16S rDNA sequence and phylogenetic position of an uncultivated spirochete from the hindgut of the termite Mastotermes darwiniensis Froggatt

Abstract We have analyzed the 16S rDNA sequence and the phylogenetic position of an uncultivated spirochete from the hindgut contents of the Australian termite Mastotermes darwiniensis Froggatt. The 16S rRNA genes of bacteria from the hindgut contents of Mastotermes darwiniensis were amplified by polymerase chain reaction. The amplification products were cloned and sequenced. The sequences were compared to known homologous primary structures. Two of the clones (MDS1 and MDS3) had an insert of 1498 nucleotides showing typical signatures of spirochete 16S rRNA sequences. The sequences of the two clones were most similar to the 16S rRNA sequence of Spirochaeta stenostrepta (89.8%) and Treponema sp. strain H1 (90.7%). Phylogenetical analysis positioned the hindgut spirochete sequence with that of the free-living anaerobic Spirochaeta stenostrepta and Treponema sp. strain H1 as its nearest relatives within the cluster of the spirochetes. We conclude that the analyzed SSU rDNA sequences originate from a spirochete related to the genus Treponema . It is possibly one of the uncultivated unique spirochetes symbiotic in termite hindguts.     

Phylogenetic analysis of Blattabacterium,endosymbiotic bacteria from the wood roach,Cryptocercus (Blattodea: Cryptocercidae), including a description of three new species

Members of the cockroach genus Cryptocercus are wood-feeding, subsocial insects that live in temperate forests of the Nearctic and Palaearctic. At present, nine species are recognized: Cryptocercus relictus and Cryptocercus kyebangensis in eastern Asia and Russia, Cryptocercus primarius and Cryptocercus matilei in southwestern China, Cryptocercus clevelandi in the western USA, and Cryptocercus darwini, Cryptocercus garciai, Cryptocercus punctulatus, and Cryptocercus wrighti in the eastern USA. Like all extant cockroaches, Cryptocercus harbor endosymbiotic bacteria, Blattabacterium, in their fat bodies. The endosymbionts in all cockroaches have been considered a single species, Blattabacterium cuenoti, since their discovery about a century ago. However, a recent analysis of DNA sequences from representatives of four cockroach families has indicated that there is considerable DNA sequence divergence among B. cuenoti from different host species. As a part of our studies on the evolution of Cryptocercus, we examined DNA sequence divergence among B. cuenoti from six of the nine known Cryptocercus species. Specifically, we sequenced approximately 2,400 bp of the 16S rRNA and 23S rRNA genes of B. cuenoti from six species of Cryptocercus. We found that B. cuenoti in Cryptocercus has differentiated into multiple monophyletic lineages distinguishable by DNA sequence of rRNA genes and host association. Our sequence divergence estimates were consistent with those reported for other, congeneric bacterial species. We propose the recognition of three new species of Blattabacterium within Cryptocercus species as follows: Blattabacterium relictus sp. nov. in C. relictus, Blattabacterium clevelandi sp. nov. in C. clevelandi, and Blattabacterium punctulatus sp. nov. in C. darwini, C. garciai, C. punctulatus, and C. wrighti.     

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.     

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.     

Spirosymplokos deltaeiberi nov. gen., nov. sp.: variable-diameter composite spirochete from microbial mats

Large (up to 100 m long), loosely coiled, free-living spirochetes with variable diameters (from 0.4 to 3 m in the same cell) were seen at least 40 times between August 1990 and January 1993. These spirochetes were observed in mud water and enrichment media from highly specific habitats in intertidal evaporite flats at three disjunct localities, one in Spain and two in Mexico. All three are sites of commerical saltworks. Associated with Microcoleus chthonoplastes, the large spirochetes from Spain display phototaxis and a composite organization. Shorter and smaller-diameter spirochetes are seen inside both healthy and spent periplasm of larger ones. Small spirochetes attached to large ones have been observed live. From two to twelve spirochete protoplasmic cylinders were seen inside a single common outer membrane. A distinctive granulated cytoplasm in which the granules are of similar diameter (20–32 nm) to that of the flagella (26 nm) was present. Granule diameters were measured in thin section and in negatively-stained whole-mount preparations. Based on their ultrastructure, large size, variable diameter, number of flagella (3 to 6), and phototactic behavior these unique spirochetes are formally named Spirosymplokos deltaeiberi. Under anoxic (or low oxygen) conditions they formed blooms in mixed culture in media selective for spirochetes. Cellobiose was the major carbon source in 80% seawater, the antibiotic rifampicin was added, mat from the original field site was present and tubes were incubated in the light at from 18–31 °C. Within 1–2 weeks populations of the large spirochete developed at 25 °C but they could not be transferred to fresh medium.     

