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  • 1
    Electronic Resource
    Electronic Resource
    An alternative approach for the molecular characterization of uncultivable protists retaining morphological information (2005)
    Oxford, UK : Blackwell Science Inc
    The @journal of eukaryotic microbiology 52 (2005), S. 0 
    Details
    ISSN: 1550-7408
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Recent culture-independent studies based on small-subunit ribosomal RNA (SSU rRNA) gene analysis revealed the existence of completely new clades of protists. The main problems with this approach are to correlate sequences from environmental rRNA genes with the organisms they belong to and then to detect the ecological role of these organisms in the environment. In order to overcome such problems we chose an alternative approach allowing us both a molecular characterization of uncultivable organisms with a low relative abundance in environmental samples, and a morphological analysis, even if restricted. The experimental protocol consists of two steps: an initial observation and photo-taking of the single cell under the DIC (Differential Interferential Contrast) microscope and then PCR amplification and direct sequencing of the 18S rRNA gene of the same cell. The advantages of this method are the possibility to: (1) establish a precise link between morphology and gene sequence; (2) detect the possible occurrence of highly similar species within the studied population; (3) avoid the insertion of Taq-polymerase errors in the gene sequence; and (4) detect possible polymorphisms in the gene under examination. Such an approach was used to sequence the 18S rRNA gene of organisms belonging to the class Karyorelictea that comprises several uncultivable ciliates with limited distinctive features. Gene sequences analysis revealed an unexpected genetic variability in trachelocercids and, in particular, the existence of polymorphisms in the SSU rRNA gene of a group of them. Such specimens show a similar morphology and, as a result of phylogenetic analysis, they form a constant clade.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1550-7408.2005.05202003_6_2.x
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  • 2
    Electronic Resource
    Electronic Resource
    Molecular phylogeny of free-living and symbiotic Verrucomicrobiales by using protein-coding genes (2005)
    Oxford, UK : Blackwell Science Inc
    The @journal of eukaryotic microbiology 52 (2005), S. 0 
    Details
    ISSN: 1550-7408
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Verrucomicrobiales are a newly proposed, deep branched division of bacteria from which only a handful of cultured isolates are available. Culture-independent analysis of environmental samples shows that members of Verrucomicrobiales are widespread and can be divided into several subdivisions. Symbiotic species of Euplotidium (Ciliophora) and Nematodes, epixenosomes and Xiphinemobacter spp. respectively, are according to the 16S sequences also members of this group. Up to now, only minimal sequence data mainly focused on 16S rRNA gene sequences are available for this group of organisms. The phylogenetic relatives of Verrucomicrobiales are still unclear with Chlamydiales and Planctomycetes being the most probable candidates. The present study describes preliminary results on phylogenetic relationships of Verrucomicrobiales by using protein-coding genes like Hsp70 and RpoC/B. The species which are used for this study are Prosthecobacter vanneervenii, P. debontii, Opitutus sp.VeGlc2, O. terrae and epixenosomes. The genes were amplified with designed consensus-degenerated primers and results are available on the Hsp70. The sequences show several differences within the group. Verrucomicrobium and Prosthecobacter sequences cluster together, but in P. debontii, two sequences were found, and the second one appears quite divergent. Also the two Opitutus genes are quite atypical and cluster together in a deep position. Preliminary attempt was performed on Euplotidium-epixenosome complex. However, amplified and cloned sequences revealed to be closely related to ciliate sequences. Further attempt will be performed applying microtiter-plate-subtractive-hybridization to characterize epixenosome genes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1550-7408.2005.05202003_6_5.x
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  • 3
    Electronic Resource
    Electronic Resource
    A multidisciplinary approach to describe protists: redescription of Chattonidium setense Villenueve, 1937 (Ciliophora, Heterotrichea) (2005)
    Oxford, UK : Blackwell Science Inc
    The @journal of eukaryotic microbiology 52 (2005), S. 0 
    Details
    ISSN: 1550-7408
