Phylogenetic diversity of termite gut spirochaetes

A molecular phylogenetic analysis was done of not-yet-cultured spirochaetes inhabiting the gut of the termite, Reticulitermes flavipes (Kollar). Ninety-eight clones of near-full-length spirochaetal 16S rDNA genes were classified by ARDRA pattern and by partial sequencing. All clones grouped within the genus Treponema , and at least 21 new species of Treponema were recognized within R. flavipes alone. Analysis of 190 additional clones from guts of Coptotermes formosanus Shiraki and Zootermopsis angusticollis (Hagen), as well as published data on clones from Cryptotermes domesticus (Haviland), Mastotermes darwiniensis Froggatt, Nasutitermes lujae (Wasmann) and Reticulitermes speratus (Kolbe), revealed a similar level of novel treponemal phylogenetic diversity in these representatives of five of the seven termite families. None of the clones was closely related (i.e. all bore ≤ 91% sequence similarity) to any previously recognized treponeme. The data also revealed the existence of two major phylogenetic groups of treponemes: one containing all of the currently known isolates of Treponema and a large number of phylotypes from the human gingival crevice, but only a minority of the termite gut spirochaete clones; another containing the majority of termite spirochaete clones and two Spirochaeta ( S. caldaria and S. stenostrepta ), which, although free living, group within the genus Treponema on the basis of 16S rRNA sequence. Signature nucleotides that almost perfectly distinguished the latter group, herein referred to as the 'termite cluster', occurred at the following ( E. coli numbering) positions: 289-G · C-311; A at 812; and an inserted nucleotide at 1273. The emerging picture is that the long-recognized and striking morphological diversity of termite gut spirochaetes is paralleled by their phylogenetic diversity and may reflect substantial physiological diversity as well.     

Phylogenetic position and in situ identification of ectosymbiotic spirochetes on protists in the termite gut

Phylogenetic relationships, diversity, and in situ identification of spirochetes in the gut of the termite Neotermes koshunensis were examined without cultivation, with an emphasis on ectosymbionts attached to flagellated protists. Spirochetes in the gut microbial community investigated so far are related to the genus Treponema and divided into two phylogenetic clusters. In situ hybridizations with a 16S rRNA-targeting consensus oligonucleotide probe for one cluster (known as termite Treponema cluster I) detected both the ectosymbiotic spirochetes on gut protists and the free-swimming spirochetes in the gut fluid of N. koshunensis. The probe for the other cluster (cluster II), which has been identified as ectosymbionts on gut protists of two other termite species, Reticulitermes speratus and Hodotermopsis sjoestedti, failed to detect any spirochete population. The absence of cluster II spirochetes in N. koshunensis was confirmed by intensive 16S ribosomal DNA (rDNA) clone analysis, in which remarkably diverse spirochetes of 45 phylotypes were identified, almost all belonging to cluster I. Ectosymbiotic spirochetes of the three gut protist species Devescovina sp., Stephanonympha sp., and Oxymonas sp. in N. koshunensis were identified by their 16S rDNA and by in situ hybridizations using specific probes. The probes specific for these ectosymbionts did not receive a signal from the free-swimming spirochetes. The ectosymbionts were dispersed in cluster I of the phylogeny, and they formed distinct phylogenetic lineages, suggesting multiple origins of the spirochete attachment. Each single protist cell harbored multiple spirochete species, and some of the spirochetes were common among protist species. The results indicate complex relationships of the ectosymbiotic spirochetes with the gut protists.     

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.     

Phylogenetic foundation of spirochetes

The spirochetes are free-living or host-associated, helical bacteria, some of which are pathogenic to man and animal. Comparisons of 16S rRNA sequences demonstrate that the spirochetes represent a monophyletic phylum within the bacteria. The spirochetes are presently classified in the Class Spirochaetes in the order Spirochetales and are divided into three major phylogenetic groupings, or families. The first family Spirochaetaceae contains species of the genera Borrelia, Brevinema, Cristispira, Spirochaeta, Spironema, and Treponema. The second family Brachyspiraceae contains the genus Brachyspira (Serpulina). The third family Leptospiraceae contains species of the genera Leptonema and Leptospira. Novel spirochetal species, or phylotypes, that can not be presently cultivated in vitro, have been identified from the human oral cavity, the termite gut, and other host-associated or free-living sources. There are now over 200 spirochetal species or phylotypes, of which more than half is presently not cultivable. It is likely that there is still a significant unrecognized spirochetal diversity that should be evaluated.     

