Sterkiella Histriomuscorum Research Articles

Shedding light on the polyphyletic behavior of the genus Sterkiella: The importance of ontogenetic and molecular phylogenetic approaches.

Present study, investigates a poorly known species of the genus Sterkiella, i.e., S. tricirrata, based on two populations isolated from soil samples collected from the Colfiorito Regional Park, Umbria Region, Italy and from the Silent Valley National Park, India. Both populations showed a highly similar morphology, however different ontogenetic pattern in between. The study confirms the validity of the species S. tricirrata which was considered to be a species within the Sterkiella histriomuscorum complex. The main ontogenetic difference between S. tricirrata and other species of the genus Sterkiella is the different mode of formation of anlagen V and VI of the proter in the former. In the phylogenetic analyses, Sterkiella tricirrata clusters with Sterkiella sinica within the stylonychine oxytrichids, in a clade away from the type species (Sterkiella cavicola) of the genus Sterkiella. The study highlights the importance of ontogenetic as well as molecular data in shedding light on the polyphyletic behavior of the genus Sterkiella. A detailed description of S. tricirrata based on morphology, ontogenesis and molecular phylogenetic methods is presented. Further, the improved diagnosis has been provided for the genus Sterkiella and the poorly known species S. tricirrata.

Characterization and Taxonomic Validity of the Ciliate Oxytricha trifallax (Class Spirotrichea) Based on Multiple Gene Sequences: Limitations in Identifying Genera Solely by Morphology

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.

Morphology and Small Subunit (SSU) rRNA Gene Sequence of the New Brackish Water Ciliate Neobakuella flava n. g., n. sp. (Ciliophora, Spirotricha, Bakuellidae) and SSU rRNA Gene Sequences of Six Additional Hypotrichs from Korea

The morphology and the small subunit (SSU) rRNA gene sequence of the hypotrich Neobakuella flava n. g., n. sp. from the estuary of the Taehwagang River (Ulsan, South Korea) were investigated. The three frontal cirri, the composition of the midventral complex of cirral pairs and rows, and the simple dorsal kinety pattern of three bipolar kineties assign it to the urostyloid taxon Bakuellidae. The increased number of buccal and parabuccal cirri, the presence of transverse cirri, and more than one left marginal row, as well as the lack of caudal cirri separate Neobakuella n. g. from the other bakuellids. Neobakuella flava n. sp. has many 0.3 μm sized green and/or yellow usually dark-green cortical granules and some sparsely distributed, 2 × 1 μm sized grass green with yellowish shimmer granules. The gene sequence data indicate a close relationship with Diaxonella and a distinct separation from the bakuellid Metaurostylopsis and parabirojimid Parabirojimia. The SSU rRNA gene sequences of four further urostyloids (i.e. Diaxonella pseudorubra, Anteholosticha monilata, Metaurostylopsis struederkypkeae, Pseudourostyla cristata) and two stylonychines (i.e. Sterkiella cavicola, Sterkiella histriomuscorum) from Korea were analyzed. Anteholosticha monilata, type of the genus, is clearly separated from the Holosticha clade, supporting the morphological separation from Holosticha. Sterkiella cavicola, type of Sterkiella, clusters within the stylonychines and is obviously closely related with S. histriomuscorum.

Multiple Uses of Lys63-Polyubiquitination in the Ciliate Sterkiella histriomuscorum

Poly-ubiquitination through Lys63, a post-translational modification system, is emerging as a mechanism implicated in a variety of non-proteolytic functions in eukaryotic cells. This modification is mainly carried out via a unique complex formed between a canonical ubiquitin-conjugating enzyme (E2), Ubc13, and an E2-variant, Uev. To date, a wealth of functional information has been obtained mostly from animals and yeast. Here, we show that the ciliate Sterkiella histriomuscorum harbors a Ubc13 gene that encodes a putative protein exhibiting 60-64% identity with Ubc13 from organisms of diverse eukaryotic phyla. A yeast double-hybrid assay and 3D-structure modelling gave evidence that ShUbc13 is able to physically interact with ShUev; the existence of such a complex in a ciliate attests for its ancient origin. Western blotting reveals that Ubc13 is present in different stages of the vegetative cycle of S. histriomuscorum. Immunolocalization of Ubc13 and K63 polyubiquitinated proteins shows three main locations, namely the nucleus, cilia and plasma membrane. Possible functions of ShUbc13 and K63 polyubiquitination in this single-celled organism are considered with reference to current knowledge. In particular their likely involvement in DNA replication and DNA damage response is especially discussed.

