The classification of free-living spirochetes

Summary The natural relationships among free-living and host-associated spirochetes were studied by investigating aspects of their morphology, physiology and DNA base composition. These studies indicated that the strictly and facultatively anaerobic, free-living spirochetes share characteristics which distinguish them from their host-associated counterparts and from the free-living leptospires.It is suggested that the strictly and facultatively anaerobic, free-living spirochetes be grouped in the genus Spirochaeta and that this genus include the following four species: S. plicatilis (type species), S. stenostrepta, S. zwelzerae (Treponema zuelzerae), and S. aurantia. The characteristics of these species are described.     

Rifampin-resistant RNA polymerase in spirochetes

Abstract Various free-living and host-associated spirochetes isolated by methods not involving rifampin were resistant to relatively high concentrations of this antibiotic. The lowest concentrations of rifampin that were inhibitory for the spirochetes ranged from 50 to more than 200 μ g/ml, depending on the species. The spirochete strains examined were at least 10-fold more resistant to rifampin than Escherichia coli and 10 000-fold more resistant than Staphylococcus aureus . The results support the conclusion that rifampin resistance is a general characteristic of spirochetes. Resistance of Spirochaeta aurantia to rifampin was not the result of detoxification of the antibiotic in the culture medium. The activity of spirochete DNA-dependent RNA polymerase in vitro was completely resistant to 10 μg of rifampin per ml, a concentration that totally inhibited E. coli RNA polymerase. Higher concentrations decreased the spirochetal activity. Thus, rifampin resistance may be due to a low affinity of spirochete RNA polymerase for the antibiotic.     

Phylogenetic position of the spirochetal genus Cristispira.

Comparative sequence analysis of 16S rRNA genes was used to determine the phylogenetic relationship of the genus Cristispira to other spirochetes. Since Cristispira organisms cannot presently be grown in vitro, 16S rRNA genes were amplified directly from bacterial DNA isolated from Cristispira cell-laden crystalline styles of the oyster Crassostrea virginica. The amplified products were then cloned into Escherichia coli plasmids. Sequence comparisons of the gene coding for 16S rRNA (rDNA) insert of one clone, designated CP1, indicated that it was spirochetal. The sequence of the 16S rDNA insert of another clone was mycoplasmal. The CP1 sequence possessed most of the individual base signatures that are unique to 16S rRNA (or rDNA) sequences of known spirochetes. CP1 branched deeply among other spirochetal genera within the family Spirochaetaceae, and accordingly, it represents a separate genus within this family. A fluorescently labeled DNA probe designed from the CP1 sequence was used for in situ hybridization experiments to verify that the sequence obtained was derived from the observed Cristispira cells.     

Phylogenetic relationships of symbiotic spirochetes in the gut of diverse termites

Phylogenetic relationships of symbiotic spirochetes in the gut of diverse termites were analyzed without cultivation of these microorganisms. A portion of the 16S rDNA (ca. 850 bp) was amplified directly from DNA of the mixed population in the gut by PCR and cloned. A total of 30 spirochetal phylotypes affiliated with the treponemes were identified from four termite species and they were compared with those already reported from other termites. They represented separate lines of descent from any known species of Treponema, and they were divided into two discrete clusters; one was related to Spirochaeta stenostrepta and S. caldaria, and the other was grouped together with members of the Treponema bryantii subgroup. Although some sequences from evolutionarily related termites showed close similarity, most of the sequences of spirochetes were dissimilar among different termite species, and spirochetal sequences from a single termite species occurred in several distinct phylogenetic positions. These findings suggest that termites constitute a rich reservoir of novel spirochetal diversity and that evolution of the symbiosis is not simple.     

Coevolution of symbiotic spirochete diversity in lower termites.

