Analysis of Extensive [FeFe] Hydrogenase Gene Diversity Within the Gut Microbiota of Insects Representing Five Families of Dictyoptera

We have designed and utilized degenerate primers in the phylogenetic analysis of [FeFe] hydrogenase gene diversity in the gut ecosystems of roaches and lower termites. H2 is an important free intermediate in the breakdown of wood by termite gut microbial communities, reaching concentrations in some species exceeding those measured for any other biological system. The primers designed target with specificity the largest group of enzymatic H domain proteins previously identified in a termite gut metagenome. “Family 3” hydrogenase sequences were amplified from the guts of lower termites, Incisitermes minor, Zootermopsis nevadensis, and Reticulitermes hesperus, and two roaches, Cryptocercus punctulatus and Periplaneta americana. Subsequent analyses revealed that all termite and Cryptocercus sequences were phylogenetically distinct from non-termiteassociated hydrogenases available from public databases. The abundance of unique sequence operational taxonomic units (as many as 21 from each species) underscores the previously demonstrated physiological importance of H2 to the gut ecosystems of these wood-feeding insects. The diversity of sequences observed might be reflective of multiple niches that the enzymes have been evolved to accommodate. Sequences cloned from Cryptocercus and the lower termite samples, all of which are wood feeding insects, clustered closely with one another in phylogenetic analyses to the exclusion of alleles from P. americana, an omnivorous cockroach, also cloned during this study. We present primers targeting a family of termite gut [FeFe] hydrogenases and provide results that are consistent with a pivotal role for hydrogen in the termite gut ecosystem and point toward unique evolutionary adaptations to the gut ecosystem

Diversity of Formyltetrahydrofolate Synthetases in the Guts of the Wood-Feeding Cockroach Cryptocercus punctulatus and the Omnivorous Cockroach Periplaneta americana

We examined the diversity of a marker gene for homoacetogens in two cockroach gut microbial communities. Formyltetrahydrofolate synthetase (FTHFS or fhs) libraries prepared from a wood-feeding cockroach, Cryptocercus punctulatus, were dominated by sequences that affiliated with termite gut treponemes. No spirochete-like sequences were recovered from the omnivorous roach Periplaneta americana, which was dominated by Firmicutes-like sequences

Genes for selenium dependent and independent formate dehydrogenase in the gut microbial communities of three lower, wood-feeding termites and a wood-feeding roach

The bacterial Wood-Ljungdahl pathway for CO_2-reductive acetogenesis is important for the nutritional mutualism occurring between wood-feeding insects and their hindgut microbiota. A key step in this pathway is the reduction of CO_2 to formate, catalysed by the enzyme formate dehydrogenase (FDH). Putative selenocysteine- (Sec) and cysteine- (Cys) containing paralogues of hydrogenase-linked FDH (FDH_H) have been identified in the termite gut acetogenic spirochete, Treponema primitia, but knowledge of their relevance in the termite gut environment remains limited. In this study, we designed degenerate PCR primers for FDH_H genes (fdhF) and assessed fdhF diversity in insect gut bacterial isolates and the gut microbial communities of termites and cockroaches. The insects examined herein represent three wood-feeding termite families, Termopsidae, Kalotermitidae and Rhinotermitidae (phylogenetically 'lower' termite taxa); the wood-feeding roach family Cryptocercidae (the sister taxon to termites); and the omnivorous roach family Blattidae. Sec and Cys FDH_H variants were identified in every wood-feeding insect but not the omnivorous roach. Of 68 novel alleles obtained from inventories, 66 affiliated phylogenetically with enzymes from T. primitia. These formed two subclades (37 and 29 phylotypes) almost completely comprised of Sec-containing and Cys-containing enzymes respectively. A gut cDNA inventory showed transcription of both variants in the termite Zootermopsis nevadensis (family Termopsidae). The gene patterns suggest that FDH_H enzymes are important for the CO_2-reductive metabolism of uncultured acetogenic treponemes and imply that the availability of selenium, a trace element, shaped microbial gene content in the last common ancestor of dictyopteran, wood-feeding insects, and continues to shape it to this day