Maintaining Cell Polarity through Vegetative Cell Pattern Dedifferentiation: Cytoskeleton and Morphogenesis in the Hypotrich Ciliate Sterkiella histriomuscorum

The morphological differentiation of ciliates is achieved through the development of a submembraneous cytoskeleton in which the cilia are anchored. In most hypotrich ciliates, this cytoskeleton is mainly constructed of microtubules. In these species, cells pass through vegetative cell pattern dedifferentiated stages during their biological cycle. In order to investigate the behaviour of the cytoskeleton during these stages, we analysed the reorganization of the cytoskeleton during the sexual cycle of Sterkiella histriomuscorum by microscopy. Sterkiella exconjugants transiently dedifferentiate to form zygocysts devoid of ciliature and infraciliature. Immunofluorescence images obtained with antibodies directed against pericentrosomal material and tubulin showed that the cells resorb their ciliature and basal bodies, but retain their submembraneous microtubular cytoskeleton during the whole process and that the body plan is maintained through vegetative cell pattern dedifferentiation: the cell polarity remains printed on the cell surface by the microtubular cytoskeleton which in turn could mark the sites of basal body assembly during zygocyst morphogenesis. The results are discussed in terms of mechanisms of cell patterning.

Differentially expressed genes during the encystment–excystment cycle of the ciliate Sterkiella histriomuscorum

Under unfavourable environmental conditions, many ciliates transform into resting cysts through a developmental process called encystment. Excystment is the reverse transformation of the resting cyst into a vegetative cell when favourable conditions are restored. In the oxytrichid Sterkiella histriomuscorum, the encystment – excystment (E–E) cycle involves extensive morphological changes since the whole cytoskeleton is disassembled during encystment. Assuming that these changes in cellular organization may be significantly reflected in the gene expression pattern, we used a “DNA macroarray” strategy to measure the transcript levels of 37 selected genes present at four distinct cellular stages (starved, encysted, excysting and vegetative cells). Differential expression was observed for 16 genes; four transcripts appeared to be markedly accumulated in a stage-specific manner. For most of the differentially expressed genes, the mRNA level was increased in cysts and excysting cells. When these mRNA are transcribed and when they are used, are still open questions. We showed that the copy number of the differentially expressed genes is the same in the macronuclei of cysts and vegetative cells ruling out a modulation of gene expression through a variation in the gene copy number upon encystment.

Conserved linkage of two genes on the same macronuclear chromosome in spirotrichous ciliates

Macronuclear chromosomes of spirotrichous ciliates are mainly "nanochromosomes" containing only a single gene. We identified a two-gene chromosome in the spirotrich Sterkiella histriomuscorum (formerly Oxytricha trifallax) which, unlike other characterized two-gene molecules, contains reading frames oriented tail to tail. These are homologs of ribosomal protein L29 (RPL29) and cyclophilin. We found that both genes are transcribed, with their polyadenylation sites on opposite strands separated by only 135 bp. Furthermore, both genes in S. histriomuscorum are present only on one macronuclear chromosome and do not occur alone or linked to other genes. The corresponding micronuclear locus is fragmented into three nonscrambled gene segments (MDSs), separated by two noncoding segments (IESs). We also found that these two genes are linked on a macronuclear chromosome, similarly arranged tail to tail, in the three spirotrichs Stylonychia lemnae, Uroleptus sp., and Holosticha sp.. In addition, single-gene macronuclear chromosomes containing only the RPL29 gene were detected in the earlier diverged Holosticha and Uroleptus. These observations suggest a possible evolutionary trend towards loss of chromosomal breakage between these two genes. This study is the first to examine gene linkage in the macronucleus of several spirotrichs and may provide insight into the evolution of multi-gene macronuclear chromosomes and chromosomal fragmentation in spirotrichs.

