Patterns of [FeFe] hydrogenase diversity in the gut microbial communities of lignocellulose-feeding higher termites

Hydrogen is the central free intermediate in the degradation of wood by termite gut microbes and can reach concentrations exceeding those measured for any other biological system. Degenerate primers targeting the largest family of [FeFe] hydrogenases observed in a termite gut metagenome have been used to explore the evolution and representation of these enzymes in termites. Sequences were cloned from the guts of the higher termites Amitermes sp. strain Cost010, Amitermes sp. strain JT2, Gnathamitermes sp. strain JT5, Microcerotermes sp. strain Cost008, Nasutitermes sp. strain Cost003, and Rhyncotermes sp. strain Cost004. Each gut sample harbored a more rich and evenly distributed population of hydrogenase sequences than observed previously in the guts of lower termites and Cryptocercus punctulatus. This accentuates the physiological importance of hydrogen for higher termite gut ecosystems and may reflect an increased metabolic burden, or metabolic opportunity, created by a lack of gut protozoa. The sequences were phylogenetically distinct from previously sequenced [FeFe] hydrogenases. Phylogenetic and UniFrac comparisons revealed congruence between host phylogeny and hydrogenase sequence library clustering patterns. This may reflect the combined influences of the stable intimate relationship of gut microbes with their host and environmental alterations in the gut that have occurred over the course of termite evolution. These results accentuate the physiological importance of hydrogen to termite gut ecosystems.     

Genomic Analysis Reveals Multiple [FeFe] Hydrogenases and Hydrogen Sensors Encoded by Treponemes from the H<Subscript>2</Subscript>-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. 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 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.     

Genetic diversity and amplification of different clostridial [FeFe] hydrogenases by group-specific degenerate primers

Aims: The aim of this study was to explore and characterize the genetic diversity of [FeFe] hydrogenases in a representative set of strains from Clostridium sp. and to reveal the existence of neither yet detected nor characterized [FeFe] hydrogenases in hydrogen‐producing strains. Methods and Results: The genomes of 57 Clostridium strains (34 different genotypic species), representing six phylogenetic clusters based on their 16S rRNA sequence analysis (cluster I, III, XIa, XIb, XIV and XVIII), were screened for different [FeFe] hydrogenases. Based on the obtained alignments, ten pairs of [FeFe] hydrogenase cluster‐specific degenerate primers were newly designed. Ten Clostridium strains were screened by PCRs to assess the specificity of the primers designed and to examine the genetic diversity of [FeFe] hydrogenases. Using this approach, a diversity of hydrogenase genes was discovered in several species previously shown to produce hydrogen in bioreactors: Clostridium sartagoforme, Clostridium felsineum, Clostridium roseum and Clostridium pasteurianum. Conclusions: The newly designed [FeFe] hydrogenase cluster‐specific primers, targeting the cluster‐conserved regions, allow for a direct amplification of a specific hydrogenase gene from the species of interest. Significance and Impact of the Study: Using this strategy for a screening of different Clostridium ssp. will provide new insights into the diversity of hydrogenase genes and should be a first step to study a complex hydrogen metabolism of this genus.     

Morphology,Phylogeny, and Diversity of Trichonympha (Parabasalia: Hypermastigida) of the Wood‐Feeding Cockroach Cryptocercus punctulatus

ABSTRACT. Trichonympha is one of the most complex and visually striking of the hypermastigote parabasalids—a group of anaerobic flagellates found exclusively in hindguts of lower termites and the wood‐feeding cockroach Cryptocercus—but it is one of only two genera common to both groups of insects. We investigated Trichonympha of Cryptocercus using light and electron microscopy (scanning and transmission), as well as molecular phylogeny, to gain a better understanding of its morphology, diversity, and evolution. Microscopy reveals numerous new features, such as previously undetected bacterial surface symbionts, adhesion of post‐rostral flagella, and a distinctive frilled operculum. We also sequenced small subunit rRNA gene from manually isolated species, and carried out an environmental polymerase chain reaction (PCR) survey of Trichonympha diversity, all of which strongly supports monophyly of Trichonympha from Cryptocercus to the exclusion of those sampled from termites. Bayesian and distance methods support a relationship between Trichonympha species from termites and Cryptocercus, although likelihood analysis allies the latter with Eucomonymphidae. A monophyletic Trichonympha is of great interest because recent evidence supports a sister relationship between Cryptocercus and termites, suggesting Trichonympha predates the Cryptocercus‐termite divergence. The monophyly of symbiotic bacteria of Trichonympha raises the intriguing possibility of three‐way co‐speciation among bacteria, Trichonympha, and insect hosts.     

