Hydrogenase Gene Research Articles

Diversity and expression of cyanobacterial hupS genes in pure cultures and in a nitrogen-limited phototrophic biofilm

A PCR was developed for conserved regions within the cyanobacterial small subunit uptake hydrogenase (hupS) gene family. These primers were used to PCR amplify partial hupS sequences from 15 cyanobacterial strains. HupS clone libraries were constructed from PCR-amplified genomic DNA and reverse-transcribed mRNA extracted from phototrophic biofilms cultivated under nitrate-limiting conditions. Partial hupS gene sequences derived from cyanobacteria, some of which were not previously known to contain hup genes were used for phylogenetic analysis. Phylogenetic trees constructed with partial hupS genes were congruent with those based on 16S rRNA genes, indicating that hupS sequences can be used to identify cyanobacteria expressing hup. Sequences from heterocystous and nonheterocystous cyanobacteria formed two separate clusters. Analysis of clone library data showed a discrepancy between the presence and the activity of cyanobacterial hupS genes in phototrophic biofilms. The results showed that the hupS gene can be used to characterize the diversity of natural populations of diazotrophic cyanobacteria, and to characterize gene expression patterns of individual species and strains.

Sequencing and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis of Four Hydrogenase Gene Clusters from an Obligately Autotrophic Hydrogen-Oxidizing Bacterium, Hydrogenobacter thermophilus TK-6

A thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, fixes carbon dioxide via the reductive tricarboxylic acid cycle and grows rapidly with a generation time of about 1 h. TK-6 is believed to have an efficient hydrogen-oxidizing ability to support such rapid growth. We cloned hydrogenase genes from TK-6 and found that this strain has at least four clusters of hydrogenase genes. Reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that all four hydrogenase gene clusters were transcribed under aerobic condition at hydrogen concentrations of 45% and 60%. One of them was not transcribed at a hydrogen concentration of 20%. All the four hydrogenase gene clusters were expressed under anaerobic denitrifying condition at a hydrogen concentration of 75%.

Molecular monitoring of microbes in a continuous hydrogen-producing system with different hydraulic retention time

An anaerobic continuous-flow fermentation system with condensed molasses fermentation soluble (CMS) from monosodium glutamate production as substrate was developed for the biohydrogen production. The production rate of the system was increased, while hydraulic retention time (HRT) was varied from 12 to 3 h and the optimal hydrogen production rate was obtained at HRT 3 h. The major bacterial composition of each HRT was analyzed by 16S rRNA gene-targeted denaturing gradient gel electrophoresis (DGGE). Acidaminococcus sp. that could evolve hydrogen from glutamate was the only hydrogen producer and could be also detected from substrate. Hydrogenase mRNA-targeted reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that except HRT 3 h, two clusters of clostridial hydrogenase gene sequences were obtained. RT-PCR-DGGE analysis was employed to illustrate the active clostridial strains confirming that clostridial strains were not present at HRT 3 h. Acidaminococcus sp. existing in substrate might be the major strain that contributes to hydrogen production at HRT 3 h.

Hydrogenase‐ and outer membrane c‐type cytochrome‐facilitated reduction of technetium(VII) by Shewanella oneidensis MR‐1

Pertechnetate, (99)Tc(VII)O(4)(-), is a highly mobile radionuclide contaminant at US Department of Energy sites that can be enzymatically reduced by a range of anaerobic and facultatively anaerobic microorganisms, including Shewanella oneidensis MR-1, to poorly soluble Tc(IV)O(2(s)). In other microorganisms, Tc(VII)O(4)(-) reduction is generally considered to be catalysed by hydrogenase. Here, we provide evidence that although the NiFe hydrogenase of MR-1 was involved in the H(2)-driven reduction of Tc(VII)O(4)(-)[presumably through a direct coupling of H(2) oxidation and Tc(VII) reduction], the deletion of both hydrogenase genes did not completely eliminate the ability of MR-1 to reduce Tc(VII). With lactate as the electron donor, mutants lacking the outer membrane c-type cytochromes MtrC and OmcA or the proteins required for the maturation of c-type cytochromes were defective in reducing Tc(VII) to nanoparticulate TcO(2) x nH(2)O((s)) relative to MR-1 or a NiFe hydrogenase mutant. In addition, reduced MtrC and OmcA were oxidized by Tc(VII)O(4)(-), confirming the capacity for direct electron transfer from these OMCs to TcO(4)(-). c-Type cytochrome-catalysed Tc(VII) reduction could be a potentially important mechanism in environments where organic electron donor concentrations are sufficient to allow this reaction to dominate.