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: This study represents the first extended report on the heterotrichid ciliate Chattonidium setense since the original description by Villeneuve in 1937. This rare marine ciliate is in fact the only representative of the family Chattonidiidae, which is the eighth, often neglected, family of order Heterotrichida. As in our previous works, here we redescribe the species combining morphological observations (live, stained, scanning, and transmission electron microscope) with behavioral and molecular data as we are convinced that both taxonomic determination and molecular characterization are necessary for the clarification of phylogenetic relationships between a species and related genera. Live observation, Feulgen procedure and SEM analysis showed, besides a number of other interesting features, the presence of a characteristic aboral ciliated cavity of undetermined function, whose presence makes ciliate's identification unambiguous. TEM observation evidenced the following main features: well-divided ecto-and endoplasm; deep insertions of the rows of somatic dikinetids in cortex invaginations; paraglycogen granules as polysaccharide reserve; large bundles of microtubules and fibers delimiting the body region surrounding the aboral cavity; two kinds of subcortical extrusomes. Since the revision of heterotrichs ciliates by Villeneuve-Brachon (1940) many genera and families have been transferred to other classes on the basis of both new molecular data and reconsidered morphological characters. Our results support previous statements that Chattonidium has the general morphology typical of Heterotrichida and that the main similarities are with genus Condylostoma. 18S rRNA molecular data firmly confirm that Chattonidium belongs to class Hetrotrichea, order Hetrotrichida (Lynn, D. H. 2003. The Ciliate Resource Archive. ) as an independent lineage. Preliminary molecular data also suggest an association between genera Chattonidium and Condylostoma. Finally, some behavioral and ecological notes on the ciliate are added.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1550-7408.2005.05202003_6_10.x
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  • 4
    Electronic Resource
    Electronic Resource
    Searching for tubulin-like genes in free-living bacteria and symbionts of ciliates. Characterization of the genomic environment of tubulin genes from Prosthecobater (2005)
    Oxford, UK : Blackwell Science Inc
    The @journal of eukaryotic microbiology 52 (2005), S. 0 
    Details
    ISSN: 1550-7408
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Tubulins are typical eucaryotic genes, which have recently been found in the free-living bacteria Prosthecobacter (Verrucomicrobiales). Nevertheless, microtubule-like structures were never observed in Prostecobacter, whereas they were reported in epixenosomes which are symbiotic Verrucomicrobiales of the ciliate Euplotidium. In the present work, an extensive search for tubulin like genes was performed with several combinations of PCR-primers, designed according to different criteria. Although some combinations of these primers are able to amplify simultaneously eukaryotic α- and β-tubulin as well as Prosthecobacter tubulin genes, no bacterial-like tubulin genes were identified in other Verrucomicrobiales species. Because of host derived contaminations, even a subtractive PCR approach, using microtiter-plate-subtractive-hybridization, was used with the epixenosomes. The characterization of the genomic environment of the Prosthecobacter tubulin genes was performed by gene walking. The sequences obtained from P. debontii revealed a so-far undiscovered gene duplication. The bacterial tubulin operon looks conserved among Prosthecobacter species and includes an α-tubulin, a β-tubulin and a kinesin like protein. Upstream of two operons a repetetive sequence could be found which could be a hint for horizontal gene transfer. The analysis of the genomic environments of the tubulin genes provided open reading frames, which have homology to Verrucomicrobium spinosum genes (ongoing genome project data), whereas up to now the blast search did not reveal tubulin-like genes in this organism. Present results suggest, that the Prosthecobacter tubulin genes are possibly of recent origin, maybe acquired by horizontal gene transfer, or lost by other so far studied Verrucomicrobiales. The origin and nature of microtubular genetic determinants from epixenosomes remains open.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1550-7408.2005.05202003_6_11.x
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  • 5
    Electronic Resource
    Electronic Resource
    Characterization of R-body genetic determinants in Caedibacter caryophilus a symbiont of Paramecium caudatum: preliminary results (2005)
    Oxford, UK : Blackwell Science Inc
    The @journal of eukaryotic microbiology 52 (2005), S. 0 
    Details
    ISSN: 1550-7408
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Refractile inclusion bodies, called R-bodies were observed within the cells of some bacterial strains. They are protein ribbons, which are typically coiled into cylindrical structures. They are produced by members of the genus Caedibacter, gram-negative rod-shaped endosymbionts of paramecia and e.g. the free-living bacteria Hydrogenophaga taeniospiralis and Acidovorax avenae. The phylogenetic relationship even between the members of the genus Caedibacter is quite low: C. taeniospiralis belongs to the Gammaproteobacteria and is related to Francisella tularensis, C. caryophilus is affiliated with the Alphaproteobacteria and clusters with the obligate endosymbiont Holospora. In the case of C. taeniospiralis 51, the genetic determinants of R-body synthesis are located on a plasmid, whereas in other strains like 7 and 562 it looks like phage particles are involved in their production. In the present study, we investigated C. caryophilus, endosymbiont of Paramecium caudatum. Separation of C. caryophilus cells was performed by Percoll™ density gradient centrifugation. The isolated DNA was separated by pulsed-field gel electrophoresis and it was possible to visualize several bands referring to one or more extrachromosomal elements. A small gene library of these extrachromosomal elements was constructed and we already identified transposition-related genes; interestingly, similar genes were reported also on the plasmid of C. taeniospiralis 51.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1550-7408.2005.05202003_6_14.x
