Genomic and physiological characterization of the Verrucomicrobia isolate Geminisphaera colitermitum gen. nov., sp. nov., reveals microaerophily and nitrogen fixation genes.

Previously we reported the cultivation of novel verrucomicrobia, including strain TAV2 (93% 16S rRNA gene identity to its nearest cultivated representative, Opitutus terreae PB90-1) from the gut of the termite Reticulitermes flavipes. To gain better insight into the Verrucomicrobia as a whole and understand the role of verrucomicrobia within the termite gut ecosystem, we analyzed a draft genome and undertook a physiological characterization of TAV2. Strain TAV2 is an autochthonous member of the R. flavipes gut microbiota and groups phylogenetically among diverse Verrucomicrobia from R. flavipes and other termites that are represented by 16S rRNA gene sequences alone. TAV2 is a microaerophile, possessing a high-affinity cbb(3)-type terminal oxidase-encoding gene and exhibiting an optimum growth rate between 2 and 8% (vol/vol) oxygen. It has the genetic potential to degrade cellulose, an important function within termite guts, but its in vitro substrate utilization spectrum was limited to starch and a few mono- and disaccharides. Growth occurred on nitrogen-free medium, and genomic screening revealed genes for dinitrogenases, heretofore detected in only a few members of the Verrucomicrobia. This represents the first (i) characterization of a verrucomicrobial species from the termite gut, (ii) report of nif and anf genes in a nonacidophilic verrucomicrobial species, and (iii) description of a microaerophilic genotype and phenotype in this phylum of bacteria. The genetic and physiological distinctiveness of TAV2 supports its recognition as the type strain of a new genus and species, for which the name Geminisphaera colitermitum gen. nov., sp. nov., is proposed.

Spirochaeta cellobiosiphila sp. nov., a facultatively anaerobic, marine spirochaete.

A facultatively anaerobic, marine spirochaete, designated strain SIP1(T), was isolated from interstitial water from a cyanobacteria-containing microbial mat. Cells of strain SIP1(T) were 0.3-0.4x10-12 mum in size, helical with a body pitch of approximately 1.4 mum and motile by means of two to four periplasmic flagella (one, or occasionally two, being inserted near each end of the cell). Cells were catalase-negative and used a variety of monosaccharides and disaccharides and pectin as energy sources, growing especially well on cellobiose. Neither organic acids nor amino acids were utilized as energy sources. One or more amino acids in tryptone and one or more components of yeast extract were required for growth. Growth was observed at 9-37 degrees C (optimally at or near 37 degrees C), at initial pH 5-8 (optimally at initial pH 7.5) and in media prepared with 20-100 % (v/v) seawater (optimally at 60-80 %) or 0.10-1.00 M NaCl (optimally at 0.30-0.40 M). The products of cellobiose fermentation were acetate, ethanol, CO(2), H(2) and small amounts of formate. Aerated cultures performed incomplete oxidation of cellobiose to acetate (and, presumably, CO(2)) plus small amounts of ethanol and formate, but exhibited a Y(cellobiose) that was only slightly greater than that of cellobiose-fermenting anoxic cultures. The G+C content of the genomic DNA of strain SIP1(T) was 41.4 mol%, the lowest among known spirochaetas. On the basis of its 16S rRNA gene sequence, strain SIP1(T) was grouped among other members of the genus Spirochaeta, but it bore only 89 % similarity with respect to its closest known relatives, Spirochaeta litoralis and Spirochaeta isovalerica, two marine obligate anaerobes. On the basis of its phenotypic properties and phylogenetic position, strain SIP1(T) represents a novel species of the genus Spirochaeta, for which the name Spirochaeta cellobiosiphila sp. nov. is proposed. The type strain is SIP1(T) (=ATCC BAA-1285(T) =DSM 17781(T)).

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.

Physiological ecology of Stenoxybacter acetivorans, an obligate microaerophile in termite guts.

Stenoxybacter acetivorans is a newly described, obligately microaerophilic beta-proteobacterium that is abundant in the acetate-rich hindgut of Reticulitermes. Here we tested the hypotheses that cells are located in the hypoxic, peripheral region of Reticulitermes flavipes hindguts and use acetate to fuel their O(2)-consuming respiratory activity in situ. Physical fractionation of R. flavipes guts, followed by limited-cycle PCR with S. acetivorans-specific 16S rRNA gene primers, indicated that cells of this organism were indeed located primarily among the microbiota colonizing the hindgut wall. Likewise, reverse transcriptase PCR of hindgut RNA revealed S. acetivorans-specific transcripts for acetate-activating enzymes that were also found in cell extracts (acetate kinase and phosphotransacetylase), as well as transcripts of ccoN, which encodes the O(2)-reducing subunit of high-affinity cbb(3)-type cytochrome oxidases. However, S. acetivorans strains did not possess typical enzymes of the glyoxylate cycle (isocitrate lyase and malate synthase A), suggesting that they may use an alternate pathway to replenish tricarboxylic acid cycle intermediates or they obtain such compounds (or their precursors) in situ. Respirometric measurements indicated that much of the O(2) consumption by R. flavipes worker larvae was attributable to their guts, and the potential contribution of S. acetivorans to O(2) consumption by extracted guts was about 0.2%, a value similar to that obtained for other hindgut bacteria examined. Similar measurements obtained with guts of larvae prefed diets to disrupt major members of the hindgut microbiota implied that most of the O(2) consumption observed with extracted guts was attributable to protozoans, a group of microbes long thought to be "strict anaerobes."

Isolation and characterization of soil bacteria that define Terriglobus gen. nov., in the phylum Acidobacteria.