Gene structure of the ciliate Sterkiella histriomuscorum based on a combined analysis of DNA and cDNA sequences from 21 macronuclear chromosomes

Macronuclear deoxyribonucleic acid (DNA) in hypotrichous ciliates consists of a set of linear molecules ranging in size from 0.5 to several tens of kilobases and typically carrying a single gene. Each minichromosome is present at a ploidy of >or=1,000 per macronucleus. These molecules are known as gene-sized molecules. Multigene molecules are also present, but are still poorly described. In analyzing the encystment-excystment cycle of Sterkiella histriomuscorum, we have characterized a set of 21 macronuclear molecules both at the DNA and complementary DNA (cDNA) levels. On a total of 23 validated coding sequences, we mapped the 5' and 3' untranslated regions for a subset of 10 and 18 transcripts, respectively. A combination of DNA and cDNA data allows us to precisely determine several structural features of macronuclear chromosomes, such as the organization of multigene molecules, an intron content higher than expected, and a conserved sequence surrounding the initiation transcription site. It also reveals one coding sequence containing a transcribed 10-bp element that displays the characteristic features of internal eliminated sequences (IES). Its presence in a fraction of the minichromosomes carrying this gene raises the possibility of an incomplete IES excision process during the development of the S. histriomuscorum macronucleus.

Proposed Function of the Accumulation of Plasma Membrane-Type Ca 2+ -ATPase mRNA in Resting Cysts of the Ciliate Sterkiella histriomuscorum

From an mRNA differential-display analysis of the encystment-excystment cycle of the ciliate Sterkiella histriomuscorum, we have isolated an expressed sequence tag encoding a plasma membrane-type Ca2+-ATPase (PMCA). PMCAs are located either in the plasma membranes or in the membranes of intracellular organelles, and their function is to pump calcium either out of the cell or into the intracellular calcium stores, respectively. The S. histriomuscorum macronuclear PMCA gene (ShPMCA) and its corresponding cDNA were cloned; it is the first member of the Ca2+-ATPase family identified in Sterkiella. The predicted protein of 1,065 amino acids exhibits 37% identity with PMCAs of diverse organisms. A phylogenetic analysis showed its relatedness to homologs of two alveolates: the ciliate Paramecium tetraurelia and the apicomplexan Toxoplasma gondii. Overexpression of the protein ShPMCA failed to rescue the wild-type phenotype of three Ca2+-ATPase-defective mutant strains of Saccharomyces cerevisiae; this failure contrasts with the reported ability of the PMCAs of parasites to complement defects in yeast. ShPMCA mRNA is markedly accumulated during encystment and in resting cysts, suggesting a function during excystment. To address the possibility of a signaling role for calcium at excystment, the capacity of calcium to induce excystment was examined.

Coding properties of Oxytricha trifallax (Sterkiella histriomuscorum) macronuclear chromosomes: analysis of a pilot genome project.

The macronuclear genomes of spirotrichous ciliates are almost entirely polyploid, single-gene chromosomes ("nanochromosomes"). We recently performed a pilot genome project for a member of this group, Oxytricha trifallax ( Sterkiella histriomuscorum), in which approximately 2000 nanochromosomes were cloned at random and end-sequenced. Here we describe the global properties of the coding regions predicted for these molecules, including nucleotide composition, codon usage, and intron properties. In identifying splice donor, acceptor and branch sites, we found that longer introns in Oxytricha have a stronger signal at the donor site than do smaller introns, as has been found for Caenorhabditis elegans and Drosophila, despite the overall small size of the introns. A systematic search for multi-gene chromosomes identified 11 candidate nanochromosomes. We compare the results from this large dataset with those obtained from earlier studies and with statistics recorded from ciliates and other eukaryotes.

Myb Genes in Ciliates: A Common Origin with the myb Protooncogene?

