed experimental studies of chromatin-mediated, RNA-guided developmental genome rearrangements.

 

 

A forward genetic screen reveals essential and non-essential RNAi factors in Parameciumtetraurelia.           

Marker S, Carradec Q, Tanty V, Arnaiz O, Meyer E.

Nucleic Acids Res. 2014 Jun;42(11):7268-80. doi: 10.1093/nar/gku223. Epub 2014 May 23.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4066745/

ABSTRACT

In most eukaryotes, small RNA-mediated gene silencing pathways form complex interacting networks. In the ciliate Paramecium tetraurelia, at least two RNA interference (RNAi) mechanisms coexist, involving distinct but overlapping sets of protein factors and producing different types of short interfering RNAs (siRNAs). One is specifically triggered by high-copy transgenes, and the other by feeding cells with double-stranded RNA (dsRNA)-producing bacteria. In this study, we designed a forward genetic screen for mutants deficient in dsRNA-induced silencing, and a powerful method to identify the relevant mutations by whole-genome sequencing. We present a set of 47 mutant alleles for five genes, revealing two previously unknown RNAi factors: a novel Paramecium-specific protein (Pds1) and a Cid1-like nucleotidyl transferase. Analyses of allelic diversity distinguish non-essential and essential genes and suggest that the screen is saturated for non-essential, single-copy genes. We show that non-essential genes are specifically involved in dsRNA-induced RNAi while essential ones are also involved in transgene-induced RNAi. One of the latter, the RNA-dependent RNA polymerase RDR2, is further shown to be required for all known types of siRNAs, as well as for sexual reproduction. These results open the way for the dissection of the genetic complexity, interconnection, mechanisms and natural functions of RNAi pathways in P. tetraurelia.

 

 

The Paramecium germline genome provides a niche for intragenic parasitic DNA: evolutionary dynamics of internal eliminated sequences. 

Arnaiz O, Mathy N, Baudry C, Malinsky S, Aury JM, Denby Wilkes C, Garnier O, Labadie K, Lauderdale BE, Le Mouël A, Marmignon A, Nowacki M, Poulain J, Prajer M, Wincker P, Meyer E, Duharcourt S, Duret L, Bétermier M, Sperling L.

PLoS Genet. 2012;8(10):e1002984. doi: 10.1371/journal.pgen.1002984. Epub 2012 Oct 4.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3464196/

Abstract

Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events. In Paramecium, these rearrangements include precise excision of unique-copy Internal Eliminated Sequences (IES) from the somatic DNA, requiring the activity of a domesticated piggyBac transposase, PiggyMac. We have sequencedParamecium tetraurelia germline DNA, establishing a genome-wide catalogue of 45,000 IESs, in order to gain insight into their evolutionary origin and excision mechanism. We obtained direct evidence that PiggyMac is required for excision of all IESs. Homology with known P. tetraurelia Tc1/mariner transposons, described here, indicates that at least a fraction of IESs derive from these elements. Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P. aureliaspecies complex, but IES invasion of the Paramecium genome appears to be an ongoing process. Once inserted, IESs decay rapidly by accumulation of deletions and point substitutions. Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a 10 bp periodicity, corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex. IESs are equally frequent within and between coding sequences; however, excision is not 100% efficient and there is selective pressure against IES insertions, in particular within highly expressed genes. We discuss the possibility that ancient domestication of a piggyBactransposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences, a fraction of the genome in which parasitic DNA is not usually tolerated.

 

 

The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): illuminating the functional diversity of eukaryotic life in the oceans through transcriptome sequencing. 

Keeling PJ, Burki F, Wilcox HM, Allam B, Allen EE, Amaral-Zettler LA, Armbrust EV, Archibald JM, Bharti AK, Bell CJ, Beszteri B, Bidle KD, Cameron CT, Campbell L, Caron DA, Cattolico RA, Collier JL, Coyne K, Davy SK, Deschamps P, Dyhrman ST, Edvardsen B, Gates RD, Gobler CJ, Greenwood SJ, Guida SM, Jacobi JL, Jakobsen KS, James ER, Jenkins B, John U, Johnson MD, Juhl AR, Kamp A, Katz LA, Kiene R, Kudryavtsev A, Leander BS, Lin S, Lovejoy C, Lynn D, Marchetti A, McManus G, Nedelcu AM, Menden-Deuer S, Miceli C, Mock T, Montresor M, Moran MA, Murray S, Nadathur G, Nagai S, Ngam PB, Palenik B, Pawlowski J, Petroni G, Piganeau G, Posewitz MC, Rengefors K, Romano G, Rumpho ME, Rynearson T, Schilling KB, Schroeder DC, Simpson AG, Slamovits CH, Smith DR, Smith GJ, Smith SR, Sosik HM, Stief P, Theriot E, Twary SN, Umale PE, Vaulot D, Wawrik B, Wheeler GL, Wilson WH, Xu Y, Zingone A, Worden AZ.

PLoS Biol. 2014 Jun 24;12(6):e1001889. doi: 10.1371/journal.pbio.1001889. eCollection 2014 Jun. No abstract available.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4068987/

Microbial ecology is plagued by problems of an abstract nature. Cell sizes are so small and population sizes so large that both are virtually incomprehensible. Niches are so far from our everyday experience as to make their very definition elusive. Organisms that may be abundant and critical to our survival are little understood, seldom described and/or cultured, and sometimes yet to be even seen. One way to confront these problems is to use data of an even more abstract nature: molecular sequence data. Massive environmental nucleic acid sequencing, such as metagenomics or metatranscriptomics, promises functional analysis of microbial communities as a whole, without prior knowledge of which organisms are in the environment or exactly how they are interacting. But sequence-based ecological studies nearly always use a comparative approach, and that requires relevant reference sequences, which are an extremely limited resource when it comes to microbial eukaryotes [1].

In practice, this means sequence databases need to be populated with enormous quantities of data for which we have some certainties about the source. Most important is the taxonomic identity of the organism from which a sequence is derived and as much functional identification of the encoded proteins as possible. In an ideal world, such information would be available as a large set of complete, well-curated, and annotated genomes for all the major organisms from the environment in question. Reality substantially diverges from this ideal, but at least for bacterial molecular ecology, there is a database consisting of thousands of complete genomes from a wide range of taxa, supplemented by a phylogeny-driven approach to diversifying genomics[2]. For eukaryotes, the number of available genomes is far, far fewer, and we have relied much more heavily on random growth of sequence databases [3],[4], raising the question as to whether this is fit for purpose.

 

 

Large-scale phylogenomic analysis reveals the phylogenetic position of the problematic taxon Protocruzia and unravels the deep phylogenetic affinities of the ciliate lineages.  

Gentekaki E, Kolisko M, Boscaro V, Bright KJ, Dini F, Di Giuseppe G, Gong Y, Miceli C, Modeo L, Molestina RE, Petroni G, Pucciarelli S, Roger AJ, Strom SL, Lynn DH.

Mol Phylogenet Evol. 2014 Sep;78:36-42. doi: 10.1016/j.ympev.2014.04.020. Epub 2014 May 6.

http://www.sciencedirect.com/science/article/pii/S1055790314001481

Abstract

The Ciliophora is one of the most studied protist lineages because of its important ecological role in the microbial loop. While there is an abundance of molecular data for many ciliate groups, it is commonly limited to the 18S ribosomal RNA locus. There is a paucity of data when it comes to availability of protein-coding genes especially for taxa that do not belong to the class Oligohymenophorea. To address this gap, we have sequenced EST libraries for 11 ciliate species. A supermatrix was constructed for phylogenomic analysis based on 158 genes and 42,158 characters and included 16 ciliates, four dinoflagellates and nine apicomplexans. This is the first multigene-based analysis focusing on the phylum Ciliophora. Our analyses reveal two robust superclades within the Intramacronucleata; one composed of the classes Spirotrichea, Armophorea and Litostomatea (SAL) and another with Colpodea and Oligohymenophorea. Furthermore, we provide corroborative evidence for removing the ambiguous taxonProtocruzia from the class Spirotrichea and placing it as incertae sedis in the phylum Ciliophora.

 

 

Survey of Paramecium duboscqui using three markers and assessment of the molecular variability in the genus Paramecium. 

Boscaro V, Fokin SI, Verni F, Petroni G.

Mol Phylogenet Evol. 2012 Dec;65(3):1004-13. doi: 10.1016/j.ympev.2012.09.001. Epub 2012 Sep 13.

http://www.sciencedirect.com/science/article/pii/S1055790312003582

Abstract

The genus Paramecium (phylum Ciliophora) is one of the best-known among protozoa. Nevertheless, the knowledge on the diversity and distribution of species within this genus was remarkably scarce until recent times. In the last years a constantly growing amount of data has formed, especially on the distribution of species and the characterization of molecular markers. Much effort has been made on detecting clades inside each morphospecies, which could suggest the presence of sibling species complexes as in the famous case of Paramecium aurelia. In this work we present new data on Paramecium duboscqui, one of the morphospecies that have not yet been surveyed employing DNA sequences as markers. We obtained data from nine strains sampled around the world, using the three most commonly employed markers (18S rRNA gene, ITS1-5.8S-ITS2 and COI gene sequences). Moreover, we compared our results with those already available for other Paramecium species, and performed phylogenetic analyses for the entire genus. We also expanded the knowledge on the ITS2 secondary structure and its usefulness in studies on Paramecium. Our approach, that considers the data of all the species together, highlighted some characteristic patterns as well as some ambiguities that should be further investigated.

 

 

Delimiting Species Boundaries within a Paraphyletic Species Complex: Insights from Morphological, Genetic, and Molecular Data on Paramecium sonneborni (Paramecium aurelia species complex, Ciliophora, Protozoa). 

Przyboś E, Tarcz S, Rautian M, Sawka N.

Protist. 2015 Sep;166(4):438-56. doi: 10.1016/j.protis.2015.07.001. Epub 2015 Jul 22.

http://www.sciencedirect.com/science/article/pii/S1434461015000449

The demarcation of boundaries between protist species is often problematic because of the absence of a uniform species definition, the abundance of cryptic diversity, and the occurrence of convergent morphology. The ciliates belonging to the Paramecium aureliacomplex, consisting of 15 species, are a good model for such systematic and evolutionary studies. One member of the complex is P. sonneborni, previously known only from one stand in Texas (USA), but recently found in two new sampling sites in Cyprus (creeks running to Salt Lake and Oroklini Lake near Larnaca). The studiedParamecium sonneborni strains (from the USA and Cyprus) reveal low viability in the F1 and F2 generations of interstrain hybrids and may be an example of ongoing allopatric speciation. Despite its molecular distinctiveness, we postulate that P. sonneborni should remain in the P. aurelia complex, making it a paraphyletic taxon. Morphological studies have revealed that some features of the nuclear apparatus of P. sonneborni correspond to the P. aurelia spp. complex, while others are similar to P. jenningsi and P. schewiakoffi. The observed discordance indicates rapid splitting of the P. aureliaP. jenningsiP. schewiakoffi group, in which genetic, morphological, and molecular boundaries between species are not congruent.

 

 

The first European stand of Paramecium sonneborni (P. aurelia complex), a species known only from North America (Texas, USA). 

Przyboś E, Tarcz S, Rautian M, Lebedeva N.

Eur J Protistol. 2014 Jun;50(3):236-47. doi: 10.1016/j.ejop.2014.03.001. Epub 2014 Mar 18.

http://www.sciencedirect.com/science/article/pii/S0932473914000182

Abstract

P. aurelia is currently defined as a complex of 15 sibling species including 14 species designated by Sonneborn (1975) and one, P. sonneborni, by Aufderheide et al. (1983). The latter was known from only one stand (Texas, USA). The main reason for the present study was a new stand of Paramecium in Cyprus, with strains recognized as P. sonneborni based on the results of strain crosses, cytological slides, and molecular analyses of three loci (ITS1-5.8S-ITS2-5′LSU rDNA, COICytB). The new stand of P. sonneborni in Europe shows that the species, previously considered endemic, may have a wider range. This demonstrates the impact of under-sampling on the knowledge of the biogeography of microbial eukaryotes. Phylogenetic trees based on all the studied fragments revealed that P. sonneborni forms a separate cluster that is closer to P. jenningsi and Pschewiakoffi than to the other members of the P. aurelia complex.

 

 

New stands of species of the Paramecium aurelia complex (Ciliophora, Protozoa) in Europe and South America (Ecuador). 

Przybós E, Rautian M, Surmacz M.

Folia Biol (Krakow). 2014;62(1):13-6.

http://www.ncbi.nlm.nih.gov/pubmed/24745144

Abstract

The occurrence of species of the P. aurelia complex has been studied at a large scale in Europe and the majority of known species of the complex have been found there. However, a different number of habitats were studied in particular zones of Europe, the greatest number in the central zone. Herein new stands of several species of the Paramecium aurelia complex are presented from Europe including P. primaurelia, P. biaurelia, P. triaurelia, P. octaurelia, P. novaurelia, and P. dodecaurelia. In South America, studies concerning the distribution of the P. aurelia species complex were carried out only occasionally and the presence of some cosmopolitan species of the complex has been recorded, i.e. P. primaurelia, P. biaurelia, and P. tetraurelia. Recently, new stands of P. primaurelia and P. septaurelia were found in Ecuador. Ciliate biogeography and distribution is also discussed.

 

 

Paramecium putrinum (Ciliophora, Protozoa): the first insight into the variation of two DNA fragments - molecular support for the existence of cryptic species. 

Tarcz S, Rautian M, Potekhin A, Sawka N, Beliavskaya A, Kiselev A, Nekrasova I, Przyboś E.

Mol Phylogenet Evol. 2014 Apr;73:140-5. doi: 10.1016/j.ympev.2014.01.019. Epub 2014 Jan 28.

http://www.sciencedirect.com/science/article/pii/S1055790314000323

Abstract

Paramecium putrinum (Claparede & Lachmann 1858) is one of the smallest (80–140 μm long) species of the genus Paramecium. Although it commonly occurs in freshwater reservoirs, no molecular studies of P. putrinum have been conducted to date. Herein we present an assessment of molecular variation in 27 strains collected from widely separated populations by using two selected DNA fragments (ITS1-5.8S-ITS2-5′LSU rDNA and COI mtDNA). Both the trees and haplotype networks reconstructed for both genome fragments show that the studied strains of P. putrinum form five main haplogroups. The mean distance between the studied strains is p-distance = 0.007/0.068 (rDNA/COI) and exhibits similar variability as that between P. bursaria syngens. Based on these data, one could hypothesize that the clusters revealed in the present study may correspond to previously reported syngens and that there are at least five cryptic species within P. putrinum.

 

 

New stands of species of the Paramecium aurelia complex in Africa and Europe.  

Rautian M, Przyboś E, Surmacz M, Lebedeva N.

Folia Biol (Krakow). 2014;62(4):361-6.

http://www.ncbi.nlm.nih.gov/pubmed/25916164

Abstract

The relevance of geographical distribution and the roles of dispersal and spatial isolation during the speciation of microorganisms are nowadays of great interest. The Paramecium aurelia species complex is a perfect model system to explore these questions given its long history as a study subject and broad distribution. However, the world-wide distribution of the Paramecium aurelia complex (Ciliophora, Protista) still needs study, e.g., sampling in the southern hemisphere has been quite limited, while Europe has been investigated for years, with the majority of aurelia species isolated from here. Recently, new stands of species of the P. aurelia complex were found in southern Europe (Malta, Bulgaria, Cyprus) and in the Czech Republic (P. primaurelia, P. triaurelia, P. octaurelia). In Africa (Republic of South Africa), new stands of P. primaurelia, P. triaurelia, and P. octaurelia were found. Interestingly, the rare species P. triaurelia, and P. octaurelia were found to co-occur both in South Africa (SA 13) and the Czech Republic (CKV 8). Newly established strains were identified to species by crossing with the test strains (the reference strains for the particular species).

 

 

The D1-D2 region of the large subunit ribosomal DNA as barcode for ciliates. 

Stoeck T, Przybos E, Dunthorn M.

Mol Ecol Resour. 2014 May;14(3):458-68. doi: 10.1111/1755-0998.12195. Epub 2013 Nov 29.

Abstract: http://www.ncbi.nlm.nih.gov/pubmed/?term=The+D1-D2+region+of+the+large+subunit+ribosomal+DNA+as+barcode+for+ciliates.