Phylogenetic analysis of the gut bacterial microflora of the fungus-growing termite Odontotermes formosanus

We constructed a bacterial 16S rRNA gene clone library from the gut microbial community of O. formosanus and phylogenetically analyzed it in order to contribute to the evolutional study of digestive symbiosis and method development for termite control. After screening by restriction fragment length polymorphism (RFLP) analysis, 56 out of 280 clones with unique RFLP patterns were sequenced and phylogenetically analyzed. The representative phylotypes were affiliated to four phylogenetic groups, Firmicutes, the Bacteroidetes/Chlorobi group, Proteobacteria, and Actinobacteria of the domain Bacteira. No one clone affiliated with the phylum Spirochaetes was identified, in contrast to the case of wood-feeding termites. The phylogenetic analysis revealed that nearly half of the representative clones (25 phylotypes) formed monophyletic clusters with clones obtained from other termite species, especially with the sequences retrieved from fungus-growing termites. These results indicate that the presence of termite-specific bacterial lineages implies a coevolutional relationship of gut microbes and host termites.     

Phylogeny of not-yet-cultured spirochetes from termite guts.

Comparisons of 16S rDNA sequences were used to determine the phylogeny of not-yet-cultured spirochetes from hindguts of the African higher termite, Nasutitermes lujae (Wasmann). The 16S rRNA genes were amplified directly from spirochete-rich hindguts by using universal primers, and the amplified products were cloned into Escherichia coli. Clones were screened with a spirochete-specific DNA probe. Analysis of 1,410 base positions of the 16S rDNA insert from one spirochete clone, designated NL1, supported its assignment to the genus Treponema, with average interspecies similarities of ca. 85%. The sequence of NL1 was most closely related (ca. 87 to 88% similarity) to sequences of Spirochaeta stenostrepta and Spirochaeta caldaria and to a previously published sequence (ca. 87% similarity) of spirochetal clone MDS1 from the Australian lower termite, Mastotermes darwiniensis (Froggatt). On the basis of 16S rRNA sequence comparisons and individual base signatures, clones NL1 and MDS1 clearly represent two novel species of Treponema, although specific epithets have not yet been proposed. The gross morphology of NL1 was determined from in situ hybridization experiments with an NL1-specific, fluorescently labeled oligonucleotide probe. Cells were approximately 0.3 to 0.4 by 30 microns in size, with a wavelength and amplitude of about 10 microns and 0.8 to 1.6 micron, respectively. Moreover, electron microscopy of various undulate cells present in gut contents confirmed that they possessed ultrastructural features typical of spirochetes, i.e., a wavy protoplasmic cylinder, periplasmic flagella, and an outer sheath. The sequence data suggest that termite gut spirochetes may represent a separate line of descent from other treponemes and that they constitute a significant reservoir of previously unrecognized spirochetal biodiversity.     

Phylogenetic analysis of the spirochetes.

The 16S rRNA sequences were determined for species of Spirochaeta, Treponema, Borrelia, Leptospira, Leptonema, and Serpula, using a modified Sanger method of direct RNA sequencing. Analysis of aligned 16S rRNA sequences indicated that the spirochetes form a coherent taxon composed of six major clusters or groups. The first group, termed the treponemes, was divided into two subgroups. The first treponeme subgroup consisted of Treponema pallidum, Treponema phagedenis, Treponema denticola, a thermophilic spirochete strain, and two species of Spirochaeta, Spirochaeta zuelzerae and Spirochaeta stenostrepta, with an average interspecies similarity of 89.9%. The second treponeme subgroup contained Treponema bryantii, Treponema pectinovorum, Treponema saccharophilum, Treponema succinifaciens, and rumen strain CA, with an average interspecies similarity of 86.2%. The average interspecies similarity between the two treponeme subgroups was 84.2%. The division of the treponemes into two subgroups was verified by single-base signature analysis. The second spirochete group contained Spirochaeta aurantia, Spirochaeta halophila, Spirochaeta bajacaliforniensis, Spirochaeta litoralis, and Spirochaeta isovalerica, with an average similarity of 87.4%. The Spirochaeta group was related to the treponeme group, with an average similarity of 81.9%. The third spirochete group contained borrelias, including Borrelia burgdorferi, Borrelia anserina, Borrelia hermsii, and a rabbit tick strain. The borrelias formed a tight phylogenetic cluster, with average similarity of 97%. THe borrelia group shared a common branch with the Spirochaeta group and was closer to this group than to the treponemes. A single spirochete strain isolated fromt the shew constituted the fourth group. The fifth group was composed of strains of Serpula (Treponema) hyodysenteriae and Serpula (Treponema) innocens. The two species of this group were closely related, with a similarity of greater than 99%. Leptonema illini, Leptospira biflexa, and Leptospira interrogans formed the sixth and most deeply branching group. The average similarity within this group was 83.2%. This study represents the first demonstration that pathogenic and saprophytic Leptospira species are phylogenetically related. The division of the spirochetes into six major phylogenetic clusters was defined also by sequence signature elements. These signature analyses supported the conclusion that the spirochetes represent a monophylectic bacterial phylum.     