Origin of termite eusociality: trophallaxis integrates the social,nutritional, and microbial environments

1. Numerous cladistic analyses have converged: termites are a monophyletic clade embedded within the paraphyletic cockroaches, and sister group to the biparental, wood‐feeding cockroach Cryptocercus. The latter is, therefore, an appropriate model for testing assumptions regarding early termite evolution. 2. The ground plan of the termite ancestor is reviewed based on shared characters of ecology, life history, and behaviour in Cryptocercus and incipient termite colonies, and includes two levels of dependence: a reliance of all individuals on gut microbiota, and dependence of early instars on parental care. Both these conditions co‐evolved with parent‐to‐offspring proctodeal trophallaxis. 3. The termite ancestor lived in a single log serving as food and nest. This ‘one‐piece’ nesting ecology prioritises nitrogen conservation and strongly influences interacting social, nutritional, and microbial environments. Each of these environments individually and in combination profoundly affect cockroach development. 4. Proctodeal trophallaxis integrates the social, nutritional, and microbial environments. A change in trophallactic behaviour, from parental to alloparental, can, therefore, shift developmental trajectories, ultimately adding a third level of dependence. The death of gut protists during the host molting period and consequent interdependence of family members shifted the hierarchical level at which selection acted; fixation of eusociality quickly followed. 5. The basic nesting ecology did not change when termites evolved eusociality, the change occurred in the allocation and use of existing resources within the social group, driven by nitrogen scarcity, mediated by trophallaxis, and made possible by a strongly lineage‐specific set of life history characteristics.     

Iron reduction in the metal-rich guts of wood-feeding termites

Termites play important roles in lignocellulose and humus turnover in diverse terrestrial ecosystems, and are significant sources of global atmospheric methane and carbon dioxide. All known termite species engage in obligate, complex nutritional symbioses with their gut microbes to carry out such processes. Several hundred microbial species, representing a broad phylogenetic and physiological diversity, are found within the well‐bounded, microliter‐in‐scale gut ecosystem of a given termite. However, most of these species have never been obtained in laboratory culture, and little can be said about their functional roles in the gut community or symbiosis. Herein, an unappreciated facet of the gut chemistry and microbiology of wood‐feeding termites is revealed: the redox metabolism of iron. Gut fluids from field‐collected termites contained millimolar amounts of ferrous iron and other heavy metals. When iron(III) hydroxides were amended to a filter paper diet of Zootermopsis nevadensis, a dampwood termite collected in the San Gabriel Mountains of Southern California, the specimens accumulated high levels of iron(II) in their guts. Additionally, iron was reduced at rapid initial rates in anoxic gut homogenates prepared from field‐collected Z. nevadensis specimens. A Clostridium sp. and a Desulfovibrio sp. were isolated from dilution‐to‐extinction enrichments of Z. nevadensis gut contents and were found to reduce iron(III), as did the termite gut spirochete Treponema primitia. The iron in the guts of wood‐feeding termites may influence the pathways of carbon‐ and electron‐flow, as well as microbial community composition in these tiny ecosystems of global importance.     

Lignocellulose degradation by microorganisms from termite hills and termite guts: A survey on the present state of art

Abstract: In several aspects termites are a fascinating group of insects having attracted the interest of many researchers. They exhibit a complex social behavior and caste differentiation occurring elsewhere only among the hymenoptera. In an enlarged part of the hindgut, the paunch, termites have established a unique symbiotic association with prokaryotic and eukaryotic microorganisms. A similar flora is also found in wood-eating roaches of the genus Cryptocercus . The study of symbiosis between termites and their intestinal microbes is of general interest, because due to this symbiotic interaction termites can feed on complex biopolymers such as wood. Flagellates and bacteria occur in the gut of lower termites, while higher termites possess only bacteria. In particular spirochetes are abundant in the termite gut. Apart from spirochetes and other more common bacteria, actinomycetes, yeasts and fungi have also been isolated from different species of termites. This review summarizes the distinct role of the intestinal flora in degradation of wood components such as cellulose, hemicellulose and lignin.     