Abstract

Ciliates are a major evolutionary lineage within the alveolates, which are distributed in nearly all habitats on our planet and are an essential component for ecosystem function, processes and stability. Accurate identification of these unicellular eukaryotes through, for example, microscopy or mating type reactions is reserved to few specialists. To satisfy the demand for a DNA barcode for ciliates, which meets the standard criteria forDNA barcodes defined by the Consortium for the Barcode of Life (CBOL), we here evaluated the D1-D2 region of the ribosomal DNA large subunit(LSU-rDNA). Primer universality for the phylum Ciliophora was tested in silico with available database sequences as well as in the laboratory with 73 ciliate species, which represented nine of 12 ciliate classes. Primers tested in this study were successful for all tested classes. To test the ability of the D1-D2 region to resolve conspecific and congeneric sequence divergence, 63 Paramecium strains were sampled from 24 mating species. The average conspecific D1-D2 variation was 0.18%, whereas congeneric sequence divergence averaged 4.83%. In pairwise genetic distance analyses, we identified a D1-D2 sequence divergence of <0.6% as an ideal threshold to discriminate Paramecium species. Using this definition, only 3.8% of all conspecific and 3.9% of all congeneric sequence comparisons had the potential of false assignments. Neighbour-joining analyses inferred monophyly for all taxa but for two Paramecium octaurelia strains. Here, we present a protocol for easy DNA amplification of single cells and voucher deposition. In conclusion, the presented data pinpoint the D1-D2 region as an excellent candidate for an official CBOL barcode for ciliated protists

 

New stands of species of the Paramecium aurelia complex (Ciliophora, Protista) in Russia (Siberia, Kamchatka). 

Przyboś E, Rautian M, Surmacz M, Bieliavskaya A.

Folia Biol (Krakow). 2013;61(1-2):41-5.

http://www.ncbi.nlm.nih.gov/pubmed/23767291

Abstract

New stands of P. primaurelia, P. biaurelia, and P. dodecaurelia were found in Russia. P. primaurelia was recorded in Tulun (Siberia, Irkutsk region) and in three stands situated on the Kamchatka peninsula: in Lake Chalaktyrskoye, in the Valley of Geysers, and Petropavlovsk Kamchatski. P. biaurelia was also found in Tulun and in two stands in the vicinity of Lake Baikal and the Buriatia region. P. dodecaurelia was recorded in Cheboksary in European Russia and in other stands situated in Asian Russia: Novosibirsk, the vicinity of Lake Baikal, Buriatia, Kamchatka (Petropavlovsk Kamchatski, Lake Chalaktyrskoye, and Nalychevo). These data extend the ranges of species of the P. aurelia complex in Russia, however, this large territory remains understudied.

 

 

An assessment of haplotype variation in ribosomal and mitochondrial DNA fragments suggests incomplete lineage sorting in some species of the Paramecium aurelia complex (Ciliophora, Protozoa).

Tarcz S, Przyboś E, Surmacz M. 

Mol Phylogenet Evol. 2013 Apr;67(1):255-65. doi: 10.1016/j.ympev.2013.01.016. Epub 2013 Feb 8.

http://www.sciencedirect.com/science/article/pii/S1055790313000535

Abstract

The Paramecium aurelia complex (Ciliophora, Protozoa) Sonneborn (1975) is composed of 15 sibling species, which are morphologically indistinguishable but sexually isolated. Therefore, the P. aurelia complex seems to be an ideal model for testing hypotheses about recent speciation events. Here we present two-locus (ITS1-5.8S-ITS2-5′LSU rDNA and COI mtDNA) analyses using over 120 strains collected from around the world and representing all currently known species of the complex. According to our findings, the studied species show different levels of haplotype variability. Some of them appear on the obtained trees as polyphyletic (e.g., P. dodecaurelia), while others as monophyletic (e.g., P. quadecaurelia), clusters. The revealed discrepancies, which are manifested by different mating behavior and haplotypes not characteristic of particular species, may be explained by incomplete lineage sorting. Furthermore, the phenomena of hybridization and introgression are considered as another explanation for our results. Despite the above discrepancies, “polyphyletic taxa” should be considered true biological species based on the results of genetic crosses. Using a combination of both strain crosses (the biological species concept) and molecular methods (the phylogenetic species concept) seems to be the appropriate way of delimiting species in closely related eukaryotic microorganisms such as the P. aurelia complex.

 

 

New stands of species of the Paramecium aurelia complex (Ciliophora, Protozoa) in the Mediterranean region (Italy, Greece, Morocco). 

Przyboś E, Rautian M.

Folia Biol (Krakow). 2012;60(3-4):147-9.

http://www.ncbi.nlm.nih.gov/pubmed/23342909

Abstract

New stands of species of the Paramecium aurelia complex are presented in the paper, P. primaurelia recorded in Italy (Pisa) and in Morocco (Marrakesh), P. biaurelia in Italy (Calabria), P. triaurelia in Morocco (Ifrane), P. pentaurelia in Greece (Kastorya), and P. dodecaurelia in Italy (Padua)

 

 

New Paramecium quadecaurelia strains (P. aurelia spp. complex, Ciliophora) identified by molecular markers (rDNA and mtDNA).  

Przyboś E, Tarcz S, Dusi E.

Eur J Protistol. 2013 Aug;49(3):477-86. doi: 10.1016/j.ejop.2012.11.001. Epub 2013 Jan 3.

http://www.sciencedirect.com/science/article/pii/S0932473912000922

Abstract

Paramecium quadecaurelia is a rare species (previously known only from two locations) belonging to the P. aurelia species complex. In the present paper, fragments of an rDNA gene (ITS1-5.8S-ITS2-5′ rDNA) and mtDNA genes (cytochrome oxidase subunit I and cytochrome b regions) were employed to assist in the identification and characterization of three new strains collected from Ecuador and Thailand. Molecular data were confirmed by mating reactions. In rDNA and mtDNA trees constructed for species of theP. aurelia complex, all Pquadecaurelia strains, including the three new strains discussed in this study and two known previously from Australia and Africa, form a monophyletic but differentiated clade. The present study shows that genetic differentiation among the strains of P. quadecaurelia is equal to or even greater than the distances between some other P. aurelia species, e.g., P. primaurelia and P. pentaurelia. Such great intra-specific differentiation may indicate a future splitting of the P. quadecaurelia species into reproductively isolated lines.

 

 

Variation in ribosomal and mitochondrial DNA sequences demonstrates the existence of intraspecific groups in Paramecium multimicronucleatum (Ciliophora, Oligohymenophorea).  

Tarcz S, Potekhin A, Rautian M, Przyboś E.

Mol Phylogenet Evol. 2012 May;63(2):500-9. doi: 10.1016/j.ympev.2012.01.024. Epub 2012 Feb 8.

http://www.sciencedirect.com/science/article/pii/S1055790312000413

Abstract

Paramecium quadecaurelia is a rare species (previously known only from two locations) belonging to the P. aurelia species complex. In the present paper, fragments of an rDNA gene (ITS1-5.8S-ITS2-5′ rDNA) and mtDNA genes (cytochrome oxidase subunit I and cytochrome b regions) were employed to assist in the identification and characterization of three new strains collected from Ecuador and Thailand. Molecular data were confirmed by mating reactions. In rDNA and mtDNA trees constructed for species of theP. aurelia complex, all Pquadecaurelia strains, including the three new strains discussed in this study and two known previously from Australia and Africa, form a monophyletic but differentiated clade. The present study shows that genetic differentiation among the strains of P. quadecaurelia is equal to or even greater than the distances between some other P. aurelia species, e.g., P. primaurelia and P. pentaurelia. Such great intra-specific differentiation may indicate a future splitting of the P. quadecaurelia species into reproductively isolated lines.

 

 

Identification of Paramecium bursaria syngens through molecular markers--comparative analysis of three loci in the nuclear and mitochondrial DNA.  

Greczek-Stachura M, Potekhin A, Przyboś E, Rautian M, Skoblo I, Tarcz S.

Protist. 2012 Sep;163(5):671-85. doi: 10.1016/j.protis.2011.10.009. Epub 2011 Dec 9.

http://www.sciencedirect.com/science/article/pii/S1434461011001052

This is the first attempt to resolve the phylogenetic relationship between different syngens of Paramecium bursaria and to investigate at a molecular level the intraspecific differentiation of strains originating from very distant geographical locations. Herein we introduce a new collection of five P. bursaria syngens maintained at St Petersburg State University, as the international collection of syngens was lost in the 1960s. To analyze the degree of speciation within Paramecium bursaria, we examined 26 strains belonging to five different syngens from distant and geographically isolated localities using rDNA (ITS1-5.8S-ITS2-5′LSU) fragments, mitochondrial cytochrome c oxidase subunit I (COI), and H4 gene fragments.

It was shown that P. bursaria strains of the same syngens cluster together in all three inferred molecular phylogenies. The genetic diversity among the studied P. bursariastrains based on rDNA sequences was rather low. The COI divergence of Paramecium bursaria was also definitely lower than that observed in the Paramecium aurelia complex. The nucleotide sequences of the H4 gene analyzed in the present study indicate the extent of genetic differences between the syngens of Paramecium bursaria. Our study demonstrates the diagnostic value of molecular markers, which are important tools in the identification of Paramecium bursaria syngens.

 

 

A two-locus molecular characterization of Paramecium calkinsi. 

Przyboś E, Tarcz S, Potekhin A, Rautian M, Prajer M.

Protist. 2012 Mar;163(2):263-73. doi: 10.1016/j.protis.2011.06.006. Epub 2011 Jul 27

http://www.sciencedirect.com/science/article/pii/S1434461011000538

Paramecium calkinsi (Ciliophora, Protozoa) is a euryhaline species which was first identified in freshwater habitats, but subsequently several strains were also collected from brackish water. It is characterized by clockwise spiral swimming movement and the general morphology of the “bursaria type.” The present paper is the first molecular characterization of P. calkinsi strains recently collected in distant regions in Russia using ITS1-5.8S- ITS2-5′LSU rDNA (1100 bp) and COI (620 bp) mtDNA sequenced gene fragments. For comparison, our molecular analysis includes P. bursaria, exhibiting a similar “bursaria morphotype” as well as species representing the “aurelia type,” i.e., P. caudatumP. multimicronucleatumP. jenningsi, and P. schewiakoffi, and some species of the P. aurelia species complex (P. primaureliaP. tetraurelia, P. sexaurelia, and P. tredecaurelia). We also use data from GenBank concerning other species in the genusParamecium and Tetrahymena (which used as an outgroup). The division of the genusParamecium into four subgenera (proposed by Fokin et al. 2004) is clearly presented by the trees. There is a clear separation between P. calkinsi strains collected from different regions (races). Consequently, given the molecular distances between them, it seems that these races may represent different syngens within the species.

 

Topic 3

 

Characterization and comparative analysis of psychrophilic and mesophilic alpha-amylases from Euplotes species: a contribution to the understanding of enzyme thermal adaptation. 

Yang G, Yang G, Aprile L, Turturo V, Pucciarelli S, Pucciarelli S, Miceli C.

Biochem Biophys Res Commun. 2013 Sep 6;438(4):715-20. doi: 10.1016/j.bbrc.2013.07.113. Epub 2013 Aug 2.

http://www.sciencedirect.com/science/article/pii/S0006291X13012989

Abstract

The eukaryotic α-amylase isolated from the psychrophilic ciliated protozoon Euplotes focardii (EfAmy) was expressed in Escherichia coli and biochemically characterized. Its enzymatic activity was compared to that of the homologous protein from the mesophilic congeneric species Euplotes crassus (EcAmy). The comparison of the amino acid composition and the surface residue composition of the two enzymes indicated a preference for tiny residues and the avoidance of charged, aromatic and hydrophobic residues in EfAmy. Our comparative homology modeling study reveals a lack of surface salt bridges, a decreased number of the surface charged residues, decreased hydrogen bonds and bound ions, and a reduction of aromatic-sulfur interactions, cationic–π interactions and disulfide interactions in EfAmy. In contrast, sequence alignment and homology modeling showed five unconserved prolines located on the surface loops ofEcAmy. By analyzing amylolytic activity towards soluble starch as the substrate, we determined the temperature and pH dependence, thermostability and kinetic parameters of these two enzymes. We demonstrated that EfAmy shows the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at low temperatures and high thermolability. In contrast, the EcAmy showed mesophilic characteristics with the highest activity at moderate temperatures and a more than 2-fold increased half-life at 50 °C compared to EfAmy. The kcat and KM values of EfAmy were higher than those of the mesophilic EcAmy at all tested temperatures. Furthermore, both EfAmy and EcAmy showed maximum activities at pH 9 and maintained high activities in the presence of surfactants. These results suggest the potential applications of EfAmy and EcAmy as ingredients in detergents for industrial applications.

 

 

Characterization of the first eukaryotic cold-adapted patatin-like phospholipase from the psychrophilic Euplotes focardii: Identification of putative determinants of thermal-adaptation by comparison with the homologous protein from the mesophilic Euplotes crassus.  

Yang G, De Santi C, de Pascale D, Pucciarelli S, Pucciarelli S, Miceli C.

Biochimie. 2013 Sep;95(9):1795-806. doi: 10.1016/j.biochi.2013.06.008. Epub 2013 Jun 22.

http://www.sciencedirect.com/science/article/pii/S0300908413001880

Abstract

The ciliated protozoon Euplotes focardii, originally isolated from the coastal seawaters of Terra Nova Bay in Antarctica, shows a strictly psychrophilic phenotype, including optimal survival and multiplication rates at 4–5 °C. This characteristic makes E. focardii an ideal model species for identifying the molecular bases of cold adaptation in psychrophilic organisms, as well as a suitable source of novel cold-active enzymes for industrial applications. In the current study, we characterized the patatin-like phospholipase fromE. focardii (EfPLP), and its enzymatic activity was compared to that of the homologous protein from the mesophilic congeneric species Euplotes crassus (EcPLP). Both EfPLP and EcPLP have consensus motifs conserved in other patatin-like phospholipases.

By analyzing both esterase and phospholipase A2 activity, we determined the thermostability and the optimal pH, temperature dependence and substrates of these enzymes. We demonstrated that EfPLP shows the characteristics of a psychrophilic phospholipase. Furthermore, we analyzed the enzymatic activity of three engineered versions of the EfPLP, in which unique residues of EfPLP, Gly80, Ala201 and Val204, were substituted through site-directed mutagenesis with residues found in the E. crassushomolog (Glu, Pro and Ile, respectively). Additionally, three corresponding mutants ofEcPLP were also generated and characterized. These analyses showed that the substitution of amino acids with rigid and bulky charged/hydrophobic side chain in the psychrophilic EfPLP confers enzymatic properties similar to those of the mesophilic patatin-like phospholipase, and vice versa.

This is the first report on the isolation and characterization of a cold-adapted patatin-like phospholipase from eukaryotes. The results reported in this paper support the idea that enzyme thermal-adaptation is based mainly on some amino acid residues that influence the structural flexibility of polypeptides and that EfPLP is an attractive biocatalyst for industrial processes at low temperatures.

 

 

Cu,Zn superoxide dismutases from Tetrahymena thermophila: molecular evolution and gene expression of the first line of antioxidant defenses. 

Ferro D, Bakiu R, De Pittà C, Boldrin F, Cattalini F, Pucciarelli S, Miceli C, Santovito G.

Protist. 2015 Feb;166(1):131-45. doi: 10.1016/j.protis.2014.12.003. Epub 2014 Dec 16.

http://www.sciencedirect.com/science/article/pii/S1434461014001254

In the present study, we describe the molecular and functional characterization of two Cu,Zn superoxide dismutase (SOD) genes, named tt-sod1a and tt-sod1b fromTetrahymena thermophila, a free-living ciliated protozoan widely used as model organism in biological research. The cDNAs and the putative amino acid sequences were compared with Cu,Zn SODs from other Alveolata. The primary sequences of T. thermophila Cu,Zn SODs are unusually long if compared to orthologous proteins, but the catalytically important residues are almost fully conserved. Both phylogenetic and preliminary homology modeling analyses provide some indications about the evolutionary relationships between the Cu,Zn SODs of Tetrahymena and the Alveolata orthologous enzymes. Copper-dependent regulation of Cu,Zn SODs expression was investigated by measuring mRNA accumulation and enzyme activity in response to chronic exposure to non-toxic doses of the metal. Our in silico analyses of the tt-sod1aand tt-sod1b promoter regions revealed putative consensus sequences similar to half Antioxidant Responsive Elements (hARE), suggesting that the transcription of these genes directly depends on ROS formation. These data emphasize the importance of complex metal regulation of tt-sod1a and tt-sod1b activation, as components of an efficient detoxification pathway allowing the survival of T. thermophila in continued, elevated presence of metals in the environment.