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  • 6
    Electronic Resource
    Electronic Resource
    Recent adaptation to symbiotic lifestyle: the Polynucleobacter–Euplotes system (2005)
    Oxford, UK : Blackwell Science Inc
    The @journal of eukaryotic microbiology 52 (2005), S. 0 
    Details
    ISSN: 1550-7408
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Bacteria belonging to the genus Polynucleobacter were previously described as endosymbionts inhabiting the cytoplasm of freshwater ciliates of the genus Euplotes having a 9 type I cirrus pattern. They were all found to be necessary for the division process of their hosts. Here, we report the finding of a Polynucleobacter bacterium as symbiont of E. harpa, a brackish species with ten frontoventral cirri. Identity of the symbionts was unambiguously determined by morphological description and by 16S rRNA gene sequence characterization. Furthermore, antibiotic treatments confirmed that these bacteria play the same functional role of the other Polynucleobacter symbionts; aposymbiotic E. harpa ceases to divide and eventually die. Phylogenetic analyses based on 18S rRNA gene show that Euplotes species harboring Polynucleobacter symbionts, included E. harpa, steadily cluster together. In order to clear whether this symbiosis was established as a single evolutionary event by a common ancestor of the ciliate hosts, or by multiple acquisition of different bacteria by the different Euplotes species, comparative phylogenetic analyses were performed within the so-called Polynucleobacter-cluster, comprising bacterial symbionts as well as strictly related free-living bacteria. Due to a high degree of conservation of 16S rRNA gene sequences within this group, phylogenetic trees based on this gene did not resolve the matter. Surprisingly, even the characterization and the analysis of the 16-23S ITS sequences, usually exhibiting faster evolutionary rates, revealed a high similarity (〉98%), thus hindering a stable and clear phylogenetic reconstruction. Data obtained cannot at present definitively confirm or reject any hypothesis on the origin of this symbiosis, revealing, at the same time, that we are dealing with a relatively young symbiotic system. As shown by the close phylogenetic relationship between free-living and symbiotic Polynucleobacter, this is an ideal model for studying development and adaptation to symbiotic lifestyle.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1550-7408.2005.05202003_6_16.x
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  • 7
    Electronic Resource
    Electronic Resource
    A Multidisciplinary Approach to Describe Protists: Redescriptions of Novistrombidium testaceum Anigstein 1914 and Strombidium inclinatumMontagnes, Taylor, and Lynn 1990 (Ciliophora, Oligotrichia) (2003)
    Oxford, UK : Blackwell Publishing Ltd
    The @journal of eukaryotic microbiology 50 (2003), S. 0 
    Details
    ISSN: 1550-7408
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: . We combined behavioral, morphological (live, stained, scanning and transmission electron-microscope), and molecular data to redescribe two common, intertidal oligotrich ciliates, Novistrombidium testaceum and Strombidium inclinatum. Both species were collected from a rocky shore near Leghorn, Ligurian Sea. A literature review revealed four morphotypes of Novistrombidium testaceum that differ in subtle ways, including oral morphology. These differences may be diagnostic, but we do not consider them sufficient to distinguish different taxa. Although other studies have synonymised Strombidium inclinatum and S. sulcatum, based on oral structures, size, and nuclear structure, there are morphological distinctions between them. In particular, the present study supports a lack of anterior protuberance in both live and preserved S. inclinatum, while S. sulcatum possesses a protuberance. The 18S rDNA molecular data, in accordance with morphological and ultrastructural observations, indicate that the Strombidiida (Oligotrichia) constitute a well-supported clade. The separation of the genera within this clade, even between Novistrombidium and Strombidium, remains unresolved, and the analysis of more species is required. Finally, we recommend that when possible, ecologists, morphological taxon-omists, and molecular biologists combine their expertise to provide comprehensive taxonomic descriptions.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1550-7408.2003.tb00114.x
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  • 8
    Electronic Resource
    Electronic Resource
    Epixenosomes: Peculiar Epibionts of the Hypotrich Ciliate Euplotidium Itoi Defend Their Host Against Predators (1999)
    Oxford, UK : Blackwell Publishing Ltd
    The @journal of eukaryotic microbiology 46 (1999), S. 0 
    Details
    ISSN: 1550-7408