Bacteria in the phylum Acidobacteria are widely distributed and abundant in soils, but their ecological roles are poorly understood, owing in part to a paucity of cultured representatives. In a molecular survey of acidobacterial diversity at the Michigan State University Kellogg Biological Station Long-Term Ecological Research site, 27% of acidobacterial 16S rRNA gene clones in a never-tilled, successional plant community belonged to subdivision 1, whose relative abundance varied inversely with soil pH. Strains of subdivision 1 were isolated from these never-tilled soils using low-nutrient medium incubated for 3 to 4 weeks under elevated levels of carbon dioxide, which resulted in a slightly acidified medium that matched the pH optima of the strains (between 5 and 6). Colonies were approximately 1 mm in diameter and either white or pink, the latter due to a carotenoid(s) that was synthesized preferentially under 20% instead of 2% oxygen. Strains were gram-negative, aerobic, chemo-organotrophic, nonmotile rods that produced an extracellular matrix. All strains contained either one or two copies of the 16S rRNA encoding gene, which along with a relatively slow doubling time (10 to 15 h at ca. 23 degrees C) is suggestive of an oligotrophic lifestyle. Six of the strains are sufficiently similar to one another, but distinct from previously named Acidobacteria, to warrant creation of a new genus, Terriglobus, with Terriglobus roseus defined as the type species. The physiological and nutritional characteristics of Terriglobus are consistent with its potential widespread distribution in soil.

Folate cross-feeding supports symbiotic homoacetogenic spirochetes.

Treponema primitia, an H2-consuming CO2-reducing homoacetogenic spirochete in termite hindguts, requires an exogenous source of folate for growth. Tetrahydrofolate (THF) acts as a C1 carrier in CO2-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 mug/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.

New strategies for cultivation and detection of previously uncultured microbes.

An integrative approach was used to obtain pure cultures of previously uncultivated members of the divisions Acidobacteria and Verrucomicrobia from agricultural soil and from the guts of wood-feeding termites. Some elements of the cultivation procedure included the following: the use of agar media with little or no added nutrients; relatively long periods of incubation (more than 30 days); protection of cells from exogenous peroxides; and inclusion of humic acids or a humic acid analogue (anthraquinone disulfonate) and quorum-signaling compounds (acyl homoserine lactones) in growth media. The bacteria were incubated in the presence of air and in hypoxic (1 to 2% O(2) [vol/vol]) and anoxic atmospheres. Some bacteria were incubated with elevated concentrations of CO(2) (5% [vol/vol]). Significantly more Acidobacteria were found on isolation plates that had been incubated with 5% CO(2). A simple, high-throughput, PCR-based surveillance method (plate wash PCR) was developed. This method greatly facilitated detection and ultimate isolation of target bacteria from as many as 1,000 colonies of nontarget microbes growing on the same agar plates. Results illustrate the power of integrating culture methods with molecular techniques to isolate bacteria from phylogenetic groups underrepresented in culture.

Description of Treponema azotonutricium sp. nov. and Treponema primitia sp. nov., the first spirochetes isolated from termite guts.

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., H(2) plus CO(2) 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 micro m), by its nonhomoacetogenic fermentation of carbohydrates to acetate, ethanol, H(2), and CO(2), 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 H(2)-consuming, CO(2)-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.

Physiology and nutrition of Treponema primitia, an H2/CO2-acetogenic spirochete from termite hindguts.

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 mono- and 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.

Phylogenetic diversity and physiology of termite gut spirochetes.

The hindgut microbiota of termites includes an abundant and morphologically diverse population of spirochetes. However, our understanding of these symbionts has remained meager since their first observation in termite guts by Leidy over a century ago, in part because none had ever been isolated in culture. Recently, this situation has changed dramatically with the application of cultivation-independent molecular methods to determine their phylogeny, and with the isolation of the first pure cultures. The emerging picture is that earth's termites constitute an enormous reservoir of novel spirochetes, which possess metabolic properties (H(2)/CO(2)-acetogenesis and N(2) fixation) hitherto unrecognized in spirochetes and which contribute to the carbon, nitrogen and energy requirements of their termite host. These discoveries help to explain the enigmatic dominance of CO(2)-reductive acetogenesis over methanogenesis in the hindgut of many termites, as well as the old observation that elimination of spirochetes from the gut results in decreased termite survival.

THE EVOLUTIONARY SIGNIFICANCE OF BACTERIA ASSOCIATED WITH RHAGOLETIS.

All Rhagoletis reportedly establish associations with one or more bacterial species, but the bases for these interactions and their implications for host race formation and speciation are poorly understood. Here we present the results of four studies designed to increase our understanding of these relationships. In the first study, we identify the bacteria associated with seven Rhagoletis taxa by surveying the inhabitants of the esophageal bulb, an organ whose major function appears to be the housing of microorganisms. The results suggest that no bacterium has entered into an obligate symbiotic relationship with any of the Rhagoletis taxa surveyed, although one bacterium, Klebsiella oxytoca, is a very common associate of six of the seven. In the second study we use horizontal starch gel electrophoresis to determine the genetic similarity of K. oxytoca clones isolated from different Rhagoletis populations. This analysis provides a rare look into the genetic structure of natural populations of an enteric bacterium and permits the construction of a dendrogram for the clones-a dendrogram which indicates that there is no clear-cut pattern to the distribution of K. oxytoca genotypes among Rhagoletis. Taken together, the above studies provide indirect evidence that the bacteria associated with Rhagoletis are not important determinants of host plant specificity. The third and fourth studies assess two possible functions associated bacteria may perform for Rhagoletis: pectic substances degradation and nitrogen fixation. Our results do not lend support to either function.