Pure cultures of termite gut spirochetes were obtained and were shown to catalyze the synthesis of acetate from H2 plus CO2. The 16S ribosomal DNA sequences of two strains were 98 percent similar and were affiliated with those of the genus Treponema. However, neither was closely related to any known treponeme. These findings imply an important role for spirochetes in termite nutrition, help to reconcile the dominance of acetogenesis over methanogenesis as an H2 sink in termite hindguts, suggest that the motility of termite gut protozoa by means of attached spirochetes may be based on interspecies H2 transfer, and underscore the importance of termites as a rich reservoir of novel microbial diversity.

Within the last several years, molecular techniques have uncovered numerous 16S rRNA gene (rDNA) sequences which represent a unique and globally distributed lineage of the kingdom Crenarchaeotathat is phylogenetically distinct from currently characterized crenarchaeotal species. rDNA sequences of members of this novel crenarchaeotal group have been recovered from low- to moderate-temperature environments (−1.5 to 32°C), in contrast to the high-temperature environments (temperature, >80°C) required for growth of the currently recognized crenarchaeotal species. We determined the diversity and abundance of the nonthermophilic members of the Crenarchaeota in soil samples taken from cultivated and uncultivated fields located at the Kellogg Biological Station’s Long-Term Ecological Research site (Hickory Corners, Mich.). Clones were generated from 16S rDNA that was amplified by using broad-specificity archaeal PCR primers. Twelve crenarchaeotal sequences were identified, and the phylogenetic relationships between these sequences and previously described crenarchaeotal 16S rDNA sequences were determined. Phylogenetic analyses included nonthermophilic crenarchaeotal sequences found in public databases and revealed that the nonthermophilic Crenarchaeota group is composed of at least four distinct phylogenetic clusters. A 16S rRNA-targeted oligonucleotide probe specific for all known nonthermophilic crenarchaeotal sequences was designed and used to determine their abundance in soil samples. The nonthermophilicCrenarchaeota accounted for as much as 1.42% ± 0.42% of the 16S rRNA in the soils analyzed.

Long after their original discovery, termite gut spirochetes were recently isolated in pure culture for the first time. They revealed metabolic capabilities hitherto unknown in the Spirochaetes division of the Bacteria, i.e., H2 plus CO2 acetogenesis (J. R. Leadbetter, T. M. Schmidt, J. R. Graber, and J. A. Breznak, Science 283:686-689, 1999) and dinitrogen fixation (T. G. Lilburn, K. S. Kim, N. E. Ostrom, K. R. Byzek, J. R. Leadbetter, and J. A. Breznak, Science 292:2495-2498, 2001). However, application of specific epithets to the strains isolated (Treponema strains ZAS-1, ZAS-2, and ZAS-9) was postponed pending a more complete characterization of their phenotypic properties. Here we describe the major properties of strain ZAS-9, which is readily distinguished from strains ZAS-1 and ZAS-2 by its shorter mean cell wavelength or body pitch (1.1 versus 2.3 μm), by its nonhomoacetogenic fermentation of carbohydrates to acetate, ethanol, H2, and CO2, and by 7 to 8% dissimilarity between its 16S rRNA sequence and those of ZAS-1 and ZAS-2. Strain ZAS-9 is proposed as the type strain of the new species, Treponema azotonutricium. Strains ZAS-1 and ZAS-2, which are H2-consuming, CO2-reducing homoacetogens, are proposed here to be two strains of the new species Treponema primitia. Apart from the salient differences mentioned above, the genomes of all three strains were similar in size (3,461 to 3,901 kb), in G+C content (50.0 to 51.0 mol%), and in possession of 2 copies of the gene encoding 16S rRNA (rrs). For comparison, the genome of the free-living spirochete Spirochaeta aurantia strain J1 was analyzed by the same methods and found to have a size of 3,719 kb, to contain 65.6 mol% G+C, and also to possess 2 copies of the rrs gene