Transcription and Regulation of the Bidirectional Hydrogenase in the Cyanobacterium Nostoc sp. Strain PCC 7120

The filamentous, heterocystous cyanobacterium Nostoc sp. strain PCC 7120 (Anabaena sp. strain PCC 7120) possesses an uptake hydrogenase and a bidirectional enzyme, the latter being capable of catalyzing both H2 production and evolution. The completely sequenced genome of Nostoc sp. strain PCC 7120 reveals that the five structural genes encoding the bidirectional hydrogenase (hoxEFUYH) are separated in two clusters at a distance of approximately 8.8 kb. The transcription of the hox genes was examined under nitrogen-fixing conditions, and the results demonstrate that the cluster containing hoxE and hoxF can be transcribed as one polycistronic unit together with the open reading frame alr0750. The second cluster, containing hoxU, hoxY, and hoxH, is transcribed together with alr0763 and alr0765, located between the hox genes. Moreover, alr0760 and alr0761 form an additional larger operon. Nevertheless, Northern blot hybridizations revealed a rather complex transcription pattern in which the different hox genes are expressed differently. Transcriptional start points (TSPs) were identified 66 and 57 bp upstream from the start codon of alr0750 and hoxU, respectively. The transcriptions of the two clusters containing the hox genes are both induced under anaerobic conditions concomitantly with the induction of a higher level of hydrogenase activity. An additional TSP, within the annotated alr0760, 244 bp downstream from the suggested translation start codon, was identified. Electrophoretic mobility shift assays with purified LexA from Nostoc sp. strain PCC 7120 demonstrated specific interactions between the transcriptional regulator and both hox promoter regions. However, when LexA from Synechocystis sp. strain PCC 6803 was used, the purified protein interacted only with the promoter region of the alr0750-hoxE-hoxF operon. A search of the whole Nostoc sp. strain PCC 7120 genome demonstrated the presence of 216 putative LexA binding sites in total, including recA and recF. This indicates that, in addition to the bidirectional hydrogenase gene, a number of other genes, including open reading frames connected to DNA replication, recombination, and repair, may be part of the LexA regulatory network in Nostoc sp. strain PCC 7120.

The isolation and microbial community analysis of hydrogen producing bacteria from activated sludge

To profile the fractions of bacteria in heat-treated activated sludge capable of producing hydrogen and subsequently to isolate those organisms and confirm their ability to produce hydrogen. Profiling the community composition of the microflora in activated sludge using 16S rRNA gene-directed polymerase chain reaction-denaturing gradient gel electrophoresis suggested that a majority of bacteria were various Clostridium species. This was confirmed by clone library analysis, where 80% of the cloned inserts were Clostridium sp. A total of five isolates were established on solid media. Three of them, designated as W1, W4 and W5, harboured the hydrogenase gene as determined by PCR and DNA sequence analysis (99% similarity). These isolates were similar to Clostridium butyricum and Clostridium diolis as determined by 16S rRNA gene sequence. A maximum hydrogen production yield of 220 ml H(2) g(-1) glucose was achieved by W5, which was grown on improved mineral medium by batch fermentation without pH adjustment and nitrogen sparging during fermentation. Accumulation of malic acid and fumaric acid during hydrogen fermentation might lead to higher hydrogen yields for W4 and W5. W1 is the first reported Clostridium species that can tolerate microaerobic conditions for producing hydrogen. Clostridium species in heat-treated activated sludge were the most commonly identified bacteria responsible for hydrogen production. Specific genetic markers for strains W1, W4 and W5 would be of great utility in investigating hydrogen production at the molecular level. Two previously described primer sets targeting hydrogenase genes were shown not to be specific, amplifying other genes from nonhydrogen producers. Clostridium species isolated from heat-treated activated sludge were confirmed as hydrogen producers during dark hydrogen fermentation. The isolates will be useful for studying hydrogen production from wastewater, including the process of gene regulation and hydrogenase activity.