Treponema saccharophilum sp. nov., a large pectinolytic spirochete from the bovine rumen.

A large, obligately anaerobic spirochete (strain PB) was isolated from bovine rumen fluid by a procedure involving rifampin as a selective agent. The helical cells measured 0.6 to 0.7 micron by 12 to 20 micron and possessed approximately 16 periplasmic flagella inserted near each end of the protoplasmic cylinder. The periplasmic flagella were arranged in a bundle wound around the cell body. Strain PB utilized as fermentable substrates various plant polysaccharides (e.g., pectin, arabinogalactan, starch, and inulin) as well as pentoses, hexoses, disaccharides, and uronic acids. Glucose was fermented to acetate, formate, and ethanol, whereas the fermentation of pectin or glucuronic acid yielded only acetate and formate as major end products. Determinations of radioactivity in end products and assays of enzymatic activities indicated that strain PB catabolized glucose via the Embden-Meyerhof pathway. Extracts of cells grown in pectin-containing media possessed relatively high levels of phospho-2-keto-3-deoxygluconate aldolase activity, an enzymatic activity typical of the Entner-Doudoroff pathway. The guanine-plus-cytosine content of the DNA of strain PB (54 mol%) was considerably higher than that of known host-associated anaerobic spirochetes. This study indicates that strain PB represents a new species of Treponema, for which we propose the name Treponema saccharophilum.     

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.     

The gut flagellate community of the termite Neotermes cubanus with special reference to Staurojoenina and Trichocovina hrdyi nov. gen. nov. sp

At least seven species of flagellates have been found to inhabit the paunch of the termite Neotermes cubanus. Staurojoenina sp. is the largest species, measuring 100-150mum in length. Three small parabasalids belong to the genera Tritrichomonas, Metadevescovina, and Foaina. A fourth small type is described as Trichocovina hrdyi nov. gen. nov. sp.; the combination of features in this flagellate, such as the presence of a costa, undulating membrane and spiralled dictyosome, does not fit to any known genus. The two oxymonad species do not possess a rostellum. One belongs to the family Polymastigidae; it has one unattached flagellum and three partially attached flagella. The second is a member of the family Pyrsonymphidae, but this one possesses ring-like surface structures, one free flagellum and three spirally attached flagella. It is the first report of a pyrsonymphid in a kalotermitid termite.     

Stenoxybacter acetivorans gen. nov., sp. nov., an Acetate-Oxidizing Obligate Microaerophile among Diverse O2-Consuming Bacteria from Termite Guts

In termite hindguts, fermentative production of acetate—a major carbon and energy source for the insect—depends on efficient removal of inwardly diffusing oxygen by microbes residing on and near the hindgut wall. However, little is known about the identity of these organisms or about the substrate(s) used to support their respiratory activity. A cultivation-based approach was used to isolate O₂-consuming organisms from hindguts of Reticulitermes flavipes. A consistently greater (albeit not statistically significant) number of colonies developed under hypoxia (2% [vol/vol] O₂) than under air, and the increase coincided with the appearance of morphologically distinct colonies of a novel, rod-shaped, obligately microaerophilic β-proteobacterium that was <95% similar (based on the 16S rRNA gene sequence) to its closest known relative (Eikenella corrodens). Nearly identical organisms (and/or their 16S rRNA genes) were obtained from geographically separated and genetically distinct populations of Reticulitermes. PCR-based procedures implied that the novel isolates were autochthonous to the hindgut of R. flavipes and comprised ca. 2 to 7% of the hindgut prokaryote community. Representative strain TAM-DN1 utilized acetate and a limited range of other organic and amino acids as energy sources and possessed catalase and superoxide dismutase. On solid medium, the optimal O₂ concentration for growth was about 2%, and no growth occurred with O₂ concentrations above 4% or under anoxia. However, cells in liquid medium could grow with higher O₂ concentrations (up to 16%), but only after proportionately extended lag phases. The genetic and physiological distinctiveness of TAM-DN1 and related strains supports their recognition as a new genus and species, for which the name Stenoxybacter acetivorans gen. nov., sp. nov. is proposed.     