 

 

Identification and analysis of two sequences encoding ice-binding proteins obtained from a putative bacterial symbiont of the psychrophilic antarctic ciliate Euplotes focardii  

Pucciarelli, S.a  , Chiappori, F.b, Devaraj, R.R.a, Yang, G.a, Yu, T.c, Ballarini, P.a, Miceli, C.a

Antarctic Science. Volume 72, 14 February 2014

Abstract

We identified two ice-binding protein (IBP) sequences, named EFsymbAFP and EFsymbIBP, from a putative bacterial symbiont of the Antarctic psychrophilic ciliate Euplotes focardii. EFsymbAFP is 57.43% identical to the antifreeze protein (AFP) from the Stigmatella aurantiaca strain DW4/3-1, which was isolated from the Victoria Valley lower glacier. EFsymbIBP is 53.38% identical to the IBP from the Flavobacteriaceae bacterium strain 3519-10, isolated from the glacial ice of Lake Vostok. EFsymbAFP and EFsymbIBP are 31.73% identical at the amino acid level and are organized in tandem on the bacterial chromosome. The relatively low sequence identity and the tandem organization, which appears unique to this symbiont, suggest an occurrence of horizontal gene transfer (HGT). Structurally, EFsymbAFP and EFsymbIBP are similar to the AFPs from the snow mould fungus Typhula ishikariensis and from the Arctic yeast Leucosporidium sp. AY30. A phylogenetic analysis showed that EFsymbAFP and EFsymbIBP cluster principally with the IBP sequences from other Antarctic bacteria, supporting the view that these sequences belong to an Antarctic symbiontic bacterium of E. focardii. These results confirm that IBPs have a complex evolutionary history, which includes HGT events, most probably due to the demands of the environment and the need for rapid adaptation. © 2014 Antarctic Science Ltd

 

High resistance of Tetrahymena thermophila to paraquat: Mitochondrial alterations, oxidative stress and antioxidant genes expression. 

Díaz S, Martín-González A, Cubas L, Ortega R, Amaro F, Rodríguez-Martín D, Gutiérrez JC.

Chemosphere. 2016 Feb;144:909-17. doi: 10.1016/j.chemosphere.2015.09.010. Epub 2015 Sep 29. No abstract available.

http://www.sciencedirect.com/science/article/pii/S0045653515300977

Highlights

Tetrahymena thermophila is an eukaryotic microorganism very resistant to paraquat.

Superoxide production is significantly increased under paraquat exposure.

ROS induction is proportional to paraquat concentration, when toxicity does not generates a high cell mortality.

Mitochondria may be the main target of paraquat toxicity in T. thermophila.

An up-regulation of gene expression encoding critical antioxidant enzymes is involved in the cell response against this toxic pollutant.

 

 

Hints for metal-preference protein sequence determinants: different metal binding features of the five tetrahymena thermophila metallothioneins. 

Espart A, Marín M, Gil-Moreno S, Palacios Ò, Amaro F, Martín-González A, Gutiérrez JC, Capdevila M, Atrian S.

Int J Biol Sci. 2015 Mar 18;11(4):456-71. doi: 10.7150/ijbs.11060. eCollection 2015.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4366644/

Abstract

The metal binding preference of metallothioneins (MTs) groups them in two extreme subsets, the Zn/Cd- and the Cu-thioneins. Ciliates harbor the largest MT gene/protein family reported so far, including 5 paralogs that exhibit relatively low sequence similarity, excepting MTT2 and MTT4. In Tetrahymena thermophila, three MTs (MTT1, MTT3 and MTT5) were considered Cd-thioneins and two (MTT2 and MTT4) Cu-thioneins, according to gene expression inducibility and phylogenetic analysis. In this study, the metal-binding abilities of the five MTT proteins were characterized, to obtain information about the folding and stability of their cognate- and non-cognate metal complexes, and to characterize the T. thermophila MT system at protein level. Hence, the five MTTs were recombinantly synthesized as Zn2+-, Cd2+- or Cu+-complexes, which were analyzed by electrospray mass spectrometry (ESI-MS), circular dichroism (CD), and UV-vis spectrophotometry. Among the Cd-thioneins, MTT1 and MTT5 were optimal for Cd2+ coordination, yielding unique Cd17- and Cd8- complexes, respectively. When binding Zn2+, they rendered a mixture of Zn-species. Only MTT5 was capable to coordinate Cu+, although yielding heteronuclear Zn-, Cu-species or highly unstable Cu-homometallic species. MTT3 exhibited poor binding abilities both for Cd2+ and for Cu+, and although not optimally, it yielded the best result when coordinating Zn2+. The two Cu-thioneins, MTT2 and MTT4 isoforms formed homometallic Cu-complexes (major Cu20-MTT) upon synthesis in Cu-supplemented hosts. Contrarily, they were unable to fold into stable Cd-complexes, while Zn-MTT species were only recovered for MTT4 (major Zn10-MTT4). Thus, the metal binding preferences of the five T. thermophila MTs correlate well with their previous classification as Cd- and Cu-thioneins, and globally, they can be classified from Zn/Cd- to Cu-thioneins according to the gradation: MTT1>MTT5>MTT3>MTT4>MTT2. The main mechanisms underlying the evolution and specialization of the MTT metal binding preferences may have been internal tandem duplications, presence of doublet and triplet Cys patterns in Zn/Cd-thioneins, and optimization of site specific amino acid determinants (Lys for Zn/Cd- and Asn for Cu-coordination).

 

 

Heavy metal whole-cell biosensors using eukaryotic microorganisms: an updated critical review.  

Gutiérrez JC, Amaro F, Martín-González A.

Front Microbiol. 2015 Feb 20;6:48. doi: 10.3389/fmicb.2015.00048. eCollection 2015.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4335268/

Abstract

This review analyzes the advantages and disadvantages of using eukaryotic microorganisms to design whole-cell biosensors (WCBs) for monitoring environmental heavy metal pollution in soil or aquatic habitats. Basic considerations for designing a eukaryotic WCB are also shown. A comparative analysis of the promoter genes used to design WCBs is carried out, and the sensitivity and reproducibility of the main reporter genes used is also reviewed. Three main eukaryotic taxonomic groups are considered: yeasts, microalgae, and ciliated protozoa. Models that have been widely analyzed as potential WCBs are the Saccharomyces cerevisiae model among yeasts, the Tetrahymena thermophila model for ciliates and Chlamydomonas model for microalgae. The advantages and disadvantages of each microbial group are discussed, and a ranking of sensitivity to the same type of metal pollutant from reported eukaryotic WCBs is also shown. General conclusions and possible future developments of eukaryotic WCBs are reported.

 

 

Functional GFP-metallothionein fusion protein from Tetrahymena thermophila: a potential whole-cell biosensor for monitoring heavy metal pollution and a cell model to study metallothionein overproduction effects.  

Amaro F, Turkewitz AP, Martín-González A, Gutiérrez JC.

Biometals. 2014 Feb;27(1):195-205. doi: 10.1007/s10534-014-9704-0. Epub 2014 Jan 16

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4707044/

Abstract

The significance of metal(oid)s as environmental pollutants has made them a priority in ecotoxicology, with the aim of minimizing exposure to animals or humans. Therefore, it is necessary to develop sensitive and inexpensive methods that can efficiently detect and monitor these pollutants in the environment. Conventional analytical techniques suffer from the disadvantages of high cost and complexity. Alternatively, prokaryotic or eukaryotic whole-cell biosensors (WCB) are one of the newest molecular tools employed in environmental monitoring that use the cell as an integrated reporter incorporating a reporter gene fused to a heavy metal responsive promoter. In the present paper, we report results from expressing, in the ciliateTetrahymena thermophila, constructs consisting of the reporter gfp gene fused to the complete MTT1 orMTT5 protein coding regions under the transcriptional control of the MTT1 metallothionein promoter, which plays a critical role in heavy metal stress in this ciliate. When exposed to Cd2+, such cells overexpress both the GFP reporter transgene and the linked metallothionein gene. We report that, for the GFPMTT5 strain, this metallothionein overexpression results in marked resistance to cadmium toxicity (24h LC50 ~ 15 µM of Cd2+), compared to wild type cells (24h LC50 ~ 1.73 µM of Cd2+). These results provide the first experimental evidence that ciliate metallothioneins, like in other organisms, function to protect the cell against toxic metal ions. Because these strains may have novel advantages as WCBs, we have compared their properties to those of other previously reported Tetrahymena WCBs.

 

 

Bioaccumulation modelling and sensitivity analysis for discovering key players in contaminated food webs: The case study of PCBs in the Adriatic Sea 

Taffi, M.ad  , Paoletti, N.b, Liò, P.c, Pucciarelli, S.a, Marini, M.d

Ecological Modelling. Volume 306, June 04, 2015, Pages 205-215

Abstract

Modelling bioaccumulation processes at the food web level is the main step to analyse the effects of pollutants at the global ecosystem level. A crucial question is understanding which species play a key role in the trophic transfer of contaminants to disclose the contribution of feeding linkages and the importance of trophic dependencies in bioaccumulation dynamics. In this work we present a computational framework to model the bioaccumulation of organic chemicals in aquatic food webs, and to discover key species in polluted ecosystems. As a result, we reconstruct the first PCBs bioaccumulation model of the Adriatic food web, estimated after an extensive review of published concentration data. We define a novel index aimed to identify the key species in contaminated networks, sensitivity centrality, and based on sensitivity analysis. The index is computed from a dynamic ODE model parametrised from the estimated PCBs bioaccumulation model and compared with a set of established trophic indices of centrality. Results evidence the occurrence of PCBs biomagnification in the Adriatic food web, and highlight the dependence of bioaccumulation on trophic dynamics and external factors like fishing activity. We demonstrate the effectiveness of the introduced sensitivity centrality in identifying the set of species with the highest impact on the total contaminant flows and on the efficiency of contaminant transport within the food web. © 2014 Elsevier B.V

 

Convergent evolution of heat-inducibility during subfunctionalization of the Hsp70 gene family. 

Krenek S, Schlegel M, Berendonk TU.

BMC Evol Biol. 2013 Feb 21;13:49. doi: 10.1186/1471-2148-13-49.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3606833/

Abstract

Background. Heat-shock proteins of the 70 kDa family (Hsp70s) are essential chaperones required for key cellular functions. In eukaryotes, four subfamilies can be distinguished according to their function and localisation in different cellular compartments: cytosol, endoplasmic reticulum, mitochondria and chloroplasts. Generally, multiple cytosol-type Hsp70s can be found in metazoans that show either constitutive expression and/or stress-inducibility, arguing for the evolution of different tasks and functions. Information about the hsp70copy number and diversity in microbial eukaryotes is, however, scarce, and detailed knowledge about the differential gene expression in most protists is lacking. Therefore, we have characterised the Hsp70 gene family of Paramecium caudatum to gain insight into the evolution and differential heat stress response of the distinct family members in protists and to investigate the diversification of eukaryotic hsp70s focusing on the evolution of heat-inducibility.

Results. Eleven putative hsp70 genes could be detected in P. caudatum comprising homologs of three major Hsp70-subfamilies. Phylogenetic analyses revealed five evolutionarily distinct Hsp70-groups, each with a closer relationship to orthologous sequences of Paramecium tetraurelia than to another P. caudatum Hsp70-group. These highly diverse, paralogous groups resulted from duplications preceding Paramecium speciation, underwent divergent evolution and were subject to purifying selection. Heat-shock treatments were performed to test for differential expression patterns among the five Hsp70-groups as well as for a functional conservation within Paramecium. These treatments induced exceptionally high mRNA up-regulations in one cytosolic group with a low basal expression, indicative for the major heat inducible hsp70s. All other groups showed comparatively high basal expression levels and moderate heat-inducibility, signifying constitutively expressed genes. Comparative EST analyses for P. tetraurelia hsp70s unveiled a corresponding expression pattern, which supports a functionally conserved evolution of the Hsp70 gene family in Paramecium.

Conclusions. Our analyses suggest an independent evolution of the heat-inducible cytosol-type hsp70s in Paramecium and in its close relative Tetrahymena, as well as within higher eukaryotes. This result indicates convergent evolution during hsp70 subfunctionalization and implies that heat-inducibility evolved several times during the course of eukaryotic evolution.

 

 

Systematics and species-specific response to pH of Oxytricha acidotolerans sp. nov. and Urosomoida sp. (Ciliophora, Hypotricha) from acid mining lakes. 

Weisse T, Moser M, Scheffel U, Stadler P, Berendonk T, Weithoff G, Berger H.

Eur J Protistol. 2013 May;49(2):255-71. doi: 10.1016/j.ejop.2012.08.001. Epub 2012 Sep 26.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3611561/

Abstract

We investigated the morphology, phylogeny of the 18S rDNA, and pH response of Oxytricha acidotoleranssp. nov. and Urosomoida sp. (Ciliophora, Hypotricha) isolated from two chemically similar acid mining lakes (pH  2.6) located at Langau, Austria, and in Lusatia, Germany. Oxytricha acidotolerans sp. nov. from Langau has 18 frontal-ventral-transverse cirri but a very indistinct kinety 3 fragmentation so that the assignment to Oxytricha is uncertain. The somewhat smaller species from Lusatia has a highly variable cirral pattern and the dorsal kineties arranged in the Urosomoida pattern and is, therefore, preliminary designated as Urosomoida sp. The pH response was measured as ciliate growth rates in laboratory experiments at pH ranging from 2.5 to 7.0. Our hypothesis was that the shape of the pH reaction norm would not differ between these closely related (3% difference in their SSU rDNA) species. Results revealed a broad pH niche for O. acidotolerans, with growth rates peaking at moderately acidic conditions (pH 5.2). Cyst formation was positively and linearly related to pH. Urosomoida sp. was more sensitive to pH and did not survive at circumneutral pH. Accordingly, we reject our hypothesis that similar habitats would harbour ciliate species with virtually identical pH reaction norm.

 

 

Coping with temperature at the warm edge--patterns of thermal adaptation in the microbial eukaryote Paramecium caudatum. 

Krenek S, Petzoldt T, Berendonk TU.

PLoS One. 2012;7(3):e30598. doi: 10.1371/journal.pone.0030598. Epub 2012 Mar 9

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3302864/

Abstract

Background. Ectothermic organisms are thought to be severely affected by global warming since their physiological performance is directly dependent on temperature. Latitudinal and temporal variations in mean temperatures force ectotherms to adapt to these complex environmental conditions. Studies investigating current patterns of thermal adaptation among populations of different latitudes allow a prediction of the potential impact of prospective increases in environmental temperatures on their fitness.

Methodology/Principal Findings. In this study, temperature reaction norms were ascertained among 18 genetically defined, natural clones of the microbial eukaryote Paramecium caudatum. These different clones have been isolated from 12 freshwater habitats along a latitudinal transect in Europe and from 3 tropical habitats (Indonesia). The sensitivity to increasing temperatures was estimated through the analysis of clone specific thermal tolerances and by relating those to current and predicted temperature data of their natural habitats.

All investigated European clones seem to be thermal generalists with a broad thermal tolerance and similar optimum temperatures. The weak or missing co-variation of thermal tolerance with latitude does not imply local adaptation to thermal gradients; it rather suggests adaptive phenotypic plasticity among the whole European subpopulation. The tested Indonesian clones appear to be locally adapted to the less variable, tropical temperature regime and show higher tolerance limits, but lower tolerance breadths.

Conclusions/Significance. Due to the lack of local temperature adaptation within the European subpopulation, P. caudatum genotypes at the most southern edge of their geographic range seem to suffer from the predicted increase in magnitude and frequency of summer heat waves caused by climate change.

 

 

New Stands of Species of the Paramecium aurelia Complex; is the Occurrence of Particular Species Limited by Temperature Barriers? 

Przyboś E, Prajer M.