Hydrogen as an energy source for the human pathogen <Emphasis Type="Italic">Bilophila wadsworthia</Emphasis>

The gram-negative anaerobic gut bacterium Bilophila wadsworthia is the third most common isolate in perforated and gangrenous appendicitis, being also found in a variety of other infections. This organism performs a unique kind of anaerobic respiration in which taurine, a major organic solute in mammals, is used as a source of sulphite that serves as terminal acceptor for the electron transport chain. We show here that molecular hydrogen, one of the major products of fermentative bacteria in the colon, is an excellent growth substrate for B. wadsworthia. We have quantified the enzymatic activities associated with the oxidation of H₂, formate and pyruvate for cells obtained in different growth conditions. The cell extracts present high levels of hydrogenase activity, and up to five different hydrogenases can be expressed by this organism. One of the hydrogenases appears to be constitutive, whereas the others show differential expression in different growth conditions. Two of the hydrogenases are soluble and are recognised by antibodies against a [FeFe] hydrogenase of a sulphate reducing bacterium. One of these hydrogenases is specifically induced during fermentative growth on pyruvate. Another two hydrogenases are membrane-bound and show increased expression in cells grown with hydrogen. Further work should be carried out to reveal whether oxidation of hydrogen contributes to the virulence of B. wadsworthia.     

Influence of Feed Components on Symbiotic Bacterial Community Structure in the Gut of the Wood-Feeding Higher Termite Nasutitermes takasagoensis

The influence of carbon sources on bacterial community structure in the gut of the wood-feeding higher termite Nasutitermes takasagoensis was investigated. 16S rRNA gene sequencing and terminal-restriction fragment length polymorphism (T-RFLP) analyses revealed that the bacterial community structure changed markedly depending on feed components at the phylum level. Spirochaetes was predominant in the clone libraries from wood- and wood powder-fed termites, whereas Bacteroidetes was the largest group in the libraries from xylan-, cellobiose-, and glucose-fed termites, and Firmicutes was predominant in the library from xylose-fed termites. In addition, clones belonging to the phylum Termite Group I (TG1) were found in the library from xylose-fed termites. Our results indicate that the symbiotic relationship between termite and gut microorganisms is not very strong or stable over a short time, and that termite gut microbial community structures vary depending on components of the feeds.     

Gut-specific actinobacterial community structure and diversity associated with the wood-feeding termite species, Nasutitermes corniger (Motschulsky) described by nested PCR-DGGE analysis

This comprehensive survey studied the actinobacterial community structure and putative representative members associated with the gut of the wood-feeding termite, Nasutitermes corniger (Motschulsky), using nested PCR-DGGE and 16S rDNA sequences analyses. The closest relatives of the actinobacteria inhabiting the gut of Nasutitermes corniger were in five families, regardless of the geographical origin of the termite colony: Propionibacteriaceae, Streptomycetaceae, Cellulomonodaceae, Corynebacteriaceae and Rubrobacteraceae. Feeding termites on beech wood did not result in substantial changes in the actinobacterial community structure as revealed by DGGE banding patterns. Most of the 16S rDNA sequences obtained after excision and sequencing of DGGE bands clustered with those previously retrieved in termite guts. These results confirm the presence of gut-specific actinobacteria. Except for the 16S rDNA sequences affiliated to Streptomycetaceae and Cellulomonodaceae, no sequence had more than 97% similarity with the closest isolated strains, indicating the presence of microorganisms that have not yet been cultivated. These results suggest that members of the Actinomycetales order account for the largest proportion of the Actinobacteria phylum inhabiting the gut of the termite N. corniger.     

Characterization of a HoxEFUYH type of [NiFe] hydrogenase from <Emphasis Type="Italic">Allochromatium vinosum</Emphasis> and some EPR and IR properties of the hydrogenase module

A soluble hydrogenase from Allochromatium vinosum was purified. It consisted of a large (M _r = 52 kDa) and a small (M _r = 23 kDa) subunit. The genes encoding for both subunits were identified. They belong to an open reading frame where they are preceded by three more genes. A DNA fragment containing all five genes was cloned and sequenced. The deduced amino acid sequences of the products characterized the complex as a member of the HoxEFUYH type of [NiFe] hydrogenases. Detailed sequence analyses revealed binding sites for eight Fe–S clusters, three [2Fe–2S] clusters and five [4Fe–4S] clusters, six of which are also present in homologous subunits of [FeFe] hydrogenases and NADH:ubiquione oxidoreductases (complex I). This makes the HoxEFUYH type of hydrogenases the one that is evolutionary closest to complex I. The relative positions of six of the potential Fe–S clusters are predicted on the basis of the X-ray structures of the Clostridium pasteurianum [FeFe] hydrogenase I and the hydrophilic domain of complex I from Thermus thermophilus. Although the HoxF subunit contains binding sites for flavin mononucleotide and NAD(H), cell-free extracts of A. vinosum did not catalyse a H₂-dependent reduction of NAD⁺. Only the hydrogenase module (HoxYH) could be purified. Its electron paramagnetic resonance (EPR) and IR spectral properties showed the presence of a Ni–Fe active site and a [4Fe–4S] cluster. Its activity was sensitive to carbon monoxide. No EPR signals from a light-sensitive Ni_a–C* state could be observed. This study presents the first IR spectroscopic data on the HoxYH module of a HoxEFUYH type of [NiFe] hydrogenase.     