Phylogenetic diversity of the intestinal bacterial community in the termite Reticulitermes speratus.

The phylogenetic diversity of the intestinal microflora of a lower termite, Reticulitermes speratus, was examined by a strategy which does not rely on cultivation of the resident microorganisms. Small-subunit rRNA genes (16S rDNAs) were directly amplified from the mixed-population DNA of the termite gut by the PCR and were clonally isolated. Analysis of partial 16S rDNA sequences showed the existence of well-characterized genera as well as the presence of bacterial species for which no 16S rDNA sequence data are available. Of 55 clones sequenced, 45 were phylogenetically affiliated with four of the major groups of the domain Bacteria: the Proteobacteria, the spirochete group, the Bacteroides group, and the low-G+C-content gram-positive bacteria. Within the Proteobacteria, the 16S rDNA clones showed a close relationship to those of cultivated species of enteric bacteria and sulfate-reducing bacteria, while the 16S rDNA clones in the remaining three groups showed only distant relationships to those of known organisms in these groups. Of the remaining 10 clones, among which 8 clones formed a cluster, there was only very low sequence similarity to known 16S rRNA sequences. None of these clones were affiliated with any of the major groups within the domain Bacteria. The 16S rDNA gene sequence data show that the majority of the intestinal microflora of R. speratus consists of new, uncultured species previously unknown to microbiologists.     

Symbiotic spirochetes in the termite hindgut: phylogenetic identification of ectosymbiotic spirochetes of oxymonad protists

Some species of protists inhabiting the hindgut of lower-termites have a large number of ectosymbiotic spirochetes on the cell surface. The phylogenetic positions of the ectosymbiotic spirochetes of three oxymonad protists, Dinenympha porteri in the gut of Reticulitermes speratus, and Pyrsonympha sp. and Dinenympha sp. in Hodotermopsis sjoestedti, were investigated without cultivation of these organisms. Protist fractions carefully collected with a micromanipulator were used as templates for the amplification of small subunit ribosomal RNA genes (SSU rDNA). The phylogenetic tree inferred from the nucleotide sequences of the SSU rDNA showed that they were affiliated with the Treponema cluster of spirochetes and they were divided into two clusters. One was grouped together with the spirochetal sequences reported previously from the gut of termites and the other was related to the Treponema bryantii subgroup of treponemes (denoted as termite Treponema clusters I and II, respectively). Whole-cell in situ hybridization using a fluorescent-labeled oligonucleotide probe specific for the group of sequences in cluster II identified most of the ectosymbiotic spirochetes of the oxymonad protists in the gut of R. speratus and H. sjoestedti. However, not all of the ectosymbiotic spirochetes could be detected by means of this cluster II group-specific probe and the population of ectosymbiotic spirochetes of cluster II was different among the oxymonad species. In the case of D. porteri, an oligonucleotide probe specific for one member of cluster II recognized a portion of the ectosymbiotic spirochetes of cluster II, and their population was also different depending on the cell-type of D. porteri in terms of the attachment of ectosymbiotic spirochetes. The results indicate that the spirochetes of cluster II and probably those of a part of cluster I can be assigned to ectosymbiotic species of oxymonad protists and that the population of ectosymbiotic spirochetes associated with a single protist consists of at least three species of phylogenetically distinct spirochetes.     