Folia Biol (Krakow). 2015;63(3):215-20.

http://www.ncbi.nlm.nih.gov/pubmed/26462333

Abstract

The occurrence of ciliates, especially the Paramecium aurelia complex, has not yet been studied in many parts of the world, or sampling was done only occasionally. Generally, the southern hemisphere still awaits investigation. In North America only the USA was studied in greater detail; the majority of species of the complex were there recorded. In Asia, more frequent sampling was performed only in Japan and Asiatic Russia. Europe was studied more carefully, however, a different number of habitats was studied in particular zones of Europe, the least in the southern zone. New stands of P. tetraurelia , P. sexaurelia, P. octaurelia, and P. novaurelia were revealed as a result of the present investigations carried out in Africa (Mozambique--P. tetraurelia, P. sexaurelia), Asia (Indonesia--P. sexaurelia), borderland of Asia and Europe (Georgia--P. octaurelia), and Europe (Macedonia--P. tetraurelia and Romania--P. novaurelia). Are climatic zones the main factor limiting the occurrence of species of the P. aurelia complex? Analysis of data on the distribution of the P. aurelia species complex in warm "tropical" zones on different continents may suggest such preferences for some species, including P. sexaurelia, P. octaurelia, P. tredecaurelia, P. quadecaurelia. The first two of these species were recorded herein in warm or "tropical" zone.

 

 

A novel robust heat-inducible promoter for heterologous gene expression in Tetrahymena thermophila. 3

Yu T, Barchetta S, Pucciarelli S, La Terza A, Miceli C.

Protist. 2012 Mar;163(2):284-95. doi: 10.1016/j.protis.2011.07.003. Epub 2011 Jul 30.

http://www.sciencedirect.com/science/article/pii/S1434461011000575

An increasing amount of data has revealed the importance of inducible promoters in ciliate research and in ciliate-related industries. However, knowledge about these promoters and related genes is relatively sparse. Here we report a novel inducible promoter from a Tetrahymena cytoplasmic Hsp70 gene member, HSP70-2. The reported promoter was able to induce the endogenous gene up to 9000-fold after a short heat shock treatment and this remarkable feature has been retained when a relatively short region of the promoter was introduced into a reporter construct followed by transformation. During the recovery period following a short heat shock, both the mRNA and protein levels of the reporter gene were maintained high up to two hours. A constant heat shock treatment to the transformed cells led to a stabilization of the reporter mRNA up to at least six hours and the reporter protein continued to accumulate up to around three hours. The promoter strength appears to be similar to that of the cadmium-induced metallothionein gene (MTT1) promoter. Therefore, the HSP70-2promoter represents an attractive alternative for the over-expression of proteins inTetrahymena, and the promoter-reporter gene construct used in this study is an ideal tool to help in understanding the regulation mechanisms of heat shock genes in ciliates.

 

 

Bioremediation in marine ecosystems: a computational study combining ecological modeling and flux balance analysis.  

Taffi M, Paoletti N, Angione C, Pucciarelli S, Marini M, Liò P.

Front Genet. 2014 Sep 12;5:319. doi: 10.3389/fgene.2014.00319. eCollection 2014.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4162388/

Abstract

The pressure to search effective bioremediation methodologies for contaminated ecosystems has led to the large-scale identification of microbial species and metabolic degradation pathways. However, minor attention has been paid to the study of bioremediation in marine food webs and to the definition of integrated strategies for reducing bioaccumulation in species. We propose a novel computational framework for analysing the multiscale effects of bioremediation at the ecosystem level, based on coupling food web bioaccumulation models and metabolic models of degrading bacteria. The combination of techniques from synthetic biology and ecological network analysis allows the specification of arbitrary scenarios of contaminant removal and the evaluation of strategies based on natural or synthetic microbial strains. In this study, we derive a bioaccumulation model of polychlorinated biphenyls (PCBs) in the Adriatic food web, and we extend a metabolic reconstruction of Pseudomonas putida KT2440 (iJN746) with the aerobic pathway of PCBs degradation. We assess the effectiveness of different bioremediation scenarios in reducing PCBs concentration in species and we study indices of species centrality to measure their importance in the contaminant diffusion via feeding links. The analysis of the Adriatic sea case study suggests that our framework could represent a practical tool in the design of effective remediation strategies, providing at the same time insights into the ecological role of microbial communities within food webs.

 

 

Ciliate communities and hidden biodiversity in freshwater biotopes of the Pistoia province (Tuscany, Italy).  

Rossi A, Boscaro V, Carducci D, Serra V, Modeo L, Verni F, Fokin SI, Petroni G.

Eur J Protistol. 2015 Dec 21;53:11-19. doi: 10.1016/j.ejop.2015.12.005. [Epub ahead of print]

http://www.sciencedirect.com/science/article/pii/S0932473915001054

Abstract

Ciliates are essential components of aquatic environments, playing a pivotal role in microbial loops. Thus, the composition and dynamics of ciliate communities have been subjected to intense studying. Morphological methods have been traditionally employed, until the development of next-generation sequencing recently allowed to explore the topic with exclusively molecular techniques. However, the results of the two approaches are hardly comparable, and the pictures they offer can be quite different. This may be due, among other reasons, to two factors: (1) morphological descriptions may miss a large portion of “hidden biodiversity” (including rare species and resistance forms) that is detected instead by molecular methods; (2) identification errors may arise due to difficulties in recognizing microbial taxa without in-depth analyses. In this survey of freshwater systems of the Pistoia province (Tuscany, Italy) we address both issues, trying to quantify the hidden diversity through prolonged observations of differentially treated sample aliquots, combining morphological identification with Sanger sequencing. We provide the first insights into the ciliate fauna of this area presenting results that are suitable for future comparisons thanks to their multidisciplinary origin, and supply the first molecular data on well-known taxa such as Linostomella and Disematostoma.

 

 

Long-term selection experiment produces breakdown of horizontal transmissibility in parasite with mixed transmission mode.  

Dusi E, Gougat-Barbera C, Berendonk TU, Kaltz O.

Evolution. 2015 Apr;69(4):1069-76. doi: 10.1111/evo.12638. Epub 2015 Apr 9.

http://onlinelibrary.wiley.com/doi/10.1111/evo.12638/full

Abstract

Evolutionary transitions from parasitism toward beneficial or mutualistic associations may encompass a change from horizontal transmission to (strict) vertical transmission. Parasites with both vertical and horizontal transmission are amendable to study factors driving such transitions. In a long-term experiment, microcosm populations of the protozoan Paramecium caudatum and its bacterial parasite Holospora undulata were exposed to three growth treatments, manipulating vertical transmission opportunities over ca. 800 host generations. In inoculation tests, horizontal transmission propagules produced by parasites from a “high-growth” treatment, with elevated host division rates increasing levels of parasite vertical transmission, showed a near-complete loss of infectivity. A similar reduction was observed for parasites from a treatment alternating between high growth and low growth (i.e., low levels of population turn-over). Parasites from a low-growth treatment had the highest infectivity on all host genotypes tested. Our results complement previous findings of reduced investment in horizontal transmission and increased vertical transmissibility of high-growth parasites. We explain the loss of horizontal transmissibility by epidemiological feedbacks and resistance evolution, reducing the frequency of susceptible hosts in the population and thereby decreasing the selective advantage of horizontal transmission. This illustrates how environmental conditions may push parasites with a mixed transmission mode toward becoming vertically transmitted nonvirulent symbionts.

 

 

Nanoparticles in wastewater treatment plants: a novel acute toxicity test for ciliates and its implementation in risk assessment. 

Burkart C, von Tümpling W, Berendonk T, Jungmann D.

Environ Sci Pollut Res Int. 2015 May;22(10):7485-94. doi: 10.1007/s11356-014-4057-3. Epub 2015 Jan 17.

Abstract: http://www.ncbi.nlm.nih.gov/pubmed/?term=Nanoparticles+in+wastewater+treatment+plants%3A+a+novel+acute+toxicity+test+for+ciliates+and+its+implementation+in+risk+assessment.

Nanomaterial (NM) release into wastewater treatment plants (WWTPs) is inevitable due to increased production and application throughout past decades and in the future. Concern arose about environmental risks and impact on activated sludge. Environmental risk assessment (ERA) for NMs according to established guidelines is considered not suitable, because NMs exhibit unique characteristics. For hazard identification on activated sludge, standard test organisms for aquatic toxicity testing are not meaningful. In this study, we developed an acute toxicity test for ciliates(Paramecium tetraurelia) as representatives of the important functional group of microbial predators and filter feeders. We chose silver nanoparticles(nAg) exemplarily for ion releasing nanoparticles and regarded toxicity by ions as well. Our results indicate that ions are more toxic (EC₅₀ 0.73 mg/L) than nanoparticles themselves (EC₅₀ 2.15 mg/L). However, nAg must be considered as a source of ions and requires size, surface coating, and compartment-specific ERA. We strived to develop such ERA based on our results, modeled environmental concentration data from literature, and surface area concentrations. Results indicated a probable risk toward activated sludge. This likely has effects on effluent water quality. We conclude that carefully modeled environmental concentrations are vital for more exact ERA for nAg and other NMs

 

 

Potential of hyperspectral imaging microscopy for semi-quantitative analysis of nanoparticle uptake by protozoa. 

Mortimer M, Gogos A, Bartolomé N, Kahru A, Bucheli TD, Slaveykova VI.

Environ Sci Technol. 2014;48(15):8760-7. doi: 10.1021/es500898j. Epub 2014 Jul 16.

http://pubs.acs.org/doi/pdf/10.1021/es500898j

Abstract

Hyperspectral imaging with enhanced darkfield microscopy (HSI-M) possesses unique advantages in its simplicity and non-invasiveness. In consideration of the urgent need for profound knowledge on the behavior and effects of engineered nanoparticles (NPs), here, we determined the capability of HSI-M for examining cellular uptake of different metal-based NPs, including nanosized metals (silver and gold, both citrate stabilized), metal oxides (copper oxide and titanium dioxide), and CdSe/ZnS core/shell quantum dots at subtoxic concentrations. Specifically, we demonstrated that HSI-M can be used to detect and semi-quantify these NPs in the ciliated protozoan Tetrahymena thermophila as a model aquatic organism. Detection and semi-quantification were achieved on the basis of spectral libraries for the NPs suspended in extracellular substances secreted by this single-celled organism, accounting for matrix effects. HSI-M was able to differentiate between NP types, provided that spectral profiles were significantly different from each other. This difference, in turn, depended upon NP type, size, agglomeration status, and position relative to the focal plane. As an exception among the NPs analyzed in this study, titanium dioxide NPs showed spectral similarities compared to cell material of unexposed control cells, leading to false positives. High biological variability resulted in highly variable uptake of NPs in cells of the same sample as well as between different exposures. We therefore encourage the development of techniques able to reduce the currently long analysis times that still hamper the acquisition of statistically strong data sets. Overall, this study demonstrates the potential and challenges of HSI-M in monitoring cellular uptake of synthetic NPs.

 

 

Uptake, localization and clearance of quantum dots in ciliated protozoa Tetrahymena thermophila. 

Mortimer M, Kahru A, Slaveykova VI.

Environ Pollut. 2014 Jul;190:58-64. doi: 10.1016/j.envpol.2014.03.021. Epub 2014 Apr 13.

http://www.sciencedirect.com/science/article/pii/S0269749114001122

Abstract

Protozoa as phagocytizing cells have been shown to integrate engineered nanoparticles (NPs), while the mechanism, dynamics and extent of such uptake are unclear. Here our fluorescence microscopy data showed that CdSe/ZnS quantum dots (QDs) with primary size of 12 nm were readily phagocytized into the food vacuoles of Tetrahymena thermophila in a time- and dose-dependent manner. Twenty hours after the exposure to QDs in sublethal concentration the clearance of the QDs from the cells was incomplete suggesting that phagocytosis of QDs into food vacuoles was not the only pathway of uptake by T. thermophila. This was further proven by the results that the inhibition of phagocytosis did not block the internalization of QDs into protozoans. This study provides a new insight into uptake and cellular trafficking of subtoxic concentrations of nanoparticles that may, due to prolonged retention times in the cells, pose risks by potentially becoming available to higher trophic levels.

 

 

Extracellular conversion of silver ions into silver nanoparticles by protozoan Tetrahymena thermophila. 

Juganson K, Mortimer M, Ivask A, Kasemets K, Kahru A.

Environ Sci Process Impacts. 2013 Jan;15(1):244-50

Abstract: http://www.ncbi.nlm.nih.gov/pubmed/?term=Extracellular+conversion+of+silver+ions+into+silver+nanoparticles+by+protozoan+Tetrahymenathermophila.

In the current study, cell-free exudates of the ciliated protozoan Tetrahymena thermophila were shown to progressively convert silver nitrate to silvernanoparticles (Ag NPs) under illumination at ambient temperature. The formation of Ag NPs in the reaction mixture was evidenced by gradual colour changes, appearance of a specific absorbance peak (420–450 nm) and visualization using scanning electron microscopy coupled to an energy-dispersive X-ray spectrometer. After 2 h of incubation the mean hydrodynamic size of the Ag NPs was 70 nm. Seven days of incubation resulted in larger agglomerates and a significant decrease in silver toxicity to T. thermophila, accompanied by about 100-fold reduction in the silver ion concentration. Protein analysis indicated an extensive extracellular protein binding by the Ag NPs formed in the protozoan exudates. As protozoa are important components in wastewater treatment, their ability to sequester silver ions into a less bioavailable and less toxic form of silver (e.g. NPs) may be one of the adaption mechanisms of ciliate survival in contaminated environments.

 

 

An interlaboratory comparison of nanosilver characterisation and hazard identification: Harmonising techniques for high quality data. 

Jemec A, Kahru A, Potthoff A, Drobne D, Heinlaan M, Böhme S, Geppert M, Novak S, Schirmer K, Rekulapally R, Singh S, Aruoja V, Sihtmäe M, Juganson K, Käkinen A, Kühnel D.

Environ Int. 2016 Feb;87:20-32. doi: 10.1016/j.envint.2015.10.014. Epub 2015 Nov 28.

http://www.sciencedirect.com/science/article/pii/S0160412015300805

Abstract

Within the FP7 EU project NanoValid a consortium of six partners jointly investigated the hazard of silver nanoparticles (AgNPs) paying special attention to methodical aspects that are important for providing high-quality ecotoxicity data. Laboratories were supplied with the same original stock dispersion of AgNPs. All partners applied a harmonised procedure for storage and preparation of toxicity test suspensions. Altogether ten different toxicity assays with a range of environmentally relevant test species from different trophic levels were conducted in parallel to AgNP characterisation in the respective test media. The paper presents a comprehensive dataset of toxicity values and AgNP characteristics like hydrodynamic sizes of AgNP agglomerates and the share (%) of Ag+-species (the concentration of Ag+-species in relation to the total measured concentration of Ag). The studied AgNP preparation (20.4 ± 6.8 nm primary size, mean total Ag concentration 41.14 mg/L, 46–68% of soluble Ag+-species in stock, 123.8 ± 12.2 nm mean z-average value in dH2O) showed extreme toxicity to crustaceansDaphnia magna, algae Pseudokirchneriella subcapitata and zebrafish Danio rerioembryos (EC50 < 0.01 mg total Ag/L), was very toxic in the in vitro assay with rainbow trout Oncorhynchus mykiss gut cells (EC50: 0.01–1 mg total Ag/L); toxic to bacteriaVibrio fischeri, protozoa Tetrahymena thermophila (EC50: 1–10 mg total Ag/L) and harmful to marine crustaceans Artemia franciscana (EC50: 10–100 mg total Ag/L). Along with AgNPs, also the toxicity of AgNO3 was analyzed. The toxicity data revealed the same hazard ranking for AgNPs and AgNO3 (i.e. the EC50 values were in the same order of magnitude) proving the importance of soluble Ag+-species analysis for predicting the hazard of AgNPs. The study clearly points to the need for harmonised procedures for the characterisation of NMs. Harmonised procedures should consider: (i) measuring the AgNP properties like hydrodynamic size and metal ions species in each toxicity test medium at a range of concentrations, and (ii) including soluble metal salt control both in toxicity testing as well as in Ag+-species measurements. The present study is among the first nanomaterial interlaboratory comparison studies with the aim to improve the hazard identification testing protocols.