The phylogenetic relationships of symbiotic spirochetes from five dry-wood feeding lower termites (Cryptotermes cavifrons, Heterotermes tenuis, Kalotermes flavicollis, Neotermes mona, and Reticulitermes grassei) was compared to those described in previous reports. The 16S rDNA bacterial genes were PCR-amplified from DNA isolated from intestinal samples using a spirochete-selective primer, and the 16S amplicons were cloned into Escherichia coli. Sequences of the cloned inserts were then used to determine closest relatives by comparison with published sequences. Clones sharing more than 97% sequence identity were grouped into the same phylotype. Forty-three new phylotypes were identified. These termite whole-gut-spirochetes fell into two previous defined clusters, designated as Treponema Clusters I and II, and one new Cluster III. Thirty-seven phylotypes were grouped in Cluster I. Cluster II comprised three phylotypes, two from Reticulitermes grassei (LJ029 and LJ012) and one from Heterotermes tenuis (LQ016). Three phylotypes, LK057, LK050 and LK028, were affiliated to Cluster III. Members of Cluster I showed the following characteristics: (i) spirochete phylotypes from a particular species of termite were more closely related to each other than to phylotypes of other termite species; (ii) spirochetes obtained from different genera of the same family, such as Cryptotermes sp., Kalotermes sp., and Neotermes sp., all from the family Kalotermitidae, were also related to each other. It was therefore concluded that spirochetes are specific symbionts that have coevolved with their respective species of termites, are stably harbored, and are closely related to members of the same termite family.     

Intra- and interspecific comparisons of bacterial diversity and community structure support coevolution of gut microbiota and termite host

We investigated the bacterial gut microbiota from 32 colonies of wood-feeding termites, comprising four Microcerotermes species (Termitidae) and four Reticulitermes species (Rhinotermitidae), using terminal restriction fragment length polymorphism analysis and clonal analysis of 16S rRNA. The obtained molecular community profiles were compared statistically between individuals, colonies, locations, and species of termites. Both analyses revealed that the bacterial community structure was remarkably similar within each termite genus, with small but significant differences between sampling sites and/or termite species. In contrast, considerable differences were found between the two termite genera. Only one bacterial phylotype (defined with 97% sequence identity) was shared between the two termite genera, while 18% and 50% of the phylotypes were shared between two congeneric species in the genera Microcerotermes and Reticulitermes, respectively. Nevertheless, a phylogenetic analysis of 228 phylotypes from Microcerotermes spp. and 367 phylotypes from Reticulitermes spp. with other termite gut clones available in public databases demonstrated the monophyly of many phylotypes from distantly related termites. The monophyletic "termite clusters" comprised of phylotypes from more than one termite species were distributed among 15 bacterial phyla, including the novel candidate phyla TG2 and TG3. These termite clusters accounted for 95% of the 960 clones analyzed in this study. Moreover, the clusters in 12 phyla comprised phylotypes from more than one termite (sub)family, accounting for 75% of the analyzed clones. Our results suggest that the majority of gut bacteria are not allochthonous but are specific symbionts that have coevolved with termites and that their community structure is basically consistent within a genus of termites.     

The coral core microbiome identifies rare bacterial taxa as ubiquitous endosymbionts

Despite being one of the simplest metazoans, corals harbor some of the most highly diverse and abundant microbial communities. Differentiating core, symbiotic bacteria from this diverse host-associated consortium is essential for characterizing the functional contributions of bacteria but has not been possible yet. Here we characterize the coral core microbiome and demonstrate clear phylogenetic and functional divisions between the micro-scale, niche habitats within the coral host. In doing so, we discover seven distinct bacterial phylotypes that are universal to the core microbiome of coral species, separated by thousands of kilometres of oceans. The two most abundant phylotypes are co-localized specifically with the corals'' endosymbiotic algae and symbiont-containing host cells. These bacterial symbioses likely facilitate the success of the dinoflagellate endosymbiosis with corals in diverse environmental regimes.     