Stenoxybacter acetivorans gen. nov., sp. nov., an acetate-oxidizing obligate microaerophile among diverse O2-consuming bacteria from termite guts

In termite hindguts, fermentative production of acetate--a major carbon and energy source for the insect--depends on efficient removal of inwardly diffusing oxygen by microbes residing on and near the hindgut wall. However, little is known about the identity of these organisms or about the substrate(s) used to support their respiratory activity. A cultivation-based approach was used to isolate O(2)-consuming organisms from hindguts of Reticulitermes flavipes. A consistently greater (albeit not statistically significant) number of colonies developed under hypoxia (2% [vol/vol] O(2)) than under air, and the increase coincided with the appearance of morphologically distinct colonies of a novel, rod-shaped, obligately microaerophilic beta-proteobacterium that was <95% similar (based on the 16S rRNA gene sequence) to its closest known relative (Eikenella corrodens). Nearly identical organisms (and/or their 16S rRNA genes) were obtained from geographically separated and genetically distinct populations of Reticulitermes. PCR-based procedures implied that the novel isolates were autochthonous to the hindgut of R. flavipes and comprised ca. 2 to 7% of the hindgut prokaryote community. Representative strain TAM-DN1 utilized acetate and a limited range of other organic and amino acids as energy sources and possessed catalase and superoxide dismutase. On solid medium, the optimal O(2) concentration for growth was about 2%, and no growth occurred with O(2) concentrations above 4% or under anoxia. However, cells in liquid medium could grow with higher O(2) concentrations (up to 16%), but only after proportionately extended lag phases. The genetic and physiological distinctiveness of TAM-DN1 and related strains supports their recognition as a new genus and species, for which the name Stenoxybacter acetivorans gen. nov., sp. nov. is proposed.     

Spirochaeta coccoides sp. nov., a novel coccoid spirochete from the hindgut of the termite Neotermes castaneus

A novel spirochete strain, SPN1, was isolated from the hindgut contents of the termite Neotermes castaneus. The highest similarities (about 90%) of the strain SPN1 16S rRNA gene sequence are with spirochetes belonging to the genus Spirochaeta, and thus, the isolate could not be assigned to the so-called termite clusters of the treponemes or to a known species of the genus Spirochaeta. Therefore, it represents a novel species, which was named Spirochaeta coccoides. In contrast to all other known validly described spirochete species, strain SPN1 shows a coccoid morphology and is immotile. The isolated strain is obligately anaerobic and ferments different mono-, di-, and oligosaccharides by forming formate, acetate, and ethanol as the main fermentation end products. Furthermore, strain SPN1 is able to grow anaerobically with yeast extract as the sole carbon and energy source. The fastest growth was obtained at 30 degrees C, the temperature at which the termites were also grown. The cells possess different enzymatic activities that are involved in the degradation of lignocellulose in the termite hindgut, such as beta-D-glucosidase, alpha-L-arabinosidase, and beta-D-xylosidase. Therefore, they may play an important role in the digestion of breakdown products from cellulose and hemicellulose in the termite gut.     

Devescovinid trichomonad with axostyle-based rotary motor ("Rubberneckia"): taxonomic assignment as Caduceia versatilis sp. nov.

An amitochondriate trichomonad cell of the family Devescovinidae (Class Parabasalia), helped demonstrate the fluid model of lipoprotein cell membranes. This wood-ingesting symbiont in the hindgut of the dry wood-eating termite Cryptotermes cavifrons is informally known to cell biologists as "Rubberneckia". As the microtubular axo-style complex generates force causing clockwise movement of the entire anterior portion of the cell at the shear zone the protist displays "head" rotation. Studies by phase contrast and videomicroscopy of live cells, of whole mounts by scanning, and thin sections by transmission electron microscopy extend the observations of Tamm and Tamm [24-26] and Tamm [19-23]. Habitat, cell shape, size, nuclear features, parabasal apparatus and other morphological details permit the assignment of "Rubberneckia" to Kirby's cosmopolitan genus Caduceia. This large-sized devescovinid has distinctive parabasal gyres, an axostylar rotary, motor, and regularly-associated nonflagellated, fusiform and flagellated rod epibiotic surface bacteria. In addition to regularly aligned epibionts intranuclear and endocytoplasmic bacteria are abundant and hydrogenosomes are Present. "Rubberneckia" is compared here to the other seven species of Caduceia. Since it is clearly sufficiently distinctive to warrant new species status, we named it C. versatilis.     