 

TOPIC 4

 

Genome-wide analysis of the phosphoinositide kinome from two ciliates reveals novel evolutionary links for phosphoinositide kinases in eukaryotic cells.  

Leondaritis G, Siokos J, Skaripa I, Galanopoulou D.

PLoS One. 2013 Nov 11;8(11):e78848. doi: 10.1371/journal.pone.0078848. eCollection 2013

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3823935/

Abstract

Background. The complexity of phosphoinositide signaling in higher eukaryotes is partly due to expansion of specific families and types of phosphoinositide kinases (PIKs) that can generate all phosphoinositides via multiple routes. This is particularly evident in the PI3Ks and PIPKs, and it is considered an evolutionary trait associated with metazoan diversification. Yet, there are limited comprehensive studies on the PIK repertoire of free living unicellular organisms.

Methodology/Principal Findings. We undertook a genome-wide analysis of putative PIK genes in two free living ciliated cells, Tetrahymenaand Paramecium. The Tetrahymena thermophila and Paramecium tetraurelia genomes were probed with representative kinases from all families and types. Putative homologs were verified by EST, microarray and deep RNA sequencing database searches and further characterized for domain structure, catalytic efficiency, expression patterns and phylogenetic relationships. In total, we identified and characterized 22 genes in theTetrahymena thermophila genome and 62 highly homologues genes in Paramecium tetraurelia suggesting a tight evolutionary conservation in the ciliate lineage. Comparison to the kinome of fungi reveals a significant expansion of PIK genes in ciliates.

Conclusions/Significance. Our study highlights four important aspects concerning ciliate and other unicellular PIKs. First, ciliate-specific expansion of PI4KIII-like genes. Second, presence of class I PI3Ks which, at least in Tetrahymena, are associated with a metazoan-type machinery for PIP3 signaling. Third, expansion of divergent PIPK enzymes such as the recently described type IV transmembrane PIPKs. Fourth, presence of possible type II PIPKs and presumably inactive PIKs (hence, pseudo-PIKs) not previously described. Taken together, our results provide a solid framework for future investigation of the roles of PIKs in ciliates and indicate that novel functions and novel regulatory pathways of phosphoinositides may be more widespread than previously thought in unicellular organisms.

 

 

An UPF3-based nonsense-mediated decay in Paramecium.  

Contreras J, Begley V, Macias S, Villalobo E.

Res Microbiol. 2014 Dec;165(10):841-6. doi: 10.1016/j.resmic.2014.10.008. Epub 2014 Oct 22

http://www.sciencedirect.com/science/article/pii/S0923250814002022

Abstract

Nonsense-mediated decay recognises mRNAs containing premature termination codons. One of its components, UPF3, is a molecular link bridging through its binding to the exon junction complex nonsense-mediated decay and splicing. In protists UPF3 has not been identified yet. We report that Paramecium tetraurelia bears an UPF3 gene and that it has a role in nonsense-mediated decay. Interestingly, the identified UPF3 has not conserved the essential amino acids required to bind the exon junction complex. Though, our data indicates that this ciliate bears genes coding for core proteins of the exon junction complex.

 

 

Identification of neutral and acidic deoxyribonuclease activities in Tetrahymena thermophila life stages.  

Aslan E, Arslanyolu M. 

Eur J Protistol. 2015 Apr;51(2):173-85. doi: 10.1016/j.ejop.2015.02.004. Epub 2015 Feb 20.

http://www.sciencedirect.com/science/article/pii/S0932473915000176

Abstract

Deoxyribonucleases (DNases) play a major role in apoptotic DNA fragmentation/degradation, and apoptotic-like DNA degradation is also observed during conjugation of the ciliate Tetrahymena thermophila; however, the characteristics of neutral and acidic DNases are still undefined in its life stages. Here, we report the biochemical characterization of DNase activities displayed in three differentTetrahymena life stages in a comparative manner. Maximum DNase activity ofTetrahymena was observed under acidic conditions, indicating that Tetrahymena has strong DNase II-like activities. Zymography revealed that Tetrahymena has at least five distinct DNase activity bands at 28, 32, 33.8, 35.5, and 69-kDa, and that the activities at 32 and 33.8-kDa were also secreted into starvation buffer. Cofactor analysis demonstrated that Mg2+ exerted inhibitory effects on neutral DNase activities. Unexpectedly, Mg2+ and Ca2+ had favorable effects on acidic DNase activities. The DNase activity profile of conjugating Tetrahymena cells revealed that the 32 and 33.8-kDa activities at pH 5.0 increased from 14 to 18 h of conjugation, corresponding to the final resorption of the old macronucleus by lysosomal enzymes during programmed nuclear death (PND). Overall, we found that Tetrahymena DNases exhibit different biochemical properties and a possible involvement of DNase II-like activities in PND.

 

 

Functional diversification of Dicer-like proteins and small RNAs required for genome sculpting. 

Sandoval PY, Swart EC, Arambasic M, Nowacki M.

Dev Cell. 2014 Jan 27;28(2):174-88. doi: 10.1016/j.devcel.2013.12.010. Epub 2014 Jan 16.

http://www.sciencedirect.com/science/article/pii/S1534580713007570

Summary

In eukaryotes, small RNAs (sRNAs) have key roles in development, gene expression regulation, and genome integrity maintenance. In ciliates, such as Paramecium, sRNAs form the heart of an epigenetic system that has evolved from core eukaryotic gene silencing components to selectively target DNA for deletion. In Paramecium, somatic genome development from the germline genome accurately eliminates the bulk of typically gene-interrupting, noncoding DNA. We have discovered an sRNA class (internal eliminated sequence [IES] sRNAs [iesRNAs]), arising later duringParamecium development, which originates from and precisely delineates germline DNA (IESs) and complements the initial sRNAs (“scan” RNAs [scnRNAs]) in targeting DNA for elimination. We show that whole-genome duplications have facilitated successive differentiations of Paramecium Dicer-like proteins, leading to cooperation between Dcl2 and Dcl3 to produce scnRNAs and to the production of iesRNAs by Dcl5. These innovations highlight the ability of sRNA systems to acquire capabilities, including those in genome development and integrity.

 

 

Differential expression of histone H3 genes and selective association of the variant H3.7 with a specific sequence class in Stylonychia macronuclear development.  

Forcob S, Bulic A, Jönsson F, Lipps HJ, Postberg J.

Epigenetics Chromatin. 2014 Feb 7;7(1):4. doi: 10.1186/1756-8935-7-4.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3918171/

Abstract

Background Regulation of chromatin structure involves deposition of selective histone variants into nucleosome arrays. Numerous histone H3 variants become differentially expressed by individual nanochromosomes in the course of macronuclear differentiation in the spirotrichous ciliate Stylonychia lemnae. Their biological relevance remains to be elucidated.

Results. We show that the differential assembly of H3 variants into chromatin is strongly correlated with the functional separation of chromatin structures in developing macronuclei during sexual reproduction inStylonychia, thus probably determining the fate of specific sequences. Specific H3 variants approximately 15 kDa or 20 kDa in length are selectively targeted by post-translational modifications. We found that only the 15 kDa H3 variants including H3.3 and H3.5, accumulate in the early developing macronucleus, and these also occur in mature macronuclei. H3.7 is a 20 kDa variant that specifically becomes enriched in macronuclear anlagen during chromosome polytenization. H3.7, acetylated at lysine-32 (probably equivalent to lysine-36 of most H3 variants), is specifically associated with a sequence class that is retained in the mature macronucleus and therefore does not undergo developmental DNA elimination. H3.8 is another 20 kDa variant that is restricted to the micronucleus. H3.8 is selectively targeted by lysine methylation and by serine or threonine phosphorylation. Intriguingly, the expression and chromatin localization of the histone variant H3.3 was impaired during macronuclear differentiation after RNA interference knock-down of Piwi expression.

Conclusions. Differential deposition of H3 variants into chromatin strongly correlates with the functional distinction of genomic sequence classes on the chromatin level, thus helping to determine the fate of specific DNA sequences during sexual reproduction in Stylonychia. Consequently, H3 variants are selectively targeted by post-translational modifications, possibly as a result of deviations within the recognition motifs, which allow binding of effector proteins. We propose that differential assembly of histone variants into chromatin of various nuclear types could contribute to nuclear identity, for example, during differential development of either new micronuclei or a macronuclear anlage from mitosis products of the zygote nucleus (synkaryon). The observation that the Piwi-non-coding RNA (ncRNA) pathway influences the expression and deposition of H3.3 in macronuclear anlagen indicates for the first time that selective histone variant assembly into chromatin might possibly depend on ncRNA.

 

 

A permissive chromatin structure is adopted prior to site-specific DNA demethylation of developmentally expressed genes involved in macronuclear differentiation.  

Bulic A, Postberg J, Fischer A, Jönsson F, Reuter G, Lipps HJ.

Epigenetics Chromatin. 2013 Mar 5;6(1):5. doi: 10.1186/1756-8935-6-5.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3608066/

Abstract

Background. DNA methylation and demethylation are important epigenetic regulatory mechanisms in eukaryotic cells and, so far, only partially understood. We exploit the minimalistic biological ciliate system to understand the crosstalk between DNA modification and chromatin structure. In the macronucleus of these cells, the DNA is fragmented into individual short DNA molecules, each representing a functional expression and replication unit. Therefore, long range epigenomic interaction can be excluded in this system.

Results. In the stichotrichous ciliate Stylonychia lemnae, cytosine methylation occurs in a small subset of macronuclear nanochromosomes expressed only during sexual reproduction. Methylation pattern shows similarity to that observed in fungi and Drosophila. Cytosine methylation correlates with gene activity and chromatin structure. Upon gene activation, cytosines become demethylated and a redistribution of histone post-translational modifications (PTMs) takes place. Evidence is presented that the formation of a permissive chromatin structure in the vicinity of the 5meCs precedes cytosine methylation and is probably a necessary prerequisite for their demethylation. Shortly after demethylation of cytosines occurs, the parental macronucleus degenerates, a new macronucleus is formed from a micronuclear derivative and the specific methylation pattern is transmitted from the germline micronucleus to the new macronucleus.

Conclusions. We show that very few, or even only one, discrete methylated cytosines are required to assign regulatory functions at a specific locus. Furthermore, evidence is provided that a permissive chromatin structure is probably a necessary prerequisite for the demethylation of specific cytosines. Our results allow us to propose a mechanistic model for the biological function of cytosine methylation in the ciliate cell and its regulation during the cell cycle.

 

 

A telomerase-associated RecQ protein-like helicase resolves telomeric G-quadruplex structures during replication.  

Postberg J, Tsytlonok M, Sparvoli D, Rhodes D, Lipps HJ.

Gene. 2012 Apr 15;497(2):147-54. doi: 10.1016/j.gene.2012.01.068. Epub 2012 Feb 2

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3650557/

Abstract

It is well established that G-quadruplex DNA structures form at ciliate telomeres and their formation throughout the cell-cycle by telomere-end-binding proteins (TEBPs) has been analyzed. During replication telomeric G-quadruplex structure has to be resolved to allow telomere replication by telomerase. It was shown that both phosphorylation of TEBPβ and binding of telomerase are prerequisites for this process, but probably not sufficient to unfold G-quadruplex structure in timely manner to allow replication to proceed. Here we describe a RecQ-like helicase required for unfolding of G-quadruplex structures in vivo. This helicase is highly reminiscent of human RecQ protein-like 4 helicase as well as other RecQ-like helicase found in various eukaryotes and E. coli. In situ analyses combined with specific silencing of either the telomerase or the helicase by RNAi and co-immunoprecipitation experiments demonstrate that this helicase is associated with telomerase during replication and becomes recruited to telomeres by this enzyme. In vitro assays showed that a nuclear extract prepared from cells in S-phase containing both the telomerase as well as the helicase resolves telomeric G-quadruplex structure. This finding can be incorporated into a mechanistic model about the replication of telomeric G-quadruplex structures during the cell cycle.

 

 

Phosphorylation of an HP1-like Protein Regulates Heterochromatin Body Assembly for DNA Elimination.

Kataoka K, Mochizuki K. 

Dev Cell. 2015 Dec 21;35(6):775-88. doi: 10.1016/j.devcel.2015.11.017. Epub 2015 Dec 10.

http://www.sciencedirect.com/science/article/pii/S1534580715007522

Summary

Heterochromatic loci are often assembled into higher-order heterochromatin bodies in diverse eukaryotes. However, the formation and biological roles of heterochromatin bodies are poorly understood. In the ciliated protozoan Tetrahymena, de novo heterochromatin body formation is accompanied by programmed DNA elimination. Here, we show that the heterochromatin body component Jub1p promotes heterochromatin body formation and dephosphorylation of the Heterochromatin Protein 1-like protein Pdd1p. Through the mutagenesis of the phosphorylated residues of Pdd1p, we demonstrate that Pdd1p dephosphorylation promotes the electrostatic interaction between Pdd1p and RNA in vitro and heterochromatin body formation in vivo. We therefore propose that heterochromatin body is assembled by the Pdd1p-RNA interaction. Pdd1p dephosphorylation and Jub1p are required for heterochromatin body formation and DNA elimination but not for local heterochromatin assembly, indicating that heterochromatin body plays an essential role in DNA elimination.

 

 

Dynamic chromatin remodelling of ciliate macronuclear DNA as determined by an optimized chromatin immunoprecipitation (ChIP) method for Paramecium tetraurelia.  

Cheaib M, Simon M. 

Appl Microbiol Biotechnol. 2013 Mar;97(6):2661-70. doi: 10.1007/s00253-013-4708-1. Epub 2013 Feb 6.

Abstract: http://www.ncbi.nlm.nih.gov/pubmed?term=dynamic%20chromatin%20remodeling%20of%20ciliate%20macronuclear%20dna%20as%20determined%20by%20an%20optimized%20chromatin%20immunoprecipitation%20(chip)%20method%20for%20paramecium%20tetraurelia&cmd=correctspelling

We report the detailed evaluation of crucial parameters for chromatin immunoprecipitation (ChIP) of macronuclear DNA in the unicellular eukaryoteParamecium tetraurelia. Optimized parameters include crosslinking conditions, chromatin sonication and antibody titration thus providing a detailed protocol for successful ChIP in P. tetraurelia. As this ciliate is bacterivorous and RNAi by feeding represents a powerful tool for analysis of gene function, we moreover determined the effects of ingested nucleic acids by food bacteria. Feasibility of our protocol is demonstrated by characterisation of chromatin remodelling at promoters of cytosolic HSP70 isoforms during transcriptional activation under heat shock conditions by analyzing RNA abundance, nucleosome occupancy and levels of H3 lysine 9 acetylation.

 

 

A Tetrahymena Hsp90 co-chaperone promotes siRNA loading by ATP-dependent and ATP-independent mechanisms.  

Woehrer SL, Aronica L, Suhren JH, Busch CJ, Noto T, Mochizuki K.

EMBO J. 2015 Feb 12;34(4):559-77. doi: 10.15252/embj.201490062. Epub 2015 Jan 14.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4331008/

Abstract

The loading of small interfering RNAs (siRNAs) and microRNAs into Argonaute proteins is enhanced by Hsp90 and ATP in diverse eukaryotes. However, whether this loading also occurs independently of Hsp90 and ATP remains unclear. We show that the Tetrahymena Hsp90 co-chaperone Coi12p promotes siRNA loading into the Argonaute protein Twi1p in both ATP-dependent and ATP-independent manners in vitro. The ATP-dependent activity requires Hsp90 and the tetratricopeptide repeat (TPR) domain of Coi12p, whereas these factors are dispensable for the ATP-independent activity. Both activities facilitate siRNA loading by counteracting the Twi1p-binding protein Giw1p, which is important to specifically sort the 26- to 32-nt siRNAs to Twi1p. Although Coi12p lacking its TPR domain does not bind to Hsp90, it can partially restore the siRNA loading and DNA elimination defects of COI12 knockout cells, suggesting that Hsp90- and ATP-independent loading of siRNA occurs in vivo and plays a physiological role in Tetrahymena.

 

 

The taming of the shrew: Regulation of a catalytically active domesticated transposase. 

Vogt A, Mochizuki K.