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: . Euplotidium itoi harbors on its dorsal surface peculiar episymbionts (referred to as epixenosomes) equipped with a complex extrusive apparatus. In the laboratory. E. itoi stocks without epixenosomes behave and reproduce like symbiotized stocks. the hypothesis that epixenosomes play a defensive role against predators was tested by comparing the behavior of Litonotus lamella when preying upon Euplotes crassus, E. itoi without epixenosomes. and E. itoi with epixenosomes. Litonotus discharges its toxicysts upon direct-cell-to cell contact, and paralyzes the three types of prey with the same efficiency. Nevertheless, Litonotus can ingest Euplotes, Euplotidium without epixenosomes, and to a certain extent, Euplotidium with epixenosomes whose ejecting capability has been inhibited. while it never eats Euplotidium with unaltered epixenosomes. In each prey-type, about 60% of the individuals attacked by Litonotus toxicyst discharge are able to recover their normal behavior once transferred into pure sea water. This percentage for E. itoi with epixenosomes that are never eaten by the predator corresponds to the probability of survival. This probability is lower for the other two prey-types in which the prey engulfed by the predator do not have the chance to recover. These data support the hypothesis and suggest the involvement of the epixenosome's ejecting apparatus in a defensive function.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1550-7408.1999.tb05125.x
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  • 9
    Electronic Resource
    Electronic Resource
    Unusual tubulins with deviant genetic code in the hypotrich ciliate Euplotidium itoi (2005)
    Oxford, UK : Blackwell Science Inc
    The @journal of eukaryotic microbiology 52 (2005), S. 0 
    Details
    ISSN: 1550-7408
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: We characterized nine macronuclear tubulin gene minichromosomes in the hypotrich ciliate Euplotidium itoi: two α-tubulin genes, five β-tubulin genes, and two γ-tubulin genes. In particular, one peculiar β-tubulin gene was identified. It shows only 81.5% amino acid similarity to the other characterized β-tubulin types and an UAG stop codon in frame. Based on this result we started to analyse stop codon usage in Euplotidium itoi macronuclear tubulin genes and some unrelated macronuclear minichromosomes. Most of the ciliates independently evolved diverse non-canonical genetic codes. Differences to the standard code consist in the reassignment of one or two of the three standard stop codons to encode a particular amino acid. Class Spirotrichea represents a particular case comprising two groups of organisms that do not share any stop codon. In oxytrichids, UAR is translated as glutamine and UGA is the single stop codon. This is in contrast to the genus Euplotes, close relatives of Euplotidium, in which UAR encodes a stop signal and UGA is translated as cysteine. Fourteen complete genes were characterized in Euplotidium itoi; in all cases UAA revealed as the single triplet used to code a stop, UGA was never and UAG rarely observed in frame. The UAG stop codon was observed in the peculiar type 3 β-tubulin gene, in two dynein partial sequences and in some other putative genes. We could confirm the active expression of three of these genes, the unusual β-tubulin, a putative lipase and a possible steroid-binding protein, by RT-PCR. Finally, a detailed analysis on the structure of the macronuclear tubulin genes, including nucleotide and amino acid differences among tubulin gene paralogs, polyadenylation sites, putative chromosome fragmentation sites, ATGC-content in coding and non-coding regions, telomere sequence and length will be presented.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1550-7408.2005.05202003_6_9.x
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  • 10
    Electronic Resource
    Electronic Resource
    Polynucleobacter Bacteria in the Brackish-Water Species Euplotes harpa (Ciliata Hypotrichia) (2005)
    Oxford, UK : Blackwell Science Inc
    The @journal of eukaryotic microbiology 52 (2005), S. 0 
    Details
    ISSN: 1550-7408
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract. We have found a Polynucleobacter bacterium in the cytoplasm of Euplotes harpa, a species living in a brackish-water habitat, with a cirral pattern not corresponding to that of the freshwater Euplotes species known to harbor this type of bacteria. The symbiont has been found in three strains of the species, obtained by clonal cultures from ciliates collected in different geographic regions. The 16S rRNA gene sequence of this bacterium identifies it as a member of the β-proteobacterial genus Polynucleobacter. This sequence shares a high similarity value (98.4–98.5%) with P. necessarius, the type species of the genus, and is associated with 16S rRNA gene sequences of environmental clones and bacterial strains included in the Polynucleobacter cluster (〉95%). An oligonucleotide probe was designed to corroborate the assignment of the retrieved sequence to the symbiont and to detect similar bacteria rapidly. Antibiotic experiments showed that the elimination of the bacteria stops the reproductive cycle in E. harpa, as has been shown for the freshwater Euplotes species.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1550-7408.2005.04-3319.x
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