Flow-FISH analysis and isolation of clostridial strains in an anaerobic semi-solid bio-hydrogen producing system by hydrogenase gene target

By using hydrogenase gene-targeted polymerase chain reaction (PCR) and reverse transcriptase PCR (RT-PCR), the predominant clostridial hydrogenase that may have contributed to biohydrogen production in an anaerobic semi-solid fermentation system has been monitored. The results revealed that a Clostridium pasteurianum-like hydrogenase gene sequence can be detected by both PCR and RT-PCR and suggested that the bacterial strain possessing this specific hydrogenase gene was dominant in hydrogenase activity and population. Whereas another Clostridium saccharobutylicum-like hydrogenase gene can be detected only by RT-PCR and suggest that the bacterial strain possessing this specific hydrogenase gene may be less dominant in population. In this study, hydrogenase gene-targeted fluorescence in situ hybridization (FISH) and flow cytometry analysis confirmed that only 6.6% of the total eubacterial cells in a hydrogen-producing culture were detected to express the C. saccharobutylicum-like hydrogenase, whereas the eubacteria that expressed the C. pasteurianum-like hydrogenase was 25.6%. A clostridial strain M1 possessing the identical nucleotide sequences of the C. saccharobutylicum-like hydrogenase gene was then isolated and identified as Clostridium butyricum based on 16S rRNA sequence. Comparing to the original inoculum with mixed microflora, either using C. butyricum M1 as the only inoculum or co-culturing with a Bacillus thermoamylovorans isolate will guarantee an effective and even better production of hydrogen from brewery yeast waste.

The organization, regulation and phylogeny of uptake hydrogenase genes in Frankia

Frankia alni ACN14a, Frankia sp. CcI3 and Frankia sp. EAN1pec, which have different host specificity and geographical distribution, have two uptake hydrogenase syntons in their genome: hydrogenase synton#1 and hydrogenase synton#2. The organization of hydrogenase genes on these syntons also varies. Phylogenetic analysis of the structural genes of these syntons showed that they were significantly divergent and that hydrogenase synton#1 subunits of these Frankia strains were probably ancestral among the actinobacteria. Hydrogenase gene duplication might have occurred long before emergence of the three Frankia lineages. The structural subunits of hydrogenase HupS2 and HupL2 (synton#2) of F. alni ACN14a and Frankia sp. CcI3, which belong to phylogenetic Frankia cluster 1, were grouped closely together but away from Frankia sp. EAN1pec, which belongs to Frankia cluster 3. Phylogenetic analysis showed the occurrence of lateral transfer of hupL2 in Frankia sp. EAN1pec to or from Geobacter sulfurreducens. The transcript levels of hupS1 and hupL1 relative to hupS2 and hupL2 were higher in F. alni ACN14a grown under free‐living conditions. Under symbiotic conditions, transcript levels of hupS2 and hupL2 were higher than those of hupS1 and hupL1. Hydrogenase subunits of synton#1 are more expressed under free‐living conditions, whereas those of synton#2 are mainly involved in symbiotic interactions.

Application of gene-shuffling for the rapid generation of novel [FeFe]-hydrogenase libraries

A gene-shuffling technique was identified, optimized and used to generate diverse libraries of recombinant [FeFe]-hydrogenases. Six native [FeFe]-hydrogenase genes from species of Clostridia were first cloned and separately expressed in Escherichia coli concomitantly with the assembly proteins required for [FeFe]-hydrogenase maturation. All enzymes, with the exception of C. thermocellum HydA, exhibited significant activity when expressed. Single-stranded DNA fragments from genes encoding the two most active [FeFe]-hydrogenases were used to optimize a gene-shuffling protocol and generate recombinant enzyme libraries. Random sampling demonstrates that several shuffled products are active. This represents the first successful application of gene-shuffling using hydrogenases. Moreover, we demonstrate that a single set of [FeFe]-hydrogenase maturation proteins is sufficient for the heterologous assembly of the bioinorganic active site of several native and shuffled [FeFe]-hydrogenases.

High Photobiological Hydrogen Production Activity of a Nostoc sp. PCC 7422 Uptake Hydrogenase-Deficient Mutant with High Nitrogenase Activity

We describe a strategy to establish cyanobacterial strains with high levels of H(2) production that involves the identification of promising wild-type strains followed by optimization of the selected strains using genetic engineering. Nostoc sp. PCC 7422 was chosen from 12 other heterocystous strains, because it has the highest nitrogenase activity. We sequenced the uptake hydrogenase (Hup) gene cluster as well as the bidirectional hydrogenase gene cluster from the strain, and constructed a mutant (Delta hupL) by insertional disruption of the hupL gene. The Delta hupL mutant produced H(2) at 100 mumoles mg chlorophyll a (-1) h(-1), a rate three times that of the wild-type. The Delta hupL cells could accumulate H(2) to about 29% (v/v) accompanied by O(2) evolution in 6 days, under a starting gas phase of Ar + 5% CO(2). The presence of 20% O(2) in the initial gas phase inhibited H(2) accumulation of the Delta hupL cells by less than 20% until day 7.