Taxonomy of spirochetes

The taxonomy of spirochetes has improved considerably over the last few years. Even non-cultivable spirochetes have been classified and identified and highly discriminating analytical methods have been used to distinguish these unique organisms. The present article reviews major characteristics and techniques that have been applied in recent molecular taxonomy and chemotaxonomy of spirochetes. These comprise cellular lipids, carbohydrates, peptidoglycan, enzymes, cell proteins, cytoplasmic fibrils, metabolites, genome size, structure and base composition, restriction endonuclease analysis, restriction fragment length polymorphism, multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, DNA-DNA hybridization, arbitrarily primed polymerase chain reaction/randomly amplified polymorphic DNA fingerprinting, ribosomal (r) RNA cataloguing and sequencing, characterization of intergenic spacer regions of rRNA genes, ospA, lipoprotein and flagellin gene sequencing, and rRNA gene organization. Characteristics and techniques such as those listed above have contributed to the recognition of new spirochetal genera and species and have made spirochetal taxonomy polyphasic. Despite these improvements, significant reservoirs of hitherto unrecognized spirochetal diversity probably still exist.     

Intra- and Interspecific Comparisons of Bacterial Diversity and Community Structure Support Coevolution of Gut Microbiota and Termite Host

We investigated the bacterial gut microbiota from 32 colonies of wood-feeding termites, comprising four Microcerotermes species (Termitidae) and four Reticulitermes species (Rhinotermitidae), using terminal restriction fragment length polymorphism analysis and clonal analysis of 16S rRNA. The obtained molecular community profiles were compared statistically between individuals, colonies, locations, and species of termites. Both analyses revealed that the bacterial community structure was remarkably similar within each termite genus, with small but significant differences between sampling sites and/or termite species. In contrast, considerable differences were found between the two termite genera. Only one bacterial phylotype (defined with 97% sequence identity) was shared between the two termite genera, while 18% and 50% of the phylotypes were shared between two congeneric species in the genera Microcerotermes and Reticulitermes, respectively. Nevertheless, a phylogenetic analysis of 228 phylotypes from Microcerotermes spp. and 367 phylotypes from Reticulitermes spp. with other termite gut clones available in public databases demonstrated the monophyly of many phylotypes from distantly related termites. The monophyletic “termite clusters” comprised of phylotypes from more than one termite species were distributed among 15 bacterial phyla, including the novel candidate phyla TG2 and TG3. These termite clusters accounted for 95% of the 960 clones analyzed in this study. Moreover, the clusters in 12 phyla comprised phylotypes from more than one termite (sub)family, accounting for 75% of the analyzed clones. Our results suggest that the majority of gut bacteria are not allochthonous but are specific symbionts that have coevolved with termites and that their community structure is basically consistent within a genus of termites.     

The lipids of four unusual non-pathogenic host-associated spirochetes.

The lipid compositions of two spirochetes isolated from the human oral cavity and two isolated from pig feces were examined. These isolates were unusual in that they did not require long-chain fatty acids for growth, as do the other host-associated spirochetes, but rather required isobutyric and valeric acids. Therefore, they could be cultured in a medium free of serum or fatty acid - albumin supplements. The major fatty acids synthesized were normal and iso fatty acids with 14 and 16 carbons. No unsaturated fatty acids were detected, nor were chain lengths longer than 16 carbons. The major complex lipids found were monogalactosyl diglyceride, phosphatidyl glycerol, and bis-phosphatidyl glycerol. Nitrogenous phospholipids, present in Treponema and Leptospira, were not synthesized by these novel strains. The data indicate an intermediate position of these isolates between Treponema and free-living Spriochaeta.     

Possible evolutionary significance of spirochaetes.

Large symbiotic spirochaetes of the family Pillotaceae (e.g. pillotinas) are found in dry wood and subterranean termites (Hollande & Garagozlou 1967). These morphologically distinctive spirochaetes comprise several genera. Some of them contain microtubules within their protoplasmic cylinders. They demonstrate a variety of relations with their termite and protist hosts. Some are free-living within the lumen of the intestine, some tend to be associated with filamentous and other bacteria, some are found regularly coursing between the numerous undulipodia ( = eukaryotic flagella, cilia, and other (9 + 2) organelles of motility) of hypermastigotes and polymastigotes. Still other smaller termite spirochaetes are regularly attached to protists via specialized attachment sites. Some even form motility symbiosis with their host protists. The analogy between the behaviour of host-associated spirochaetes and the possible steps in the origin of the undulipodia and mitotic system of eukaryotes is discussed briefly.     