Molecular phylogenetic diversity of the bacterial community in the gut of the termite Coptotermes formosanus

The phylogenetic diversity of the bacterial community in the gut of the termite Coptotermes formosanus was investigated using a 16S rRNA gene clone library constructed by PCR. After screening by restriction fragment length polymorphism (RFLP) analysis, 49 out of 261 clones with unique RFLP patterns were sequenced and phylogenetically analyzed. Many of the clones (94%) were derived from Bacteroidales, Spirochaetes, and low G + C content gram-positive bacteria consisting of Clostridiales, Mycoplasmatales, Bacillales, and Lactobacillales. In addition, a few clones derived from Actinobacteria, Proteobacteria, Planctomycetes, Verrucomicrobia, and the candidate phylum "Synergistes" were also found. The most frequently identified RFLP type, BCf1-03, was assigned to the order Bacteroideales, and it constituted about 70% of the analyzed clones. The phylogenetic analysis revealed that the representative clones found in this study tended to form some clusters with the sequences cloned from the termite gut in several other studies, suggesting the existence of termite-specific bacterial lineages.     

Phylogenetic and morphological diversity of Bacteroidales members associated with the gut wall of termites

The microbial community adherent directly or indirectly to the gut wall of termites is distinct from that of the other habitats in the gut. The bacterial 16S rRNA genes were identified from the fractionated gut walls of two termite species, Hodotermopsis sjoestedti and Neotermes koshunensis, and compared with those previously identified from Reticulitermes speratus. Surprisingly, the bacterial constituents were almost entirely different among the termites at the phylotype level (the criterion of the phylotype was >97% nucleotide identity). Bacteria in the order Bacteroidales, which were commonly abundant symbionts on gut walls, were phylogenetically analyzed. They were dispersed in a number of clusters formed by phylotypes from the guts of various termites. In situ hybridization with probes specific for some phylotypes and a phylogenetic cluster detected the cells of several Bacteroidales members with a significant variety of cell morphology in the gut wall fractions, which reflects the phylogenetic diversity of this order.     

Physiological diversity of rumen spirochetes.

Bovine rumen fluid contained relatively large numbers of spirochetes capable of fermenting polymers commonly present in plant materials. Polymers such as xylan, pectin, and arabinogalactan served as fermentable substrates for the spirochetes, whereas cellulose did not. Furthermore, spirochetes cultured from rumen fluid utilized as growth substrates hydrolysis products of plant polymers (e.g., D-xylose, L-arabinose, D-galacturonic acid, D-glucuronic acid, cellobiose), but did not ferment amino acids. Viable cell counts of spirochetes capable of fermenting individual plant polymers or their hydrolysis products yielded minimum values ranging from 0.2 X 10(6) to 4 X 10(6) cells per ml of rumen fluid. Thirteen strains of rumen spirochetes were characterized in terms of their fermentation products from glucose, the guanine plus cytosine content of their DNA, their ultrastructure, and their ability to ferment pectin, starch, or arabinogalactan. Of the 13 strains, 6 fermented glucose mainly to formate, acetate, and succinate, whereas the remaining 7 strains did not produce succinate, but instead formed ethanol, in addition to formate and acetate. The succinate-forming strains had two periplasmic (axial) fibrils per cell, measured 0.2 to 0.3 by 5 to 8 micrograms, had a guanine plus cytosine content of the DNA ranging from 36 to 38 mol%, and lacked the ability to ferment pectin, starch, or arabinogalactan. The ethanol-forming strains had from 8 to more than 32 periplasmic fibrils per cell, tended to be larger in cell size than the succinate-forming strains, and had a guanine plus cytosine content of the DNA ranging from 41 to 54 mol%. Some of the ethanol-forming strains fermented pectin, starch, or arabinogalactan. The results of this study indicate that the bovine rumen is inhabited by a physiologically and morphologically diverse population of spirochetes. It is likely that these spirochetes contribute significantly to the degradation of plant materials ingested by the ruminants.     

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 cellulolytic system of the termite gut

The demand for the usage of natural renewable polymeric material is increasing in order to satisfy the future needs for energy and chemical precursors. Important steps in the hydrolysis of polymeric material and bioconversion can be performed by microorganisms. Over about 150 million years, termites have optimized their intestinal polysaccharide-degrading symbiosis. In the ecosystem of the “termite gut,” polysaccharides are degraded from lignocellulose, such as cellulose and hemicelluloses, in 1 day, while lignin is only weakly attacked. The understanding of the principles of cellulose degradation in this natural polymer-degrading ecosystem could be helpful for the improvement of the biotechnological hydrolysis and conversion of cellulose, e.g., in the case of biogas production from natural renewable plant material in biogas plants. This review focuses on the present knowledge of the cellulose degradation in the termite gut.     