Mob Genet Elements. 2014 May 27;4:e29383. eCollection 2014.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4091102/

Abstract

Transposons are mobile genetic elements that can be harmful for the host when mobilized. However, they are also genomic reservoirs for novel genes that can be evolutionarily beneficial. There are many examples of domesticated transposases, which play important roles in the hosts. In most cases domesticated transposases have lost their endonuclease activities and the hosts utilize their DNA-binding properties. However, some other domesticated transposases perform endonuclease activities for host biological processes. Because such a catalytically active transposase is potentially harmful for the integrity of the host genome, its activity should be tightly regulated. The catalytically active domesticated piggyBac transposase Tpb2p catalyzes programmed DNA elimination in the ciliate Tetrahymena. Here, we discuss the regulatory mechanism that prevents unintended DNA cleavage by Tpb2p and compare it to another well-studied catalytically active domesticated transposase, the RAG recombinase in V(D)J recombination. The regulatory mechanisms involve the temporarily regulated expression of the transposases, the target sequence preference of the endonuclease, and the recruitment of the transposases to locally restricted chromatin environments.

 

 

A domesticated PiggyBac transposase interacts with heterochromatin and catalyzes reproducible DNA elimination in Tetrahymena.   

Vogt A, Mochizuki K.

PLoS Genet. 2013;9(12):e1004032. doi: 10.1371/journal.pgen.1004032. Epub 2013 Dec 12.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3861120/

Abstract

The somatic genome of the ciliated protist Tetrahymena undergoes DNA elimination of defined sequences called internal eliminated sequences (IESs), which account for 30% of the germline genome. During DNA elimination, IES regions are heterochromatinized and assembled into heterochromatin bodies in the developing somatic nucleus. The domesticated piggyBac transposase Tpb2p is essential for the formation of heterochromatin bodies and DNA elimination. In this study, we demonstrate that the activities of Tpb2p involved in forming heterochromatin bodies and executing DNA elimination are genetically separable. The cysteine-rich domain of Tpb2p, which interacts with the heterochromatin-specific histone modifications, is necessary for both heterochromatin body formation and DNA elimination, whereas the endonuclease activity of Tpb2p is only necessary for DNA elimination. Furthermore, we demonstrate that the endonuclease activity of Tpb2p in vitro and the endonuclease activity that executes DNA elimination in vivo have similar substrate sequence preferences. These results strongly indicate that Tpb2p is the endonuclease that directly catalyzes the excision of IESs and that the boundaries of IESs are at least partially determined by the combination of Tpb2p-heterochromatin interaction and relaxed sequence preference of the endonuclease activity of Tpb2p.

 

 

Selective and programmed cleavage of GPI-anchored proteins from the surface membrane by phospholipase C.  

Müller A, Klöppel C, Smith-Valentine M, Van Houten J, Simon M.

Biochim Biophys Acta. 2012 Jan;1818(1):117-24. doi: 10.1016/j.bbamem.2011.10.009. Epub 2011 Oct 14.

http://www.sciencedirect.com/science/article/pii/S0005273611003592

Abstract

Many surface proteins of eukaryotic cells are tethered to the membrane by a GPI-anchor which is enzymatically cleavable. Here, we investigate cleavage and release of different GPI-proteins by phospholipase C from the outer membrane of the ciliate Paramecium tetraurelia. Our data indicate that different GPI-proteins are not equally cleaved as proteins of the surface antigen family are preferentially released in vitro compared to several smaller GPI-proteins. Likewise, the analysis of culture medium indicates exclusive in vivo release of surface antigens by two phospholipase C isoforms (PLC2 and PLC6). This suggests that phospholipase C shows affinity for select groups of GPI-anchored proteins. Our data also reveal an up-regulation of PLC isoforms in GPI-anchored protein cleavage during antigenic switching. As a consequence, silencing of these PLCs leads to a drastic decrease of antigen concentration in the medium. These results suggest a higher order of GPI-regulation by phospholipase C as cleavage occurs programmed and specific for single GPI-proteins instead of an unspecific shedding of the entire surface membrane GPI-content.

 

 

Mob1: defining cell polarity for proper cell division.  

Tavares A, Gonçalves J, Florindo C, Tavares AA, Soares H.

J Cell Sci. 2012 Jan 15;125(Pt 2):516-27. doi: 10.1242/jcs.096610. Epub 2012 Feb 13

http://jcs.biologists.org/content/125/2/516.long

Summary

Mob1 is a component of both the mitotic exit network and Hippo pathway, being required for cytokinesis, control of cell proliferation and apoptosis. Cell division accuracy is crucial in maintaining cell ploidy and genomic stability and relies on the correct establishment of the cell division axis, which is under the control of the cell's environment and its intrinsic polarity. The ciliate Tetrahymena thermophila possesses a permanent anterior–posterior axis, left–right asymmetry and divides symmetrically. These unique features of Tetrahymena prompted us to investigate the role ofTetrahymena Mob1. Unexpectedly, we found that Mob1 accumulated in basal bodies at the posterior pole of the cell, and is the first molecular polarity marker so far described in Tetrahymena. In addition, Mob1 depletion caused the abnormal establishment of the cell division plane, providing clear evidence that Mob1 is important for its definition. Furthermore, cytokinesis was arrested and ciliogenesis delayed in Tetrahymena cells depleted of Mob1. This is the first evidence for an involvement of Mob1 in cilia biology. In conclusion, we show that Mob1 is an important cell polarity marker that is crucial for correct division plane placement, for cytokinesis completion and for normal cilia growth rates.

 

 

Ku-mediated coupling of DNA cleavage and repair during programmed genome rearrangements in the ciliate Paramecium tetraurelia.  

Marmignon A, Bischerour J, Silve A, Fojcik C, Dubois E, Arnaiz O, Kapusta A, Malinsky S, Bétermier M.

PLoS Genet. 2014 Aug 28;10(8):e1004552. doi: 10.1371/journal.pgen.1004552. eCollection 2014 Aug.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4148214/

Abstract

During somatic differentiation, physiological DNA double-strand breaks (DSB) can drive programmed genome rearrangements (PGR), during which DSB repair pathways are mobilized to safeguard genome integrity. Because of their unique nuclear dimorphism, ciliates are powerful unicellular eukaryotic models to study the mechanisms involved in PGR. At each sexual cycle, the germline nucleus is transmitted to the progeny, but the somatic nucleus, essential for gene expression, is destroyed and a new somatic nucleus differentiates from a copy of the germline nucleus. In Paramecium tetraurelia, the development of the somatic nucleus involves massive PGR, including the precise elimination of at least 45,000 germline sequences (Internal Eliminated Sequences, IES). IES excision proceeds through a cut-and-close mechanism: a domesticated transposase, PiggyMac, is essential for DNA cleavage, and DSB repair at excision sites involves the Ligase IV, a specific component of the non-homologous end-joining (NHEJ) pathway. At the genome-wide level, a huge number of programmed DSBs must be repaired during this process to allow the assembly of functional somatic chromosomes. To understand how DNA cleavage and DSB repair are coordinated during PGR, we have focused on Ku, the earliest actor of NHEJ-mediated repair. Two Ku70 and three Ku80 paralogs are encoded in the genome of P. tetraurelia: Ku70a and Ku80c are produced during sexual processes and localize specifically in the developing new somatic nucleus. Using RNA interference, we show that the development-specific Ku70/Ku80c heterodimer is essential for the recovery of a functional somatic nucleus. Strikingly, at the molecular level, PiggyMac-dependent DNA cleavage is abolished at IES boundaries in cells depleted for Ku80c, resulting in IES retention in the somatic genome. PiggyMac and Ku70a/Ku80c co-purify as a complex when overproduced in a heterologous system. We conclude that Ku has been integrated in the Paramecium DNA cleavage factory, enabling tight coupling between DSB introduction and repair during PGR.

 

 

Is non-homologous end-joining really an inherently error-prone process?  

Bétermier M, Bertrand P, Lopez BS.

PLoS Genet. 2014 Jan;10(1):e1004086. doi: 10.1371/journal.pgen.1004086. Epub 2014 Jan 16. Review.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3894167/

Abstract

DNA double-strand breaks (DSBs) are harmful lesions leading to genomic instability or diversity. Non-homologous end-joining (NHEJ) is a prominent DSB repair pathway, which has long been considered to be error-prone. However, recent data have pointed to the intrinsic precision of NHEJ. Three reasons can account for the apparent fallibility of NHEJ: 1) the existence of a highly error-prone alternative end-joining process; 2) the adaptability of canonical C-NHEJ (Ku- and Xrcc4/ligase IV–dependent) to imperfect complementary ends; and 3) the requirement to first process chemically incompatible DNA ends that cannot be ligated directly. Thus, C-NHEJ is conservative but adaptable, and the accuracy of the repair is dictated by the structure of the DNA ends rather than by the C-NHEJ machinery. We present data from different organisms that describe the conservative/versatile properties of C-NHEJ. The advantages of the adaptability/versatility of C-NHEJ are discussed for the development of the immune repertoire and the resistance to ionizing radiation, especially at low doses, and for targeted genome manipulation.

 

 

International Congress on Transposable Elements (ICTE) 2012 in Saint Malo and the sea of TE stories.

Ainouche A, Bétermier M, Chandler M, Cordaux R, Cristofari G, Deragon JM, Lesage P, Panaud O, Quesneville H, Vaury C, Vieira C, Vitte C.

Mob DNA. 2012 Oct 30;3(1):17. doi: 10.1186/1759-8753-3-17.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3520747/

Abstract

An international conference on Transposable Elements (TEs) was held 21–24 April 2012 in Saint Malo, France. Organized by the French Transposition Community (GDR Elements Génétiques Mobiles et Génomes, CNRS) and the French Society of Genetics (SFG), the conference’s goal was to bring together researchers from around the world who study transposition in diverse organisms using multiple experimental approaches. The meeting drew more than 217 attendees and most contributed through poster presentations (117), invited talks and short talks selected from poster abstracts (48 in total). The talks were organized into four scientific sessions, focused on: impact of TEs on genomes, control of transposition, evolution of TEs and mechanisms of transposition. Here, we present highlights from the talks given during the platform sessions. The conference was sponsored by Alliance pour les sciences de la vie et de la santé (Aviesan), Centre national de la recherche scientifique (CNRS), Institut national de la santé et de la recherche médicale (INSERM), Institut de recherche pour le développement (IRD), Institut national de la recherche agronomique (INRA), Université de Perpignan, Université de Rennes 1, Région Bretagne and Mobile DNA.

 

Topic 5

Genome-defence small RNAs exapted for epigenetic mating-type inheritance.  

Singh DP, Saudemont B, Guglielmi G, Arnaiz O, Goût JF, Prajer M, Potekhin A, Przybòs E, Aubusson-Fleury A, Bhullar S, Bouhouche K, Lhuillier-Akakpo M, Tanty V, Blugeon C, Alberti A, Labadie K, Aury JM, Sperling L, Duharcourt S, Meyer E.

Nature. 2014 May 22;509(7501):447-52. doi: 10.1038/nature13318. Epub 2014 May 7.

http://www.nature.com/nature/journal/v509/n7501/full/nature13318.html

Abstract

In the ciliate Paramecium, transposable elements and their single-copy remnants are deleted during the development of somatic macronuclei from germline micronuclei, at each sexual generation. Deletions are targeted by scnRNAs, small RNAs produced from the germ line during meiosis that first scan the maternal macronuclear genome to identify missing sequences, and then allow the zygotic macronucleus to reproduce the same deletions. Here we show that this process accounts for the maternal inheritance of mating types in Paramecium tetraurelia, a long-standing problem in epigenetics. Mating type E depends on expression of the transmembrane protein mtA, and the default type O is determined during development by scnRNA-dependent excision of themtA promoter. In the sibling species Paramecium septaurelia, mating type O is determined by coding-sequence deletions in a different gene, mtB, which is specifically required for mtAexpression. These independently evolved mechanisms suggest frequent exaptation of the scnRNA pathway to regulate cellular genes and mediate transgenerational epigenetic inheritance of essential phenotypic polymorphisms.

Topic 6

 

Distinct functional roles of β-tubulin isotypes in microtubule arrays of Tetrahymena thermophila, a model single-celled organism. 

Pucciarelli S, Ballarini P, Sparvoli D, Barchetta S, Yu T, Detrich HW 3rd, Miceli C.

PLoS One. 2012;7(6):e39694. doi: 10.1371/journal.pone.0039694. Epub 2012 Jun 22.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3382179/

Abstract

Background. The multi-tubulin hypothesis proposes that each tubulin isotype performs a unique role, or subset of roles, in the universe of microtubule function(s). To test this hypothesis, we are investigating the functions of the recently discovered, noncanonical β-like tubulins (BLTs) of the ciliate, Tetrahymena thermophila.Tetrahymena forms 17 distinct microtubular structures whose assembly had been thought to be based on single α- and β-isotypes. However, completion of the macronuclear genome sequence of Tetrahymenademonstrated that this ciliate possessed a β-tubulin multigene family: two synonymous genes (BTU1 andBTU2) encode the canonical β-tubulin, BTU2, and six genes (BLT1-6) yield five divergent β-tubulin isotypes. In this report, we examine the structural features and functions of two of the BLTs (BLT1 and BLT4) and compare them to those of BTU2.

Methodology/Principal Findings. With respect to BTU2, BLT1 and BLT4 had multiple sequence substitutions in their GTP-binding sites, in their interaction surfaces, and in their microtubule-targeting motifs, which together suggest that they have specialized functions. To assess the roles of these tubulins in vivo, we transformed Tetrahymena with expression vectors that direct the synthesis of GFP-tagged versions of the isotypes. We show that GFP-BLT1 and GFP-BLT4 were not detectable in somatic cilia and basal bodies, whereas GFP-BTU2 strongly labeled these structures. During cell division, GFP-BLT1 and GFP-BLT4, but not GFP-BTU2, were incorporated into the microtubule arrays of the macronucleus and into the mitotic apparatus of the micronucleus. GFP-BLT1 also participated in formation of the microtubules of the meiotic apparatus of the micronucleus during conjugation. Partitioning of the isotypes between nuclear and ciliary microtubules was confirmed biochemically.

Conclusion/Significance. We conclude that Tetrahymena uses a family of distinct β-tubulin isotypes to construct subsets of functionally different microtubules, a result that provides strong support for the multi-tubulin hypothesis.

 

 

Tubulin folding: The special case of a beta-tubulin isotype from the Antarctic psychrophilic ciliate Euplotes focardii 

Pucciarelli, S.a  , Chiappori, F.b, Sparvoli, D.a, Milanesi, L.b, Miceli, C.a, Melki, R.c

Polar Biology. Volume 36, Issue 12, 1 April 2013, Pages 1833-1838

Abstract

Folding assistance is a fundamental requirement of certain proteins, and it may be subjected to physicochemical constraints in case of organisms adapted to polar temperatures. Limited information is available about protein folding in the polar environment. Folding of tubulin provides one of the few studied cases. Here, we report a pilot folding analysis of a divergent beta-tubulin isotype, named EFBT3, from the Antarctic psychrophilic ciliate Euplotes focardii. To attain its native monomeric structure, beta-tubulin needs the assistance of the eukaryotic class II chaperonin CCT and cofactor A (CofA). The in vitro folding reaction of EFBT3 with CCT and CofA purified from rabbit did not generate any folded product. In contrast, the reaction performed with the rabbit reticulocyte lysate, that contains all the chaperones required for efficient tubulin folding, was productive, suggesting that additional factors besides purified CCT and CofA are required for EFBT3 to attain its monomeric structure. We also demonstrated that the rare Cys281 of EFBT3 is critical for the folding reaction. Model predictions indicate that EFBT3 binds to CofA differently from yeast beta-tubulin, suggesting a diverse folding mechanism that may be correlated with microtubule cold adaptation. © Springer-Verlag Berlin Heidelberg 2013

 

 

Structural thermal adaptation of β-tubulins from the Antarctic psychrophilic protozoan Euplotes focardii 

Chiappori, F.a  , Pucciarelli, S.b  , Merelli, I.a, Ballarini, P.b, Miceli, C.b, Milanesi, L.a

Proteins: Structure, Function and Bioinformatics. Volume 80, Issue 4, April 2012, Pages 1154-1166

Abstract

Tubulin dimers of psychrophilic eukaryotes can polymerize into microtubules at 4°C, a temperature at which microtubules from mesophiles disassemble. This unique capability requires changes in the primary structure and/or in post-translational modifications of the tubulin subunits. To contribute to the understanding of mechanisms responsible for microtubule cold stability, here we present a computational structural analysis based on molecular dynamics (MD) and experimental data of three β-tubulin isotypes, named EFBT2, EFBT3, and EFBT4, from the Antarctic protozoon Euplotes focardii that optimal temperature for growth and reproduction is 4°C. In comparison to the β-tubulin from E. crassus, a mesophilic Euplotes species, EFBT2, EFBT3, and EFBT4 possess unique amino acid substitutions that confer different flexible properties of the polypeptide, as well as an increased hydrophobicity of the regions involved in microtubule interdimeric contacts that may overcome the microtubule destabilizing effect of cold temperatures. The structural analysis based on MD indicated that all isotypes display different flexibility properties in the regions involved in the formation of longitudinal and lateral contacts during microtubule polymerization. We also investigated the role of E. focardii β-tubulin isotypes during the process of cilia formation. The unique characteristics of the primary and tertiary structures of psychrophilic β-tubulin isotypes seem responsible for the formation of microtubules with distinct dynamic and functional properties. © 2011 Wiley Periodicals, Inc.