Characterization and phylogenomic analysis of Breznakiella homolactica gen. nov. sp. nov. indicate that termite gut treponemes evolved from non-acetogenic spirochetes in cockroaches

Spirochetes of the genus Treponema are surprisingly abundant in termite guts, where they play an important role in reductive acetogenesis. Although they occur in all termites investigated, their evolutionary origin is obscure. Here, we isolated the first representative of ‘termite gut treponemes’ from cockroaches, the closest relatives of termites. Phylogenomic analysis revealed that Breznakiella homolactica gen. nov. sp. nov. represents the most basal lineage of the highly diverse ‘termite cluster I', a deep-branching sister group of Treponemataceae (fam. ‘Termitinemataceae’) that was present already in the cockroach ancestor of termites and subsequently coevolved with its host. Breznakiella homolactica is obligately anaerobic and catalyses the homolactic fermentation of both hexoses and pentoses. Resting cells produced acetate in the presence of oxygen. Genome analysis revealed the presence of pyruvate oxidase and catalase, and a cryptic potential for the formation of acetate, ethanol, formate, CO₂ and H₂ - the fermentation products of termite gut isolates. Genes encoding key enzymes of reductive acetogenesis, however, are absent, confirming the hypothesis that the ancestral metabolism of the cluster was fermentative, and that the capacity for acetogenesis from H₂ plus CO₂ - the most intriguing property among termite gut treponemes - was acquired by lateral gene transfer.     

Effects of antibiotics on hydrogen production and gut symbionts in the Formosan subterranean termite Coptotermes formosanus (Isoptera: Rhinotermitidae)

Abstract Symbiotic microorganisms that inhabit the gut of Coptotermes formosanus enable this termite to degrade lignocelluloses and further produce hydrogen as an important intermediate to be recycled in its hindgut or as a byproduct to be emitted to the atmosphere. Both symbiotic protists and prokaryotes in the guts of termites demonstrated some different roles with respect to hydrogen production. In this study, the effects of two antibiotics, ampicillin and tetracycline, on hydrogen emission and the gut symbionts of C. formosanus were investigated. Hydrogen emission from termite guts was significantly enhanced when termites fed on wood diets treated with either ampicillin or tetracycline. The greatest H₂ emission rates, 2 519 ± 74 and 2 080 ± 377 nmol/h/g body weight, were recorded with the treatments of ampicillin and tetracycline, respectively, which showed 6–7 times more H₂ production than that of controls. Antibiotic‐treated diets negatively affected the prokaryotic communities and reduced their abundances, particularly on those ectosymbionts inhabiting the gut walls or in the gut fluid of C. formosanus, such as spirochetes. However, no significant reductions in the counts of gut cellulolytic protists, Pseudotrichonympha grassii and Holomastigotoids hartmanni, were recorded; and with a further observation by confocal laser scanning microscopy, the endosymbionts inhabiting P. grassii generally survived the antibiotic treatments. These results suggest that some prokaryotes may serve as the main hydrogen consumers, while P. grassii, together with its endosymbionts, may function as the main contributors for hydrogen production in the hindgut of C. formosanus.     