The motility symbiont of the termite gut flagellate Caduceia versatilis is a member of the "Synergistes" group

The flagellate Caduceia versatilis in the gut of the termite Cryptotermes cavifrons reportedly propels itself not by its own flagella but solely by the flagella of ectosymbiotic bacteria. Previous microscopic observations have revealed that the motility symbionts are flagellated rods partially embedded in the host cell surface and that, together with a fusiform type of ectosymbiotic bacteria without flagella, they cover almost the entire surface. To identify these ectosymbionts, we conducted 16S rRNA clone analyses of bacteria physically associated with the Caduceia cells. Two phylotypes were found to predominate in the clone library and were phylogenetically affiliated with the "Synergistes" phylum and the order Bacteroidales in the Bacteroidetes phylum. Probes specifically targeting 16S rRNAs of the respective phylotypes were designed, and fluorescence in situ hybridization (FISH) was performed. As a result, the "Synergistes" phylotype was identified as the motility symbiont; the Bacteroidales phylotype was the fusiform ectobiont. The "Synergistes" phylotype was a member of a cluster comprising exclusively uncultured clones from the guts of various termite species. Interestingly, four other phylotypes in this cluster, including the one sharing 95% sequence identity with the motility symbiont, were identified as nonectosymbiotic, or free-living, gut bacteria by FISH. We thus suggest that the motility ectosymbiont has evolved from a free-living gut bacterium within this termite-specific cluster. Based on these molecular and previous morphological data, we here propose a novel genus and species, "Candidatus Tammella caduceiae," for this unique motility ectosymbiont of Caducaia versatilis.     

GAPDH gene diversity in spirochetes: a paradigm for genetic promiscuity.

In this study we have determined gap sequences from nine different spirochetes. Phylogenetic analyses of these sequences in the context of all other available eubacterial and a selection of eukaryotic Gap sequences demonstrated that the eubacterial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene diversity encompasses at least five highly distinct gene families. Within these gene families, spirochetes show an extreme degree of sequence divergence that is probably the result of several lateral gene transfer events between spirochetes and other eubacterial phyla, and early gene duplications in the eubacterial ancestor. A Gap1 sequence from the syphilis spirochete Treponema pallidum has recently been shown to be closely related to GapC sequences from Euglenozoa. Here we demonstrate that several other spirochetal species are part of this cluster, supporting the conclusion that an interkingdom gene transfer from spirochetes to Euglenozoa must have occurred. Furthermore, we provide evidence that the GAPDH genes present in the protists Parabasalia may also be of spirochetal descent.     

Diverse bacteria related to the bacteroides subgroup of the CFB phylum within the gut symbiotic communities of various termites

Phylogenetically diverse clones of the partial 16S rDNA (ca. 850 bp) of bacteria belonging to the bacteroides subgroup of the cytophaga-flavobacter-bacteroides phylum were collected from the symbiotic microbial communities in the guts of six termite species without cultivation. Combined with the sequences reported previously, a total of thirty phylotypes of the subgroup were identified and classified into five phylogenetic clusters. One that was comprised of the phylotypes from a single termite species was related to the genus Rikenella. Two were clustered each with some cultured strains, genera of which have not been clearly defined yet. The remaining two clusters had no culturable representatives, suggesting the presence of yet-uncultivated genera within the termite guts. From these sequence data, we designed a specific primer for the bacteroides subgroup, which was successful in the terminal-restriction fragment length polymorphism analysis to detect the phylotypes of the subgroup in the termite gut.     