<Emphasis Type="Italic">Sporotalea propionica</Emphasis> gen. nov. sp. nov., a hydrogen-oxidizing,oxygen-reducing,propionigenic firmicute from the intestinal tract of a soil-feeding termite

An unusual propionigenic bacterium was isolated from the intestinal tract of the soil-feeding termite Thoracotermes macrothorax. Strain TmPN3 is a motile, long rod that stains gram-positive, but reacts gram-negative in the KOH test. It forms terminal endospores and ferments lactate, glucose, lactose, fructose, and pyruvate to propionate and acetate via the methyl-malonyl-CoA pathway. Propionate and acetate are formed at a ratio of 2:1, typical of most propionigenic bacteria. Under a H₂/CO₂ atmosphere, the fermentation product pattern of glucose, fructose, and pyruvate shifts towards propionate formation at the expense of acetate. Cell suspensions reduce oxygen with lactate, glucose, glycerol, or hydrogen as electron donor. In the presence of oxygen, the product pattern of lactate fermentation shifts from propionate to acetate production. 16S rRNA gene sequence analysis showed that strain TmPN3 is a firmicute that clusters among the Acidaminococcaceae, a subgroup of the Clostridiales comprising obligately anaerobic, often endospore-forming bacteria that possess an outer membrane. Based on phenotypic differences and less than 92% sequence similarity to the 16S rRNA gene sequence of its closest relative, the termite hindgut isolate Acetonema longum, strain TmPN3^T is proposed as the type species of a new genus, Sporotalea propionica gen. nov. sp. nov. (DSM 13327^T, ATCC BAA-626^T).     

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.     

Spherical body formation in the spirochaete Brachyspira hyodysenteriae

When cultures of Brachyspira hyodysenteriae were grown under a wide range of in vitro conditions, at least 1% of the cells formed spherical bodies different to the normal helical form. This percentage increased considerably in aging cultures or following their incubation in caramelized media. Spherical body formation was initiated from a terminal localized swelling of the outer sheath followed by a retraction of the protoplasmic cylinder into the resulting swollen vesicle. As this occurred, the periplasmic flagella seemed to unwind from the protoplasmic cylinder. Once retracted, the protoplasmic cylinder was found to be wrapped in an organized manner around the inner surface of the membrane of the swollen vesicle. Although most were 2-3 microm in diameter, some much larger spherical bodies (6-12 microm diameter) were occasionally seen, with a corresponding increase in the visible number of peripheral protoplasmic cylinder cross-sections. Spherical bodies from older cultures did not contain protoplasmic cylinders arranged around the periphery, but instead were characterized by the presence of a centrally located, electron-dense body c. 0.5-0.8 mum in diameter. Brachyspira hyodysenteriae spherical bodies differ in both their structural organization and probable method of formation from similar structures described in other spirochaete genera.     

Treponema saccharophilum sp. nov., a large pectinolytic spirochete from the bovine rumen

A large, obligately anaerobic spirochete (strain PB) was isolated from bovine rumen fluid by a procedure involving rifampin as a selective agent. The helical cells measured 0.6 to 0.7 micron by 12 to 20 micron and possessed approximately 16 periplasmic flagella inserted near each end of the protoplasmic cylinder. The periplasmic flagella were arranged in a bundle wound around the cell body. Strain PB utilized as fermentable substrates various plant polysaccharides (e.g., pectin, arabinogalactan, starch, and inulin) as well as pentoses, hexoses, disaccharides, and uronic acids. Glucose was fermented to acetate, formate, and ethanol, whereas the fermentation of pectin or glucuronic acid yielded only acetate and formate as major end products. Determinations of radioactivity in end products and assays of enzymatic activities indicated that strain PB catabolized glucose via the Embden-Meyerhof pathway. Extracts of cells grown in pectin-containing media possessed relatively high levels of phospho-2-keto-3-deoxygluconate aldolase activity, an enzymatic activity typical of the Entner-Doudoroff pathway. The guanine-plus-cytosine content of the DNA of strain PB (54 mol%) was considerably higher than that of known host-associated anaerobic spirochetes. This study indicates that strain PB represents a new species of Treponema, for which we propose the name Treponema saccharophilum.     

Actinobacillus rossii sp. nov., Actinobacillus seminis sp. nov., nom. rev., Pasteurella bettii sp. nov., Pasteurella lymphangitidis sp. nov., Pasteurella mairi sp. nov., and Pasteurella trehalosi sp. nov

Evidence from numerical taxonomic analysis and DNA-DNA hybridization supports the proposal of new species in the genera Actinobacillus and Pasteurella. The following new species are proposed: Actinobacillus rossii sp. nov., from the vaginas of postparturient sows; Actinobacillus seminis sp. nov., nom. rev., associated with epididymitis of sheep; Pasteurella bettii sp. nov., associated with human Bartholin gland abscess and finger infections; Pasteurella lymphangitidis sp. nov. (the BLG group), which causes bovine lymphangitis; Pasteurella mairi sp. nov., which causes abortion in sows; and Pasteurella trehalosi sp. nov., formerly biovar T of Pasteurella haemolytica, which causes septicemia in older lambs.