Treponema primitia strains ZAS-1 and ZAS-2, the first spirochetes to be isolated from termite hindguts (J. R. Leadbetter, T. M. Schmidt, J. R. Graber, and J. A. Breznak, Science 283:686-689, 1999), were examined for nutritional, physiological, and biochemical properties relevant to growth and survival in their natural habitat. In addition to using H 2 plus CO 2 as substrates, these strains were capable of homoacetogenic growth on monoand disaccharides and (in the case of ZAS-2) methoxylated benzenoids. Cells were also capable of mixotrophic growth (i.e., simultaneous utilization of H 2 and organic substrates). Cell extracts of T. primitia possessed enzyme activities of the Wood/Ljungdahl (acetyl coenzyme A) pathway of acetogenesis, including tetrahydrofolate-dependent enzymes of the methyl group-forming branch. However, a folate compound was required in the medium for growth. ZAS-1 and ZAS-2 growing on H 2 plus CO 2 displayed H 2 thresholds of 650 and 490 ppmv, respectively. Anoxic cultures of ZAS-1 and ZAS-2 maintained growth after the addition of as much as 0.5% (vol/vol) O 2 to the headspace atmosphere. Cell extracts exhibited NADH and NADPH peroxidase and NADH oxidase activities but neither catalase nor superoxide dismutase activity. Results indicate that (i) T. primitia is able to exploit a variety of substrates derived from the food of its termite hosts and in so doing contributes to termite nutrition via acetogenesis, (ii) in situ growth of T. primitia is likely dependent on secretion of a folate compound(s) by other members of the gut microbiota, and (iii) cells possess enzymatic adaptations to oxidative stress, which is likely to be encountered in peripheral regions of the termite hindgut.Spirochetes are among the most abundant microbial groups in termite hindguts, accounting for as much as one-half of the prokaryotic community (42). For more than a century, however, our knowledge of these organisms was largely limited to sporadic reports of their presence in various termite species, their morphological diversity, and their physical association with termite gut protozoa (8). Although elimination of spirochetes from the termite gut led to a decrease in termite survival (20), the specific roles of spirochetes and the factors contributing to their abundance in the hindgut have remained obscure.Over the past 10 years, our understanding of termite hindgut spirochetes has advanced dramatically. Cultivation-independent molecular approaches revealed that they group within the genus Treponema and that the large majority of 16S rRNA gene clones form a phylogenetically discrete cluster (the "termite cluster") within this genus (34). These studies also revealed a striking degree of phylogenetic diversity among termite gut treponemes, with as many as 21 distinct species occurring within a single termite host species (34). A few years ago, the first pure cultures of these organisms were isolated in our laboratory and were found to possess metabolic capabilities hitherto unknown in the Spirochaetes division of the Bac...

Treponema primitia, an H 2 -consuming CO 2 -reducing homoacetogenic spirochete in termite hindguts, requires an exogenous source of folate for growth. Tetrahydrofolate (THF) acts as a C 1 carrier in CO 2 -reductive acetogenesis, a microbially mediated process important to the carbon and energy requirements of termites. To examine the hypothesis that other termite gut microbes probably supply some form of folate to T. primitia in situ, we used a bioassay to screen for and isolate folate-secreting bacteria from hindguts of Zootermopsis angusticollis, which is the host of T. primitia. Based on morphology, physiology, and 16S rRNA gene sequences, the major folate secretors were identified as strains of Lactococcus lactis and Serratia grimesii. During growth, these isolates secreted 5-formyl-THF at levels up to 146 ng/ml, and their cell-free culture fluids satisfied the folate requirement of T. primitia strains in vitro. Analysis of Z. angusticollis hindgut fluid revealed that 5-formyl-THF was the only detectable folate compound and occurred at an in situ concentration (1.3 g/ml) which was more than sufficient to support the growth of T. primitia. These results imply that cross-feeding of 5-formyl-THF by other community members is important for growth of symbiotic hindgut spirochetes and thus termite nutrition and survival.Spirochetes are a major component of the hindgut microbiota of termites, accounting for as many as 50% of all prokaryotic cells (37), but for many years their role in the gut remained obscure as none had ever been isolated and studied in vitro. Recently, the first pure cultures of the following termite gut spirochetes were obtained: Treponema primitia strains ZAS-1 and ZAS-2 and Treponema azotonutricium strain 29,30). These organisms are believed to be important for termite nutrition because of their ability to fix dinitrogen and produce acetate, a major carbon and energy source for termites. In fact, T. primitia is a homoacetogen and possesses the Wood-Ljungdahl (acetyl coenzyme A) pathway for CO 2 -reductive acetogenesis (14). This enables T. primitia to conserve energy by homoacetogenesis from H 2 plus CO 2 , as well as from carbohydrates and methoxylated benzenoids, and to use CO 2 as a carbon source for growth. The Wood-Ljungdahl pathway is undoubtedly important in situ, because a significant portion of acetate formed in the hindgut arises from CO 2 reduction (5, 56). Given the abundance and species diversity of treponemes in termite hindguts (31), it is likely that many of the other not-yet-cultivated spirochetes are also CO 2 -reducing acetogens. This notion is supported by the large number of formyltetrahydrofolate synthetase (a key enzyme of the WoodLjungdahl pathway) genes similar to those of T. primitia detected in hindguts of Zootermopsis angusticollis, the termite that is the host of T. primitia (45).During recent studies of the physiology and nutrition of T. primitia, cells were found to require folate for growth (14). Requirements for exogenous folate compounds are not uncommon among...