 

Unicellular eukaryotes as models in cell and molecular biology: critical appraisal of their past and future value. 

Simon M, Plattner H.

Int Rev Cell Mol Biol. 2014;309:141-98. doi: 10.1016/B978-0-12-800255-1.00003-X. Review.

Abstract: http://www.sciencedirect.com/science/article/pii/B978012800255100003X

Unicellular eukaryotes have been appreciated as model systems for the analysis of crucial questions in cell and molecular biology. This includes Dictyostelium (chemotaxis, amoeboid movement, phagocytosis), Tetrahymena (telomere structure, telomerase function), Paramecium (variant surface antigens, exocytosis, phagocytosis cycle) or both ciliates (ciliary beat regulation, surface pattern formation), Chlamydomonas (flagellar biogenesis and beat), and yeast (Scerevisiae) for innumerable aspects. Nowadays many problems may be tackled with “higher” eukaryotic/metazoan cells for which full genomic information as well as domain databases, etc., were available long before protozoa. Established molecular tools, commercial antibodies, and established pharmacology are additional advantages available for higher eukaryotic cells. Moreover, an increasing number of inherited genetic disturbances in humans have become elucidated and can serve as new models. Among lower eukaryotes, yeast will remain a standard model because of its peculiarities, including its reduced genome and availability in the haploid form. But do protists still have a future as models? This touches not only the basic understanding of biology but also practical aspects of research, such as fund raising. As we try to scrutinize, due to specific advantages some protozoa should and will remain favorable models for analyzing novel genes or specific aspects of cell structure and function. Outstanding examples are epigenetic phenomena—a field of rising interest.

 

 

In silico identification and characterization of the MAPK family members of unicellular model eukaryote Tetrahymena thermophila.  

Yıldız MT, Arslanyolu M.

Eur J Protistol. 2014 Oct;50(5):538-50. doi: 10.1016/j.ejop.2014.08.005. Epub 2014 Sep 8.

http://www.sciencedirect.com/science/article/pii/S0932473914000674

Abstract

The biological function and evolutionary diversity of the mitogen-activated protein kinase (MAPK) family have mostly been studied in fungi, animals and plants, with very limited information from lower eukaryotes. This study aimed to describe the MAPKs of unicellular Tetrahymena thermophila. Eight members of the T. thermophila MAPK (TtMPK) gene family, in addition to previously reported TtMPK1, TtMPK2 and TtMPK3, were identified bioinformatically using a T. thermophila genome database. Phylogenetic analysis assigned the TtMPKs into two major groups, ERK1/2-like (TtMPK1, 2, 3, 5, 6, 7, 8, and 9) as stress-responsive MAPKs for biotic and abiotic stresses, and ERK7/8-like (TtMPK4, 10, and 11) as cell-cycle-associated protein kinases for biotic factors. Semi-quantitative RT-PCR analysis of the TtMPKs showed high mRNA expression at 30 °C; however, only TtMPK5 and TtMPK6 showed high expression at 37 °C. Osmotic shock by 100 mM NaCl only increased the expression of TtMPK2, whereas 20 mM NaCl reduced the expression of all MPKs to almost zero. The results suggested that T. thermophilaMAPKs are among the closest representatives of the ancestors of the eukaryotic MAPK family. Although no functional characterization of MPKs was performed, this study is the first report of the genome-wide MAPK family in T. thermophila.

 

 

Cloning, expression and characterization of a gene encoding mitogen activated protein kinase 2 (MPK2) from Tetrahymena thermophila. 

Arslanyolu M, Yıldız MT.

Gene. 2014 Aug 1;546(1):40-9. doi: 10.1016/j.gene.2014.05.041. Epub 2014 May 22

http://www.sciencedirect.com/science/article/pii/S0378111914006040

Abstract

Environmental effects and mitogens determine cell phenotype in eukaryotes mainly through MAPK pathways. However, MAPK signaling pathways in T. thermophila have not been studied comprehensively. This study aims to express recombinant MPK2, a MAPK from T. thermophila, in E. coli to characterize its kinase activity. MPK2 was cloned by RT-PCR using degenerate oligonucleotide primers and RACE method. The full-length cDNA of the MPK2 gene is 1705 bp that includes 1281 bp ORF coding for a putative protein of 426 amino acids having a mass of 50.2 kDa. The putative MPK2 protein contains all eleven conserved subdomains that are characteristics of serine/threonine protein kinases, and a TDY motif, which is a putative dual phosphorylation site common in Protista. MPK2 displays highest 48% overall identity to human ERK5 (MAPK7). The expression vector pGEX4T-1-MPK2 was constructed by inserting the coding region of MPK2 cDNA into pGEX4T-1 after introducing the nine point mutations, and then transformed into E. coli BL21(DE3). Autophosphorylation of 76 kDa GST-MPK2 at tyrosine residues was confirmed not only by Western blot using anti-phosphotyrosine monoclonal antibody but also by in vitro kinase assay. GST-MPK2 was also able to phosphorylate the artificial substrate myelin basic protein. This study concludes that the free-living unicellular protist T. thermophila MPK2 has commonly conserved MAPK enzyme features, possibly involved in the regulation of cell survival responding to abiotic or biotic stressors, and the production and movement of haploid gametic nuclei between pairs during conjugation.

Topic 7

 

Microbial Consortium Associated with the Antarctic Marine Ciliate Euplotes focardii: An Investigation from Genomic Sequences. 

Pucciarelli S, Devaraj RR, Mancini A, Ballarini P, Castelli M, Schrallhammer M, Petroni G, Miceli C.

Microb Ecol. 2015 Aug;70(2):484-97. doi: 10.1007/s00248-015-0568-9. Epub 2015 Feb 24.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4494151/

Abstract

We report the characterization of the bacterial consortium associated to Euplotes focardii, a strictly psychrophilic marine ciliate that was maintained in laboratory cultures at 4 °C after its first isolation from Terra Nova Bay, in Antarctica. By Illumina genome analyser, we obtained 11,179 contigs of potential prokaryotic origin and classified them according to the NCBI’s prokaryotic attributes table. The majority of these sequences correspond to either Bacteroidetes (16 %) or Proteobacteria (78 %). The latter were dominated by gamma- (39 %, including sequences related to the pathogenic genus Francisella), and alpha-proteobacterial (30 %) sequences. Analysis of the Pfam domain family and Gene Ontology term variation revealed that the most frequent terms that appear unique to this consortium correspond to proteins involved in “transmembrane transporter activity” and “oxidoreductase activity”. Furthermore, we identified genes that encode for enzymes involved in the catabolism of complex substance for energy reserves. We also characterized members of the transposase and integrase superfamilies, whose role in bacterial evolution is well documented, as well as putative antifreeze proteins. Antibiotic treatments of E. focardii cultures delayed the cell division of the ciliate. To conclude, our results indicate that this consortium is largely represented by bacteria derived from the original Antarctic sample and may contribute to the survival of E. focardii in laboratory condition. Furthermore, our results suggest that these bacteria may have a more general role in E. focardii survival in its natural cold and oxidative environment.

 

 

A House for Two-Double Bacterial Infection in Euplotes woodruffi Sq1 (Ciliophora, Euplotia) Sampled in Southeastern Brazil.  

Senra MV, Dias RJ, Castelli M, Silva-Neto ID, Verni F, Soares CA, Petroni G.

Microb Ecol. 2015 Sep 17. [Epub ahead of print]

Abstract: http://www.ncbi.nlm.nih.gov/pubmed/?term=A+House+for+Two-Double+Bacterial+Infection+in+Euplotes+woodruffi+Sq1+(Ciliophora%2C+Euplotia)+Sampled+in+Southeastern+Brazil

Several ciliated protists form symbiotic associations with a diversity of microorganisms, leading to drastic impact on their ecology and evolution. In this work, two Euplotes spp. sampled in Rio de Janeiro, Brazil, were identified based on morphological and molecular features as Euplotes woodruffistrain Sq1 and E. encysticus strain Sq2 and investigated for the presence of endosymbionts. While E. woodruffi Sq1 stably hosts two bacterialpopulations, namely Polynucleobacter necessarius (Betaproteobacteria) and a new member of the family "Candidatus Midichloriaceae" (Alphaproteobacteria, Rickettsiales), here described as "Candidatus Bandiella woodruffii," branching with a broad host range bacterial group found in association with cnidarians, sponges, euglenoids, and some arthropods; in E. encysticus Sq2 no symbiotic bacterium could be detected. The dispersion ability of this novel bacterium was tested by co-incubating E. woodruffi Sq1 with three different ciliate species. Among the tested strains "Ca. B. woodruffii" could only be detected in association with E. encysticus Sq2 with a prevalence of 20 % after 1 week and 40 % after 2 weeks, maintaining this level for up to 6 months. Nevertheless, this apparent in vitro association was abolished when E. woodruffi Sq1 donor was removed from the microcosm, suggesting that this bacterium has the capacity for at least a short-term survival outside its natural host and the aptitude to ephemerally interact with other organisms. Together, these findings strongly suggest the need for more detailed investigations to evaluate the host range for "Ca. B. woodruffii" and any possible pathogenic effect of this bacterium on other organisms including humans.

 

 

Response of the bacterial symbiont Holospora caryophila to different growth conditions of its host.  7

Castelli M, Lanzoni O, Fokin SI, Schrallhammer M, Petroni G.

Eur J Protistol. 2015 Feb;51(1):98-108. doi: 10.1016/j.ejop.2014.11.006. Epub 2014 Dec 16.

http://www.sciencedirect.com/science/article/pii/S0932473914000844

Abstract

Previous studies on bacterial symbionts of ciliates have shown that some symbionts can be maintained relatively well under standard laboratory conditions whereas others are frequently lost, especially when the host is cultivated at a high division rate. In this study, the variation in infection level by the endosymbiont Holospora caryophila within its host population Paramecium octaurelia was investigated in response to three alimentary treatments and a subsequent starvation phase. The response of the ciliates was determined as a nearly exponential growth rate with different slopes in each treatment, proportional to the amount of food received. The initial infection level was higher than 90%. After 24 days of exponential host's growth, the prevalence remained stable at approximately 90% in all treatments, even after a subsequent starvation phase of 20 days. However, at intermediate time-points in both the feeding and the starvation phase, fluctuations in the presence of the intracellular bacteria were observed. These results show that H. caryophila is able to maintain its infection under the tested range of host growth conditions, also due to the possibility of an effective re-infection in case of partial loss.

 

 

Focusing on genera to improve species identification: revised systematics of the ciliateSpirostomum.  

Boscaro V, Carducci D, Barbieri G, Senra MV, Andreoli I, Erra F, Petroni G, Verni F, Fokin SI.

Protist. 2014 Aug;165(4):527-41. doi: 10.1016/j.protis.2014.05.004. Epub 2014 Jun 2.

http://www.sciencedirect.com/science/article/pii/S1434461014000510

Although many papers dealing with the description of new ciliate taxa are published each year, species taxonomy and identification in most groups of the phylum Ciliophora remain confused. This is largely due to a scarcity of surveys on the systematics of immediately higher levels (genera and families) providing data for old and new species together. Spirostomum is a common and distinctive inhabitant of fresh- and brackish water environments, including artificial and eutrophic ones, and is a good model for applied ecology and symbiosis research. Despite this, only 3 of the numerous species are commonly cited, and no studies have yet confirmed their monophyly, with the consequence that reproducibility of the results may be flawed. In this paper we present morphological and molecular data for 30 Spirostomum populations representing 6 different morphospecies, some of which were collected in previously unreported countries. We performed a detailed revision of Spirostomum systematics combining literature surveys, new data on hundreds of organisms and statistical and phylogenetic analyses; our results provide insights on the evolution, ecology and distribution of known morphospecies and a novel one: Spirostomum subtilis sp. n. We also offer tools for quick species identification.

 

 

Free-living ciliates as potential reservoirs for eukaryotic parasites: occurrence of a trypanosomatid in the macronucleus of Euplotes encysticus.  

Fokin SI, Schrallhammer M, Chiellini C, Verni F, Petroni G.

Parasit Vectors. 2014 Apr 28;7:203. doi: 10.1186/1756-3305-7-203.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4022238/

Abstract

Background. Flagellates of the family Trypanosomatidae are obligate endoparasites, which can be found in various hosts. Several genera infect insects and occur as monoxenous parasites especially in representatives of Diptera and Hemiptera. These trypanosomatid flagellates probably share the worldwide distribution of their hosts, which are often infested by large numbers of endoparasites. Traditionally, their taxonomy was based on morphology, host origin, and life cycle. Here we report the characterization of a trypanosomatid infection detected in a protozoan, a ciliate collected from a polluted freshwater pond in a suburb of New Delhi (India).

Methods. Live observations and morphological studies applying light, fluorescence and transmission electron microscopy were conducted. Molecular analyses of host and parasite were performed and used for phylogenetic reconstructions and species (host) or genus level (parasite) identification.

Results. Although the morphological characteristics were not revealing, a high similarity of the trypanosomatids 18S rRNA gene sequence to Herpetomonas ztiplika and Herpetomonas trimorpha (Kinetoplastida, Trypanosomatidae), both parasites of biting midges (Culicoides kibunensis and Culicoides truncorum,respectively) allowed the assignment to this genus. The majority of the host population displayed a heavy infection that significantly affected the shape of the host macronucleus, which was the main site of parasite localization. In addition, the growth rate of host cultures, identified as Euplotes encysticus according to cell morphology and 18S rRNA gene sequence, was severely impacted by the infection.

Conclusions. The host-parasite system described here represents a recent example of free-living protists acting as environmental reservoirs for parasitic eukaryotic microorganisms.

 

 

Rediscovering the genus Lyticum, multiflagellated symbionts of the order Rickettsiales.   7

Boscaro V, Schrallhammer M, Benken KA, Krenek S, Szokoli F, Berendonk TU, Schweikert M, Verni F, Sabaneyeva EV, Petroni G.

Sci Rep. 2013 Nov 22;3:3305. doi: 10.1038/srep03305.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3837311/

Abstract

Among the bacterial symbionts harbored by the model organism Paramecium, many still lack a recent investigation that includes a molecular characterization. The genus Lyticum consists of two species of large-sized bacteria displaying numerous flagella, despite their inability to move inside their hosts' cytoplasm. We present a multidisciplinary redescription of both species, using the deposited type strains as well as newly collected material. On the basis of 16S rRNA gene sequences, we assigned Lyticum to the orderRickettsiales, that is intensely studied because of its pathogenic representatives and its position as the extant group most closely related to the mitochondrial ancestor. We provide conclusive proofs that at least someRickettsiales possess actual flagella, a feature that has been recently predicted from genomic data but never confirmed. We give support to the hypothesis that the mitochondrial ancestor could have been flagellated, and provide the basis for further studies on these ciliate endosymbionts.

 

 

Polynucleobacter necessarius, a model for genome reduction in both free-living and symbiotic bacteria.  

Boscaro V, Felletti M, Vannini C, Ackerman MS, Chain PS, Malfatti S, Vergez LM, Shin M, Doak TG, Lynch M, Petroni G.