Phylogenetic diversity and whole-cell hybridization of oxymonad flagellates from the hindgut of the wood-feeding lower termite Reticulitermes flavipes

SSU rRNA genes of oxymonad protists from the hindgut of the wood-feeding termite Reticulitermes flavipes were PCR-amplified using a newly designed oxymonad-specific forward primer and a newly designed reverse primer specific for termite gut flagellates. After cloning, the clone library was sorted into four groups by RFLP analysis and nearly full-length SSU rRNA gene sequences were obtained for representative clones from each group. Phylogenetic analysis revealed that sequences of all four groups formed a monophyletic cluster with the only other existing SSU rRNA gene sequence of oxymonads. Using whole-cell hybridization with clone-specific fluorescently labeled probes, each of the four clone groups could be assigned to a specific morphotype, which were identified as Dinenympha gracilis, Dinenympha fimbriata, and so-far undescribed species of Pyrsonympha and Dinenympha. Our results demonstrate that the morphological variety of oxymonads is not caused by the presence of different developmental stages of the same organism, but that the various morphotypes represent different species.     

Hemicellulose-degrading bacteria and yeasts from the termite gut

Termites play a major role in the recycling of photosynthetically fixed carbon. With the aid of their symbiotic intestinal flora, they are able to degrade extensively wood constituents such as cellulose and hemicellulose. Nevertheless, the microbial species involved in the degradation of hemicelluloses are poorly defined. The purpose of this paper was to examine the microflora involved in hemicellulose degradation. Different aerobic and facultatively anaerobic bacteria and yeasts were isolated using xylan, arabinogalactan and carboxymethylcellulose as substrates. Gram-positive isolates belonged to the genera Bacillus, Paenibacillus, Streptomyces or the actinobacteria group, while the Gramnegative strains were assigned to the genera Pseudomonas, Acinetobacter, Ochrobactrum , and to genera belonging to the family Enterobacteriaceae. The spectrum and activity of xylan- and arabinogalactan-hydrolysing glycosidases of these new isolates, together with additional bacterial strains originally obtained from enrichments with aromatic compounds were determined.     

Ectobiotic spirochetes of flagellates from the termite Mastotermes darwiniensis: attachment and cyst formation

The association of the gut flagellates Mixotricha paradoxa and Deltotrichonympha sp. from the termite Mastotermes darwiniensis with ectobiotic spirochetes and bacterial rods is investigated with light and electron microscopy. Treatment with different chemicals disturbing molecular interactions and use of the freeze-fracture and freeze-etch technique show that hydrophobic interactions and integral membrane proteins seem to be involved in the firm attachment at the contact sites. Application of antibiotics reduces the number of ectobionts and leads to a disintegration of the cortical attachment systems. As a result Mixotricha becomes spherical and immotile. In both flagellates the antibiotics have a further effect: they lead to a transformation of some of the spirochetes into cystic bodies. Cyst formation of ectobiotic spirochetes is here reported for the first time. Starvation has a similar but less dramatic influence than antibiotics. The cysts contain protoplasmic cylinders in the periphery and sometimes larger central bodies. Production of dormant cystic forms may be a survival mechanism under hostile conditions.     

Phylogenetic position and physiology of Cerinosterus cyanescens

Partial 25S rRNA sequencing of Cerinosterus cyanescens showed it to be a close relative of Microstroma juglandis, a member of the basidiomycetous order Microstromatales. It is unrelated to the generic type species, C. luteoalba, which is a member of the order Dacrymycetales. The clinical occurrence of C. cyanescens is possibly explained by its thermotolerance and lipolytic activity. The species' nutritional profile is established. Growth on n-hexadecane is rapid; it grows well on typical plant constituents like gallic, tannic, vanillic, quinic and p-coumaric acids, but not on 3-hydroxybenzoic acid, phenol and hydroquinone. The failure to assimilate D-galactose, L-sorbose and ethylamine, the presence of urease and sensitivity to cycloheximide are diagnostic for the species.