Synthetic biology for improved hydrogen production in Chlamydomonas reinhardtii

Hydrogen is a clean alternative to fossil fuels. It has applications for electricity generation and transportation and is used for the manufacturing of ammonia and steel. However, today, H₂ is almost exclusively produced from coal and natural gas. As such, methods to produce H₂ that do not use fossil fuels need to be developed and adopted. The biological manufacturing of H₂ may be one promising solution as this process is clean and renewable. Hydrogen is produced biologically via enzymes called hydrogenases. There are three classes of hydrogenases namely [FeFe], [NiFe] and [Fe] hydrogenases. The [FeFe] hydrogenase HydA1 from the model unicellular algae Chlamydomonas reinhardtii has been studied extensively and belongs to the A1 subclass of [FeFe] hydrogenases that have the highest turnover frequencies amongst hydrogenases (21,000 ± 12,000 H₂ s⁻¹ for CaHydA from Clostridium acetobutyliticum). Yet to date, limitations in C. reinhardtii H₂ production pathways have hampered commercial scale implementation, in part due to O₂ sensitivity of hydrogenases and competing metabolic pathways, resulting in low H₂ production efficiency. Here, we describe key processes in the biogenesis of HydA1 and H₂ production pathways in C. reinhardtii. We also summarize recent advancements of algal H₂ production using synthetic biology and describe valuable tools such as high-throughput screening (HTS) assays to accelerate the process of engineering algae for commercial biological H₂ production.     

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.     

The exchange activities of [Fe] hydrogenase (iron–sulfur-cluster-free hydrogenase) from methanogenic archaea in comparison with the exchange activities of [FeFe] and [NiFe] hydrogenases

[Fe] hydrogenase (iron–sulfur-cluster-free hydrogenase) catalyzes the reversible reduction of methenyltetrahydromethanopterin (methenyl-H₄MPT⁺) with H₂ to methylene-H₄MPT, a reaction involved in methanogenesis from H₂ and CO₂ in many methanogenic archaea. The enzyme harbors an iron-containing cofactor, in which a low-spin iron is complexed by a pyridone, two CO and a cysteine sulfur. [Fe] hydrogenase is thus similar to [NiFe] and [FeFe] hydrogenases, in which a low-spin iron carbonyl complex, albeit in a dinuclear metal center, is also involved in H₂ activation. Like the [NiFe] and [FeFe] hydrogenases, [Fe] hydrogenase catalyzes an active exchange of H₂ with protons of water; however, this activity is dependent on the presence of the hydride-accepting methenyl-H₄MPT⁺. In its absence the exchange activity is only 0.01% of that in its presence. The residual activity has been attributed to the presence of traces of methenyl-H₄MPT⁺ in the enzyme preparations, but it could also reflect a weak binding of H₂ to the iron in the absence of methenyl-H₄MPT⁺. To test this we reinvestigated the exchange activity with [Fe] hydrogenase reconstituted from apoprotein heterologously produced in Escherichia coli and highly purified iron-containing cofactor and found that in the absence of added methenyl-H₄MPT⁺ the exchange activity was below the detection limit of the tritium method employed (0.1 nmol min⁻¹ mg⁻¹). The finding reiterates that for H₂ activation by [Fe] hydrogenase the presence of the hydride-accepting methenyl-H₄MPT⁺ is essentially required. This differentiates [Fe] hydrogenase from [FeFe] and [NiFe] hydrogenases, which actively catalyze H₂/H₂O exchange in the absence of exogenous electron acceptors.     

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.The guts of wood-feeding termites and Cryptocercus punctulatus cockroaches share an unusual pattern of electron flow, as high rates of CO₂-reductive acetogenesis typically supplant methanogenesis as the terminal electron sink (2, 3). Past studies have shown that from 10 to 30% of gut acetate produced in environments of termites and wood-feeding cockroaches is microbially generated from CO₂ (3, 28), ultimately powering 18 to 26% of the host insect''s own respiratory energy metabolism (25). Nevertheless, most termites emit methane (2), and termite emissions constitute approximately 4% of the global methane budget (27). Cockroaches have been proposed to represent an additional source of note (9). Interestingly, methanogenic termites and cockroaches exhibit increased acetogenesis following addition of exogenous H₂ (3, 29). This suggests that these insects are host to a robust population of bacteria that are capable of homoacetogenesis but may be primarily using alternative electron donors (and other substrates and pathways) in vivo.Acetogenic bacteria belonging to two bacterial phyla, Firmicutes and Spirochaetes, have been isolated from the guts of termites (1, 4, 11, 12, 14). Several surveys have targeted and used the gene for formyltetrahydrofolate synthetase (FTHFS), a key gene in the Wood-Ljungdahl pathway of acetogenesis (16), as a potential marker for the pathway (15, 18). For the wood-feeding termites that have been examined, the studies have revealed an abundance of FTHFS sequences that form a coherent phylogenetic cluster, together with genes from homoacetogenic termite gut spirochetes belonging to the genus Treponema (24, 26, 30). This suggests that treponemes may be among the more abundant of the homoacetogens active in these environments.Little is known about the population structure and biology of CO₂-reducing, acetogenic bacteria in the guts of either omnivorous or wood-feeding cockroaches. The wood-feeding cockroach Cryptocercus hosts an abundance of flagellate protozoa closely related to those believed to dominate polysaccharide fermentation in the guts of termites (5, 6, 22), suggesting that at least one key environmental niche is filled by similar microbes in both termites and Cryptocercidae. Additionally, Cryptocercidae cockroaches, like termites, house diverse spirochetes and are the site of intense CO₂ reduction into acetate (3, 7). Possibly, spirochetes capable of CO₂ reduction into acetate are present in the hindguts of cockroaches. However, no evidence has yet been presented for the existence of homoacetogenic treponemes in environments other than the guts of termites, and FTHFS surveys of human (21) or horse (15) fecal matter and bovine rumen samples (20) revealed only Firmicutes-like and other FTHFS alleles that are distinct from those comprising the termite treponeme cluster.Here, by examining FTHFS gene diversity in Cryptocercus punctulatus and Periplaneta americana guts, we endeavored to learn more about the distribution and origins of homoacetogenic treponemes (and their genes) that are found in wood-feeding termites. In particular, we wished to ascertain whether FTHFS genes present in either of the two cockroaches are termite treponeme-like and, if so, whether analysis reveals any obvious signal indicating recent or ancient lateral community transfer events between insect lineages.     