Impact of alterations near the [NiFe] active site on the function of the H2 sensor from Ralstonia eutropha

In proteobacteria capable of H(2) oxidation under (micro)aerobic conditions, hydrogenase gene expression is often controlled in response to the availability of H(2). The H(2)-sensing signal transduction pathway consists of a heterodimeric regulatory [NiFe]-hydrogenase (RH), a histidine protein kinase and a response regulator. To gain insights into the signal transmission from the Ni-Fe active site in the RH to the histidine protein kinase, conserved amino acid residues in the L0 motif near the active site of the RH large subunit of Ralstonia eutropha H16 were exchanged. Replacement of the strictly conserved Glu13 (E13N, E13L) resulted in loss of the regulatory, H(2)-oxidizing and D(2)/H(+) exchange activities of the RH. According to EPR and FTIR analysis, these RH derivatives contained fully assembled [NiFe] active sites, and para-/ortho-H(2) conversion activity showed that these centres were still able to bind H(2). This indicates that H(2) binding at the active site is not sufficient for the regulatory function of H(2) sensors. Replacement of His15, a residue unique in RHs, by Asp restored the consensus of energy-linked [NiFe]-hydrogenases. The respective RH mutant protein showed only traces of H(2)-oxidizing activity, whereas its D(2)/H(+)-exchange activity and H(2)-sensing function were almost unaffected. H(2)-dependent signal transduction in this mutant was less sensitive to oxygen than in the wild-type strain. These results suggest that H(2) turnover is not crucial for H(2) sensing. It may even be detrimental for the function of the H(2) sensor under high O(2) concentrations.

Occurrence of Hydrogenases in Cyanobacteria and Anoxygenic Photosynthetic Bacteria: Implications for the Phylogenetic Origin of Cyanobacterial and Algal Hydrogenases

Hydrogenases are important enzymes in the energy metabolism of microorganisms. Therefore, they are widespread in prokaryotes. We analyzed the occurrence of hydrogenases in cyanobacteria and deduced a FeFe-hydrogenase in three different heliobacterial strains. This allowed the first phylogenetic analysis of the hydrogenases of all five major groups of photosynthetic bacteria (heliobacteria, green nonsulfur bacteria, green sulfur bacteria, photosynthetic proteobacteria, and cyanobacteria). In the case of both hydrogenases found in cyanobacteria (uptake and bidirectional), the green nonsulfur bacterium Chloroflexus aurantiacus was found to be the closest ancestor. Apart from a close relation between the archaebacterial and the green sulfur bacterial sulfhydrogenase, we could not find any evidence for horizontal gene transfer. Therefore, it would be most parsimonious if a Chloroflexus-like bacterium was the ancestor of Chloroflexus aurantiacus and cyanobacteria. After having transmitted both hydrogenase genes vertically to the different cyanobacterial species, either no, one, or both enzymes were lost, thus producing the current distribution. Our data and the available data from the literature on the occurrence of cyanobacterial hydrogenases show that the cyanobacterial uptake hydrogenase is strictly linked to the occurrence of the nitrogenase. Nevertheless, we did identify a nitrogen-fixing Synechococcus strain without an uptake hydrogenase. Since we could not find genes of a FeFe-hydrogenase in any of the tested cyanobacteria, although strains performing anoxygenic photosynthesis were also included in the analysis, a cyanobacterial origin of the contemporary FeFe-hydrogenase of algal plastids seems unlikely.