In animals, the protooncogene myb family is characterized by a DNA-binding domain (so-called MYB domain), which consists of 3 imperfect tandem repeats of a helix-turn-helix motif. Homologous genes have been characterized in plants and also in Dictyostelium discoideum. However, in plants, the myb family is more diverse and displays 2 types of MYB domains: the animal-like 3 repeats (MYB-3R) and the 2 repeats (MYB-2R) domains. The question is therefore raised as to the putative existence of genes with MYB-3R and/or MYB-2R domains in their last common unicellular ancestor. Here, we present evidence that in ciliates like in plants, both types of domain exist. A gene having a MYB-3R domain has been identified in the oxytrichid Sterkiella histriomuscorum and a gene having a MYB-2R domain has been identified in the euplotid Euplotes aediculatus. Both genes are expressed during the vegetative growth of the cells. A conserved intron exists in the gene of Sterkiella and phylogenetical analyses show that the 2 ciliate genes belong to the myb protooncogene family as deeply split lineages. This is the first report of a myb homolog in a ciliated protist, thus, confirming its origin in strict unicellular eukaryotes.

Histone H4 genes in some stichotrichous ciliates

Two or more macronuclear molecules encode histone H4 among eight stichotrichs ( Sterkiella nova , Sterkiella histriomuscorum , Stylonychia mytilus , Stylonychia lemnae , Pleurotricha lanceolata , Oxytricha granulifera , Sterkiella sp. (Aspen), and Paraurostyla viridis ). Only one macronuclear molecule encodes histone H4 in Uroleptus sp. Histone H4 molecules have been sequenced in five of the stichotrichs. Codon bias is extreme. The encoded amino acid sequences (104 amino acids) are identical. They differ significantly from the histone H4 sequences encoded by the hypotrichs, Euplotes aediculatus and Moneuplotes crassus (both 107 amino acids). Three major and five minor blocks of conserved nucleotide sequences are present throughout the ∼1150 bp 5′ noncoding leaders in the five stichotrichs. These conserved sequences may serve in regulation of gene function. However, all five 5′ leaders also contain different ORFs encoding amino acid sequence with similarity to sequences in GenBank. Except possibly for the ORF in Stylonychia mytilus and an ORF in Pleurotricha lanceolata , the ORFs appear not to be significant. The 50 bp in the ends of the macronuclear molecules contain an anomalous base composition that may be important in genome processing. The micronuclear histone H4 gene in Sterkiella nova contains an IES in the 5 leader, and the ORF in Sterkiella histriomuscorum contains a single IES.

Searching for excystment-regulated genes in Sterkiella histriomuscorum (Ciliophora, Oxytrichidae): a mRNA differential display analysis of gene expression in excysting cells.

In the absence of food, the oxytrichid Sterkiella histriomuscorum transforms like many ciliates into resting cysts. When transferred back into feeding medium, the cyst re-transforms into a vegetative cell. The entry into and exit from the dormant cyst stage are complex developmental processes still poorly investigated at the molecular level. Assuming that these changes in state could involve changes in gene expression, we have used the technique of mRNA differential display to detect differentially expressed genes in cysts and two different stages of excysting cell. Variation in the temporal expression pattern of transcripts could be detected and, in using an inverse-PCR strategy on circularized macronuclear DNA, we have sequenced the macronuclear genes of three of the isolated cDNAs. which correspond to 1) a nucleotide-binding domain-encoding gene, 2) a DHHC-domain-carrying gene, and 3) a phosphatase type 2C-encoding gene. For the first two genes, Northern blot analyses supported an excystment-associated regulated gene expression. We discuss their possible role during excystment and we show that the combination of differential display and inverse PCR constitutes a powerful approach to isolate excystment-regulated genes in hypotrichs.