Genomic and metagenomic surveys of hydrogenase distribution indicate H2 is a widely utilised energy source for microbial growth and survival

Recent physiological and ecological studies have challenged the long-held belief that microbial metabolism of molecular hydrogen (H₂) is a niche process. To gain a broader insight into the importance of microbial H₂ metabolism, we comprehensively surveyed the genomic and metagenomic distribution of hydrogenases, the reversible enzymes that catalyse the oxidation and evolution of H₂. The protein sequences of 3286 non-redundant putative hydrogenases were curated from publicly available databases. These metalloenzymes were classified into multiple groups based on (1) amino acid sequence phylogeny, (2) metal-binding motifs, (3) predicted genetic organisation and (4) reported biochemical characteristics. Four groups (22 subgroups) of [NiFe]-hydrogenase, three groups (6 subtypes) of [FeFe]-hydrogenases and a small group of [Fe]-hydrogenases were identified. We predict that this hydrogenase diversity supports H₂-based respiration, fermentation and carbon fixation processes in both oxic and anoxic environments, in addition to various H₂-sensing, electron-bifurcation and energy-conversion mechanisms. Hydrogenase-encoding genes were identified in 51 bacterial and archaeal phyla, suggesting strong pressure for both vertical and lateral acquisition. Furthermore, hydrogenase genes could be recovered from diverse terrestrial, aquatic and host-associated metagenomes in varying proportions, indicating a broad ecological distribution and utilisation. Oxygen content (pO₂) appears to be a central factor driving the phylum- and ecosystem-level distribution of these genes. In addition to compounding evidence that H₂ was the first electron donor for life, our analysis suggests that the great diversification of hydrogenases has enabled H₂ metabolism to sustain the growth or survival of microorganisms in a wide range of ecosystems to the present day. This work also provides a comprehensive expanded system for classifying hydrogenases and identifies new prospects for investigating H₂ metabolism.     

Electron transfer between hydrogenases and mono- and multiheme cytochromes in Desulfovibrio ssp

 A comparative study of electron transfer between the 16 heme high molecular mass cytochrome (Hmc) from Desulfovibrio vulgaris Hildenborough and the [Fe] and [NiFe] hydrogenases from the same organism was carried out, both in the presence and in the absence of catalytic amounts of cytochrome c ₃. For comparison, this study was repeated with the [NiFe] hydrogenase from D. gigas. Hmc is very slowly reduced by the [Fe] hydrogenase, but faster by either of the two [NiFe] hydrogenases. In the presence of cytochrome c ₃, in equimolar amounts to the hydrogenases, the rates of electron transfer are significantly increased and are similar for the three hydrogenases. The results obtained indicate that the reduction of Hmc by the [Fe] or [NiFe] hydrogenases is most likely mediated by cytochrome c ₃. A similar study with D. vulgaris Hildenborough cytochrome c ₅₅₃ shows that, in contrast, this cytochrome is reduced faster by the [Fe] hydrogenase than by the [NiFe] hydrogenases. However, although catalytic amounts of cytochrome c ₃ have no effect in the reduction by the [Fe] hydrogenase, it significantly increases the rate of reduction by the [NiFe] hydrogenases. Received: 14 April 1998 / Accepted: 25 June 1998