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., 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

Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones

LuxR-type transcriptional regulators play key roles in quorum-sensing systems that employ acyl-homoserine lactones (acyl-HSLs) as signal molecules. These proteins mediate quorum control by changing their interactions with RNA polymerase and DNA in response to binding their cognate acyl-HSL. The evolutionarily related LuxR-type proteins exhibit considerable diversity in primary sequence and in their response to acyl-HSLs having acyl groups of differing length and composition. Little is known about which residues determine acyl-HSL specificity, and less about the evolutionary time scales required to forge new ones. To begin to examine such issues, we have focused on the LuxR protein from Vibrio fischeri, which activates gene transcription in response to binding its cognate quorum signal, 3-oxohexanoyl-homoserine lactone (3OC6HSL). Libraries of luxR mutants were screened for variants exhibiting increased gene activation in response to octanoyl-HSL (C8HSL), with which wild-type LuxR interacts only weakly. Eight LuxR variants were identified that showed a 100-fold increase in sensitivity to C8HSL; these variants also displayed increased sensitivities to pentanoyl-HSL and tetradecanoyl-HSL, while maintaining a wild-type or greater response to 3OC6HSL. The most sensitive variants activated gene transcription as strongly with C8HSL as the wild type did with 3OC6HSL. With one exception, the amino acid residues involved were restricted to the N-terminal, 'signal-binding' domain of LuxR. These residue positions differed from critical positions previously identified via 'loss-of-function' mutagenesis. We have demonstrated that acyl-HSL-dependent quorum-sensing systems can evolve rapidly to respond to new acyl-HSLs, suggesting that there may be an evolutionary advantage to maintaining such plasticity

Bacterial chemolithoautotrophy via manganese oxidation

Manganese is one of the most abundant elements on Earth. The oxidation of manganese has long been theorized—yet has not been demonstrated—to fuel the growth of chemolithoautotrophic microorganisms. Here we refine an enrichment culture that exhibits exponential growth dependent on Mn(II) oxidation to a co-culture of two microbial species. Oxidation required viable bacteria at permissive temperatures, which resulted in the generation of small nodules of manganese oxide with which the cells associated. The majority member of the culture—which we designate ‘Candidatus Manganitrophus noduliformans’—is affiliated to the phylum Nitrospirae (also known as Nitrospirota), but is distantly related to known species of Nitrospira and Leptospirillum. We isolated the minority member, a betaproteobacterium that does not oxidize Mn(II) alone, and designate it Ramlibacter lithotrophicus. Stable-isotope probing revealed ¹³CO₂ fixation into cellular biomass that was dependent upon Mn(II) oxidation. Transcriptomic analysis revealed candidate pathways for coupling extracellular manganese oxidation to aerobic energy conservation and autotrophic CO₂ fixation. These findings expand the known diversity of inorganic metabolisms that support life, and complete a biogeochemical energy cycle for manganese that may interface with other major global elemental cycles

Bacterial chemolithoautotrophy via manganese oxidation

Manganese is one of the most abundant elements on Earth. The oxidation of manganese has long been theorized—yet has not been demonstrated—to fuel the growth of chemolithoautotrophic microorganisms. Here we refine an enrichment culture that exhibits exponential growth dependent on Mn(II) oxidation to a co-culture of two microbial species. Oxidation required viable bacteria at permissive temperatures, which resulted in the generation of small nodules of manganese oxide with which the cells associated. The majority member of the culture—which we designate ‘Candidatus Manganitrophus noduliformans’—is affiliated to the phylum Nitrospirae (also known as Nitrospirota), but is distantly related to known species of Nitrospira and Leptospirillum. We isolated the minority member, a betaproteobacterium that does not oxidize Mn(II) alone, and designate it Ramlibacter lithotrophicus. Stable-isotope probing revealed ¹³CO₂ fixation into cellular biomass that was dependent upon Mn(II) oxidation. Transcriptomic analysis revealed candidate pathways for coupling extracellular manganese oxidation to aerobic energy conservation and autotrophic CO₂ fixation. These findings expand the known diversity of inorganic metabolisms that support life, and complete a biogeochemical energy cycle for manganese that may interface with other major global elemental cycles