Low-diversity fungal assemblage in an Antarctic Dry Valleys soil

The McMurdo Dry Valleys of Antarctica present extreme environmental challenges. Life is restricted to patchy occurrence of lichens, mosses and invertebrates, plus soil microbial communities. Fungi have been described in lichen symbioses but relatively little is known about the occurrence of free-living soil fungi in the Dry Valleys. A challenge in estimating fungal species richness has been the extent to which estimates based on either cultivation or environmental DNA reflect the active assemblage in cold-arid soils. Here, we describe analysis for inland Dry Valleys soil of environmental DNA and RNA (cDNA) to infer total and putative metabolically active assemblages, respectively, plus cultivation approaches using a variety of laboratory growth conditions. Environmental sequences indicated a highly restricted assemblage of just seven phylotypes that affiliated phylogenetically within two known genera, Helicodendron and Zalerion, plus previously unidentified fungal phylotypes. None of the commonly encountered molds and mitosporic genera recorded from maritime Antarctic locations were encountered. A striking difference was observed in the frequency of recovery for phylotypes between libraries. This suggests that both species richness and beta diversity estimates based on DNA libraries have the potential to misinform putatively active assemblages. Cultivation yielded a cold-tolerant Zalerion strain that affiliated with DNA and RNA library clones, and a psychrotrophic yeast (Debaryomyces hansenii), which was not detected using either culture-independent approach.     

Candidatus Symbiothrix dinenymphae: bristle-like Bacteroidales ectosymbionts of termite gut protists

Many reports have stated that flagellated protists in termite guts harbour ectosymbiotic spirochetes on their cell surface. In this study, we describe another bristle-like ectosymbiont affiliated with the order Bacteroidales. The 16S rRNA phylotype Rs-N74 predominates among Bacteroidales clones obtained from the gut of the termite Reticulitermes speratus. An Rs-N74 phylotype-specific probe was designed in this study and used for detection of the corresponding bacteria in the gut by fluorescence in situ hybridization (FISH) analysis. Surprisingly, the signals were detected specifically from the bristle-like 'appendages' of various flagellate species belonging to the genus Dinenympha; these 'appendages' had been believed to be spirochetal ectosymbionts or structures of the protists. The Rs-N74 bacteria attached to the cell surface of the protists by a tip and coexisted with the spirochetal ectosymbionts. An electron micrograph revealed their morphology to be similar to a typical Bacteroidales bacterium. This bacterium is proposed to represent a novel genus and species, 'Candidatus Symbiothrix dinenymphae', phylogenetically affiliated with a cluster consisting exclusively of uncultured strains from termite guts. A Bacteroidales-specific probe for FISH further revealed that this type of symbiosis exists also in various other protists, including parabasalids and oxymonads, and is widespread in termite guts.     

Cultivation of particle-associated heterotrophic bacteria during a spring phytoplankton bloom in the North Sea

Seawater contains free-living and particle-attached bacteria. Only a small fraction is cultivable on plates. As free-living and particle-associated bacteria differ in their physiological traits, their cultivability on plates may coincide with particle association. Using filtration and Imhoff sedimentation cones, particles were collected during a spring phytoplankton bloom off Helgoland (North Sea) in order to obtain particle-associated bacteria as inocula. Direct dilution plating resulted in 526 strains from 3 µm filtration retentates and 597 strains from settled particles. Motile Gammaproteobacteria from the genera Pseudoalteromonas, Shewanella, Psychrobacter, Vibrio and Colwellia, as well as particle-attached Flavobacteriia affiliating with the genera Tenacibaculum and Gramella, were frequently isolated. As a result, a diverse collection comprised of 266 strains was deposited. Two strains were most likely to represent novel genera and 78 strains were probably novel species. Recently, a high-throughput cultivation study from the same site using seawater as an inoculum had retrieved 271 operational phylogenetic units (OPUs) that represented 88% of the 4136 characterized strains at the species level. A comparison of 16S rRNA gene sequences revealed that the collection obtained matched 104 of the 271 seawater OPUs at the species level and an additional 113 at the genus level. This large overlap indicated a significant contribution of particle-associated bacteria to the cultivable microbiome from seawater. The presence of 49 genera not identified in the larger seawater study suggested that sample fractionation was an efficient strategy to cultivate rare members of the planktonic microbiome. The diverse collection of heterotrophic bacteria retrieved in this study will be a rich source for future studies on the biology of particle-associated bacteria.