Messenger RNA in Dormant Cells of Sterkiella histriomuscorum (Oxytrichidae): Identification of Putative Regulatory Gene Transcripts

In the absence of food, the oxytrichid Sterkiella histriomuscorum, like many ciliates, enters into dormancy and transforms into a round and walled encysted cell. When transferred back into a feeding medium, the cyst re-transforms into a vegetative cell in a few hours. This encystment-excystment pathway, which is common to many free-living and parasitic protists, is still poorly understood at the molecular level. In order to identify potential dormant transcripts in the cysts of Sterkiella, we have constructed cDNA libraries from mature cysts. Transcripts have been isolated confirming the presence of a mRNA pool in the dormant cells. The sequence analysis of two cDNA indicates open reading frames which show significant similarities to known proteins involved in mechanisms of regulation: 1) nifR3, an element of the nitrogen regulatory system in bacteria and 2) CROC-1, a newly identified human transcription factor. The two corresponding macronuclear genes represent the first putative regulatory genes isolated in ciliates. From a differential screening of the cDNA library against vegetative cDNA, one cyst-specific (and very abundant) transcript has been isolated but the product has not yet been identified. The possible involvment of these new ciliate genes in the excystment process is discussed.

Comparison of divisional morphogenesis in four morphologically different clones of the genus Gonostomum and update of the natural hypotrich system (Ciliophora, Hypotrichida)

Summary Divisional morphogenesis of a clone of the multimacronucleate Gonostomum kuehnelti Foissner, 1987 (clone I), with four transverse cirri and of three clones of Gonostomum affine (Stein, 1859) Sterki, 1878, with two macronuclear segments are described and compared using protargol impregnation. The three clones of G. affine differ in having two (clone II), four (clone III) and six (clone IV) transverse cirri, whereas all clones completely lack ventral cirri between proximal membranelles and transverse cirri. Early morphogenetic processes show that all four clones develop the family characters of the Oxytrichidae Ehrenberg, 1838: “neokinetal 3” anlagen and long primary primordia. Only clone I shows a significant morphogenetic difference by generating proter's first (left) frontal cirrus by the oral primordium. Clones II to IV generate this cirrus de novo. Biometry of middle dividers and specimens in interphase reveals an invariable cirral pattern generated from six ventral anlagen in clone I, whereas six or seven ventral anlagen, one or two left marginal rows and two to four macronuclear segments develop in clones II–IV. All four clones are, however, stable in the lack of ventral cirri and, surprisingly, clones II–IV are also stable (not overlapping) in their different number of transverse cirri. Nevertheless, morphologic differences in clones II–IV are considered to be probably polymorphic states of G. affine . Recently published morphogenetic data are analyzed, shown in schematized computer drawings and added to the natural hypotrich system: Lamtostyla edaphoni is transferred to the genus Amphisiella Gourret and Roeser, 1888 within the family Oxytrichidae because of neokinetal 3 anlagen and long primary primordia development. Onychodromopsis flexilis is also assigned to the Oxytrichidae because of neokinetal 3 and long primary primordia, whereas Sterkiella histriomuscorum generates “neokinetal 1” anlagen and long primary primordia are absent and therefore it is assigned to the family Parakahliellidae. A computer generated tree is presented using the programs MacClade and PAUP and consisting of 42 morphogenetically investigated hypotrich species with 6 characters in 15 states. For the homonym Clara the genus name Tetmemena nom. nov. is suggested.

Timing of life cycle morphogenesis in synchronous samples of Sterkiella histriomuscorum I. The vegetative cell cycle

Summary We describe standardized growth conditions and protocols for manipulating the life cycle of a common stichotrich ciliate, Sterkiella histriomuscorum (Oxytrichidae), previously isolated as Oxytricha fallax . High density cultures were obtained with short cell cycles and with predictable kinetics of growth or differentiation. By manipulating the culture conditions we could obtain mass cultures undergoing conjugation, excystment, encystment or cannibalism. Under poor growth conditions, cells down-size by rescaling the cytostome and macronuclear DNA content, as do other Oxytrichidae. Using synchronous samples obtained from cultures in each of the life cycle phases, we describe in detail the timing of morphogenesis stages in the vegetative cell cycle, in encysting cells and in excysting cells. The timing of the nuclear and cortical events are correlated to obtain a cell cycle diagram. This establishes a firm base for study and manipulation of the life cycle differentiation and cell cycle regulation of a stichotrich ciliate at the genetic and physiological level.