Identification of QuiP, the Product of Gene PA1032, as the Second Acyl-Homoserine Lactone Acylase of Pseudomonas aeruginosa PAO1

The relevance of the acyl homoserine lactone (acyl-HSL) quorum signals N-3-oxododecanoyl-homoserine lactone (3OC12HSL) and N-butanoyl-homoserine lactone to the biology and virulence of Pseudomonas aeruginosa is well investigated. Previously, P. aeruginosa was shown to degrade long-chain, but not short-chain, acyl-HSLs as sole carbon and energy sources (J. J. Huang, J.-I. Han, L.-H. Zhang, and J. R. Leadbetter, Appl. Environ. Microbiol. 69:5941-5949, 2003). A gene encoding an enzyme with acyl-HSL acylase activity, pvdQ (PA2385), was identified, but it was not required for acyl-HSL utilization. This indicated that P. aeruginosa encodes another acyl-HSL acylase, which we identify here. A comparison of total cell proteins of cultures grown with long-acyl acyl-HSLs versus other substrates implicated the involvement of a homolog of PvdQ, the product of gene PA1032, for which we propose the name QuiP. Transposon mutants of quiP were defective for growth when P. aeruginosa was cultured in medium containing decanoyl-HSL as a sole carbon and energy source. Complementation with a functional copy of quiP rescued this growth defect. When P. aeruginosa was grown in buffered lysogeny broth, constitutive expression of QuiP in P. aeruginosa led to decreased accumulations of the quorum signal 3OC12HSL, relative to the wild type. Heterologous expression of QuiP was sufficient to confer long-chain acyl-HSL acylase activity upon Escherichia coli. Examination of gene expression patterns during acyl-HSL-dependent growth of P. aeruginosa further supported the involvement of quiP in signal decay and revealed other genes also possibly involved. It is not yet known under which "natural" conditions quiP is expressed or how P. aeruginosa balances the expression of its quorum-sensing systems with the expression of its acyl-HSL acylase activities

Genomic Analysis Reveals Multiple [FeFe] Hydrogenases and Hydrogen Sensors Encoded by Treponemes from the H_2-Rich Termite Gut

We have completed a bioinformatic analysis of the hydrogenases encoded in the genomes of three termite gut treponeme isolates: hydrogenotrophic, homoacetogenic Treponema primitia strains ZAS-1 and ZAS-2, and the hydrogen-producing, sugar-fermenting Treponema azotonutricium ZAS-9. H_2 is an important free intermediate in the breakdown of wood by termite gut microbial communities, reaching concentrations in some species exceeding those measured for any other biological system. The spirochetes encoded 4, 8, and 5 [FeFe] hydrogenase-like proteins, identified by their H domains, respectively, but no other recognizable hydrogenases. The [FeFe] hydrogenases represented many sequence families previously proposed in an analysis of termite gut metagenomic data. Each strain encoded both putative [FeFe] hydrogenase enzymes and evolutionarily related hydrogen sensor/transducer proteins likely involved in phosphorelay or methylation pathways, and possibly even chemotaxis. A new family of [FeFe] hydrogenases (FDH-Linked) is proposed that may form a multimeric complex with formate dehydrogenase to provide reducing equivalents for reductive acetogenesis in T. primitia. The many and diverse [FeFe] hydrogenase-like proteins encoded within the sequenced genomes of the termite gut treponemes has enabled the discovery of a putative new class of [FeFe] hydrogenase proteins potentially involved in acetogenesis and furthered present understanding of many families, including sensory, of H domain proteins beyond what was possible through the use of fragmentary termite gut metagenome sequence data alone, from which they were initially defined