Proc Natl Acad Sci U S A. 2013 Nov 12;110(46):18590-5. doi: 10.1073/pnas.1316687110. Epub 2013 Oct 28.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3831957/

ABSTRACT

We present the complete genomic sequence of the essential symbiont Polynucleobacter necessarius(Betaproteobacteria), which is a valuable case study for several reasons. First, it is hosted by a ciliated protist, Euplotes; bacterial symbionts of ciliates are still poorly known because of a lack of extensive molecular data. Second, the single species P. necessarius contains both symbiotic and free-living strains, allowing for a comparison between closely related organisms with different ecologies. Third, free-living P. necessarius strains are exceptional by themselves because of their small genome size, reduced metabolic flexibility, and high worldwide abundance in freshwater systems. We provide a comparative analysis of P. necessarius metabolism and explore the peculiar features of a genome reduction that occurred on an already streamlined genome. We compare this unusual system with current hypotheses for genome erosion in symbionts and free-living bacteria, propose modifications to the presently accepted model, and discuss the potential consequences of translesion DNA polymerase loss.

 

 

'Candidatus Megaira polyxenophila' gen. nov., sp. nov.: considerations on evolutionary history, host range and shift of early divergent rickettsiae.  

Schrallhammer M, Ferrantini F, Vannini C, Galati S, Schweikert M, Görtz HD, Verni F, Petroni G.

PLoS One. 2013 Aug 20;8(8):e72581. doi: 10.1371/journal.pone.0072581. eCollection 2013.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3748036/

Abstract

“Neglected Rickettsiaceae” (i.e. those harboured by non-hematophagous eukaryotic hosts) display greater phylogenetic variability and more widespread dispersal than pathogenic ones; yet, the knowledge about their actual host range and host shift mechanism is scarce. The present work reports the characterization following the full-cycle rRNA approach (SSU rRNA sequence, specific in situ hybridization, and ultrastructure) of a novel rickettsial bacterium, herewith proposed as 'Candidatus Megaira polyxenophila' gen. nov., sp. nov. We found it in association with four different free-living ciliates (Diophrys oligothrix, Euplotes octocarinatus, Paramecium caudatum, and Spirostomum sp., all belonging to Alveolata, Ciliophora); furthermore it was recently observed as intracellular occurring in Carteria cerasiformis and Pleodorina japonica(Chlorophyceae, Chlorophyta). Phylogenetic analyses demonstrated the belonging of the candidate new genus to the family Rickettsiaceae (AlphaproteobacteriaRickettsiales) as a sister group of the genusRickettsiaIn situ observations revealed the ability of the candidate new species to colonize either nuclear or cytoplasmic compartments, depending on the host organism. The presence of the same bacterial species within different, evolutionary distant, hosts indicates that 'Candidatus Megaira polyxenophila' recently underwent several distinct host shifts, thus suggesting the existence of horizontal transmission pathways. We consider these findings as indicative of an unexpected spread of rickettsial infections in aquatic communities, possibly by means of trophic interactions, and hence propose a new interpretation of the origin and phylogenetic diversification of rickettsial bacteria.

 

 

Morphology, ultrastructure, and molecular phylogeny of the ciliate Sonderia vorax with insights into the systematics of order Plagiopylida.  

Modeo L, Fokin SI, Boscaro V, Andreoli I, Ferrantini F, Rosati G, Verni F, Petroni G.

BMC Microbiol. 2013 Feb 18;13:40. doi: 10.1186/1471-2180-13-40.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3626617/

Abstract

Background. Ciliates of the family Sonderiidae are common members of the eukaryotic communities in various anoxic environments. They host both ecto- and endosymbiotic prokaryotes (the latter associated with hydrogenosomes) and possess peculiar morpho-ultrastructural features, whose functions and homologies are not known. Their phylogenetic relationships with other ciliates are not completely resolved and the available literature, especially concerning electron microscopy and molecular studies, is quite scarce.

Results. Sonderia vorax Kahl, 1928 is redescribed from an oxygen-deficient, brackish-water pond along the Ligurian Sea coastlines of Italy. Data on morphology, morphometry, and ultrastructure are reported. S. vorax is ovoid-ellipsoid in shape, dorsoventrally flattened, 130 x 69 μm (mean in vivo); it shows an almost spherical macronucleus, and one relatively large micronucleus. The ventral kinetom has a “secant system” including fronto-ventral and fronto-lateral kineties. A distinctive layer of bacteria laying between kineties covers the ciliate surface. Two types of extrusomes and hydrogenosomes-endosymbiotic bacteria assemblages are present in the cytoplasm. The phylogeny based on 18S rRNA gene sequences places S. vorax among Plagiopylida; Sonderiidae clusters with Plagiopylidae, although lower-level relationships remain uncertain. The studied population is fixed as neotype and the ciliate is established as type species of the genus, currently lacking.

Conclusions. This is the first description of a representative of Sonderiidae performed with both morphological and molecular data. To sum up, many previous hypotheses on this interesting, poorly known taxon are confirmed but confusion and contradictory data are as well highlighted.

 

 

"Candidatus Defluviella procrastinata" and "Candidatus Cyrtobacter zanobii", two novel ciliateendosymbionts belonging to the "Midichloria clade".  

Boscaro V, Petroni G, Ristori A, Verni F, Vannini C.

Microb Ecol. 2013 Feb;65(2):302-10. doi: 10.1007/s00248-012-0170-3. Epub 2013 Jan 8.

Abstract: http://www.ncbi.nlm.nih.gov/pubmed?term="Candidatus%20Defluviella%20procrastinata"%20and%20"Candidatus%20Cyrtobacter%20zanobii"%20,%20two%20novel%20ciliate%20endosymbionts%20belonging%20to%20the%20"Midichloria%20clade"&cmd=correctspelling

The "Midichloria clade" is a recently discovered but well-established evolutionary lineage clustering inside the order Rickettsiales (Alphaproteobacteria). Not much is known about the biology of these organisms. The best characterized ones are endocellular symbionts of very different eukaryotic hosts, ranging from arthropods to protists. "Candidatus Midichloria mitochondrii", the most studied organism of the group, is an interesting object of study because of its unique capability to infect metazoans' mitochondria and the presence of flagellar genes in its genome. With this work, we aim at increasing the knowledge on the biodiversity and phylogeny of the "Midichloria group". We characterized according to the "full cycle rRNA approach" two novel endosymbionts of ciliated protozoa, i.e. Paramecium nephridiatum and Euplotes aediculatus. According to the nomenclatural rules for uncultivated prokaryotes, we established the novel taxa "Candidatus Defluviella procrastinata" and "Candidatus Cyrtobacterzanobii" for the two bacterial symbionts. Our phylogenetic analysis based on 16S rRNA gene sequences confirms that the evolutionary histories of "Midichloria clade" representatives and of their hosts are very different. This suggests that the symbiotic processes arose many times independently, perhaps through ways of transmission still not described in Rickettsiales.

 

 

Morphological, ultrastructural, and molecular characterization of Euplotidium rosati n. sp. (Ciliophora, Euplotida) from Guam. 

Modeo L, Petroni G, Lobban CS, Verni F, Vannini C.

J Eukaryot Microbiol. 2013 Jan-Feb;60(1):25-36. doi: 10.1111/jeu.12003. Epub 2012 Nov 29.

http://www.ncbi.nlm.nih.gov/pubmed/23194274

Abstract

We combined morphological (i.e. live, stained, scanning, and transmission electron microscopy) with morphometric and molecular analysis to describe a ciliate species collected from shallow reefs in Guam, grown, and maintained in our laboratory. The species was recognized as a member of Euplotidium, and compared with established species of the genus: Euplotidium itoi Ito 1958; Euplotidium psammophilus (Vacelet 1961) Borror 1972; Euplotidium arenarium Magagnini and Nobili 1964; Euplotidium helgae Hartwig 1980; Euplotidium prosaltans Tuffrau 1985, and Euplotidium smalli Lei, Choi and Xu, 2002. To obtain more elements to compare the species, new morphometric data and additional SSU rRNA gene sequences of E. itoi and of E. arenarium are reported. On the basis of this comparison, we established the new species Euplotidium rosati that has a cirral pattern composed of 12 frontoventral and six transverse cirri, and lacks the left marginal cirrus. Euplotidium rosati harbors on its dorsal surface epixenosomes, the peculiar extrusive symbionts described in other Euplotidium species. The whole body of our observations together with the analysis of the data available in the literature leads us to propose a redefinition of the genus. The results may also be useful to clarify the tangled relationship between Euplotidium and Gastrocirrhus.

 

 

Revised systematics of Holospora-like bacteria and characterization of "Candidatus Gortzia infectiva", a novel macronuclear symbiont of Paramecium jenningsi.  

Boscaro V, Fokin SI, Schrallhammer M, Schweikert M, Petroni G.

Microb Ecol. 2013 Jan;65(1):255-67. doi: 10.1007/s00248-012-0110-2. Epub 2012 Sep 1.

Abstract: http://www.ncbi.nlm.nih.gov/pubmed/?term=Revised+systematics+of+Holospora-like+bacteria+and+characterization+of+"Candidatus+Gortzia+infectiva"%2C+a+novel+macronuclear+symbiont+of+Paramecium+jenningsi

The genus Holospora (Rickettsiales) includes highly infectious nuclear symbionts of the ciliate Paramecium with unique morphology and life cycle. To date, nine species have been described, but a molecular characterization is lacking for most of them. In this study, we have characterized anovel Holospora-like bacterium (HLB) living in the macronuclei of a Paramecium jenningsi population. This bacterium was morphologically and ultrastructurally investigated in detail, and its life cycle and infection capabilities were described. We also obtained its 16S rRNA gene sequence and developed a specific probe for fluorescence in situ hybridization experiments. A new taxon, "Candidatus Gortzia infectiva", was established for this HLB according to its unique characteristics and the relatively low DNA sequence similarities shared with other bacteria. The phylogeny of the order Rickettsiales based on 16S rRNA gene sequences has been inferred, adding to the available data the sequence of the novel bacterium and those of two Holospora species (Holospora obtusa and Holospora undulata) characterized for the purpose. Our phylogenetic analysis provided molecular support for the monophyly of HLBs and showed a possible pattern of evolution for some of their features. We suggested to classify inside the family Holosporaceae only HLBs, excluding other more distantly related and phenotypically different Paramecium endosymbionts.

 

 

Characterization of "Candidatus Nebulobacter yamunensis" from the cytoplasm of Euplotes aediculatus (Ciliophora, Spirotrichea) and emended description of the family Francisellaceae.  

Boscaro V, Vannini C, Fokin SI, Verni F, Petroni G.

Syst Appl Microbiol. 2012 Oct;35(7):432-40. doi: 10.1016/j.syapm.2012.07.003. Epub 2012 Aug 29.

http://www.sciencedirect.com/science/article/pii/S0723202012000963

@bstract

Our knowledge of ciliate endosymbionts occurrence and diversity greatly expanded in the last decades, due to the development of characterization methods for uncultivable bacteria. Symbionts related to human pathogens such as rickettsiae and francisellae have been detected inside the cytoplasm of different ciliate species. In the present work, we have characterized a novel Francisella-related bacterium inside the rich prokaryotic community harbored by a population of Euplotes aediculatus (Ciliophora, Spirotrichea). Following the “Full-Cycle rRNA Approach” we obtained the almost full-length 16S rRNA gene sequence of this bacterium, and developed probes for diagnostic fluorescence in situ hybridizations. Attempts to culture the endosymbiont outside of its host failed. We classified this novel organism in a new taxon for which we propose the name “CandidatusNebulobacter yamunensis”. In order to investigate its evolutionary relationships, we have also performed phylogenetic analyses on the class Gammaproteobacteria and the orderThiotrichales, which include the monogeneric family Francisellaceae. We found highly supported evidences for the establishment of a new monophyletic taxon includingFrancisella species, other organisms currently incertae sedis, and “CandidatusNebulobacter yamunensis”. These organisms form a clade sharing a signature sequence not present in other Thiotrichales bacteria. Moreover, most of them have developed an intracellular life cycle inside eukaryotic organisms. We emended the original description of family Francisellaceae in order to encompass all members of the described clade.

 

 

Betaproteobacterial symbionts of the ciliate Euplotes: origin and tangled evolutionary path of an obligate microbial association. 

Vannini C, Ferrantini F, Ristori A, Verni F, Petroni G.

Environ Microbiol. 2012 Sep;14(9):2553-63. doi: 10.1111/j.1462-2920.2012.02760.x. Epub 2012 Apr 26.

http://www.ncbi.nlm.nih.gov/pubmed/22533449

Abstract

The Polynucleobacter-Euplotes association is an obligatory symbiotic system between a monophyletic group of ciliate species belonging to the genus Euplotes and bacteria of the species Polynucleobacter necessarius (Betaproteobacteria). Both organisms are unable to survive independently. Several studies revealed the existence of free-living populations of Polynucleobacter bacteria which are phylogenetically closely related to the endosymbiotic ones, but never share associations with Euplotes in the natural environment. Hence, following the most parsimonious explanation on the origin of the association, this symbiosis should represent a synapomorphic character for the hosts' clade. Nevertheless, phylogenetic analyses performed on an increased number of strains here presented suggest that Euplotes species, during their evolution, recruited Polynucleobacter bacteria as symbionts more than once. Moreover, in three cases, we observed different bacteria as obligate symbionts. These symbionts are the first characterized representatives of a phylogenetic lineage branching in a basal position with respect to the genus Polynucleobacter. The hypothesis that the original obligate symbionts belonged to this newly discovered clade and that, only subsequently, in most cases they have been replaced by Polynucleobacter bacteria recruited from the environment is proposed and discussed. The evolutionary path of this association seems anyway to have been more complex than so far supposed.

 

Tracing the role of R-bodies in the killer trait: absence of toxicity of R-body producing recombinant E. coli on paramecia. 

Schrallhammer M, Galati S, Altenbuchner J, Schweikert M, Görtz HD, Petroni G.

Eur J Protistol. 2012 Nov;48(4):290-6. doi: 10.1016/j.ejop.2012.01.008. Epub 2012 Feb 20

http://www.sciencedirect.com/science/article/pii/S0932473912000193

Abstract

R-bodies are coiled proteinaceous ribbons produced by Paramecium endosymbionts belonging to the genus Caedibacter. These intracellular bacteria confer upon their hosts a phenomenon called the killer trait. It is the ability to kill symbiont-free competitors called sensitives. The R-body is the crucial element of this process, but despite many efforts, the actual role of R-bodies in killing sensitive paramecia is still not satisfactory clarified. The open question is whether the R-body acts as transmitter for a yet unidentified toxin or whether it directly kills sensitive paramecia having intrinsic cytotoxic effects. In the present study, this problem is addressed by heterologous expression of Caedibacter taeniospiralis R-body in Escherichia coli followed by a detailed analysis of its potential intrinsic toxic effect on feeding sensitive Paramecium tetraurelia. Using this approach, we can exclude any eventual effects of additional, unidentified factors produced by C. taeniospiralis and thus observe the impact of the recombinant R-body itself. No cytotoxic effects of recombinant R-bodies were detected following this approach, strengthening the hypothesis that R-bodies act as releasing system for an unidentified C. taeniospiralistoxin.

 

 

Vertically transmitted symbiont reduces host fitness along temperature gradient. 

Dusi E, Krenek S, Schrallhammer M, Sachse R, Rauch G, Kaltz O, Berendonk TU.

J Evol Biol. 2014 Apr;27(4):796-800.

Abstract: http://www.ncbi.nlm.nih.gov/pubmed/?term=Vertically+transmitted+symbiont+reduces+host+fitness+along+temperature+gradient.

Parasites with exclusive vertical transmission from host parent to offspring are an evolutionary puzzle. With parasite fitness entirely linked to hostreproduction, any fitness cost for infected hosts risks their selective elimination. Environmental conditions likely influence parasite impact and thereby the success of purely vertical transmission strategies. We tested for temperature-dependent virulence of Caedibacter taeniospiralis, avertically transmitted bacterial symbiont of the protozoan Paramecium tetraurelia. We compared growth of infected and cured host populations at five temperatures (16–32 °C). Infection reduced host density at all temperatures, with a peak of −30% at 28 °C. These patterns were largely consistent across five infected Paramecium strains. Similar to Wolbachia symbionts, C. taeniospiralis may compensate fitness costs by conferring to the hosta ‘killer trait’, targeting uninfected competitors. Considerable loss of infection at 32 °C suggests that killer efficacy is not universal and that limited heat tolerance restricts the conditions for persistence of C. taeniospiralis.

 

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