Regulation of Uptake Hydrogenase and Effects of Hydrogen Utilization on Gene Expression in Rhodopseudomonas palustris

Rhodopseudomonas palustris is a purple, facultatively phototrophic bacterium that uses hydrogen gas as an electron donor for carbon dioxide fixation during photoautotrophic growth or for ammonia synthesis during nitrogen fixation. It also uses hydrogen as an electron supplement to enable the complete assimilation of oxidized carbon compounds, such as malate, into cell material during photoheterotrophic growth. The R. palustris genome predicts a membrane-bound nickel-iron uptake hydrogenase and several regulatory proteins to control hydrogenase synthesis. There is also a novel sensor kinase gene (RPA0981) directly adjacent to the hydrogenase gene cluster. Here we show that the R. palustris regulatory sensor hydrogenase HupUV acts in conjunction with the sensor kinase-response regulator protein pair HoxJ-HoxA to activate hydrogenase expression in response to hydrogen gas. Transcriptome analysis indicated that the HupUV-HoxJA regulatory system also controls the expression of genes encoding a predicted dicarboxylic acid transport system, a putative formate transporter, and a glutamine synthetase. RPA0981 had a small effect in repressing hydrogenase synthesis. We also determined that the two-component system RegS-RegR repressed expression of the uptake hydrogenase, probably in response to changes in intracellular redox status. Transcriptome analysis indicated that about 30 genes were differentially expressed in R. palustris cells that utilized hydrogen when growing photoheterotrophically on malate under nitrogen-fixing conditions compared to a mutant strain that lacked uptake hydrogenase. From this it appears that the recycling of reductant in the form of hydrogen does not have extensive nonspecific effects on gene expression in R. palustris.

The rice field cyanobacteria Anabaena azotica and Anabaena sp. CH1 express vanadium-dependent nitrogenase

Anabaena azotica FACHB-118 and Anabaena sp. CH1, heterocystous cyanobacteria isolated from Chinese and Taiwanese rice fields, expressed vanadium-containing nitrogenase when under molybdenum deficiency. This is the second direct observation of an alternative nitrogenase in cyanobacteria. The vanadium nitrogenase-specific genes vnfDG are fused and clustered in a phylogenetic tree next to the corresponding genes of Methanosarcina. The expression of vnfH in cells cultured in Mo-free medium and of nifH in Mo-grown cells was shown for the first time by sequencing cDNA derived from cultures of A. azotica and Anabaena sp. CH1. The vnfH sequences clustered with that of Anabaena variabilis. The vnf genes were strongly transcribed only in cultures grown either in Mo-free medium, or in W-containing medium, but also weakly in Mo-containing medium. NifH was transcribed in all media. On-line measurements of acetylene reduction by Mo-free A. azotica cultures demonstrated that the V-nitrogenase was active. Ethane was formed continuously at a rate of 2.1% of that of ethylene. Acetylene reduction of cultures grown either with or without Mo had a high temperature optimum of 42.5 degrees C. The uptake hydrogenase gene hupL was expressed in Mo-free medium concomitantly with vnfDG in A. azotica, Anabaena sp. CH1, and A. variabilis.

Temporal expression of respiratory genes in an enrichment culture containing Dehalococcoides ethenogenes.

Multiple reductive dehalogenase (RDase), hydrogenase (H2ase), and other respiration-associated (RA) oxidoreductase genes have been identified in cultured representatives of Dehalococcoides. Although their products are likely to play key roles in the environmentally important process of reductive dechlorination, very little information is available about their regulation and specific functions. Here we show increased expression and temporal variability in the expression of five RDase genes and in the expression of genes for a putative formate dehydrogenase (Fdh) and two H2ases, including a periplasmic [Ni/Fe] H2ase (Hup) and a cytoplasmic [Fe] H2ase (Vhu). mRNA transcripts extracted from tetrachloroethene-dechlorinating mixed cultures corresponding to Fdh, the H2ase Hup, and the RDase targets TceA and DET0162 were expressed most highly, with average levels 34 (+/- 7.5)-, 23 (+/- 6.7)-, 16 (+/- 3.3)-, and 13 (+/- 3.3)-fold higher, respectively, than that for RNA polymerase (RpoB). H2ase and RA transcripts reached their respective expression maxima within the first 2 h after feeding. RDase transcripts, however, were most highly expressed after 3 h and exhibited greater temporal variability than other transcripts. Comparison with D. ethenogenes strain 195 pure culture expression levels indicated that RDase DET1545 was more highly expressed in mixed cultures, where, on average, its transcript level was sixfold higher than that of RpoB. While the specific functions of several of these gene products remain elusive, the high expression levels and temporal variability reported here suggest that these groups of enzymes are metabolically important for the respiration of chlorinated ethenes in mixed cultures containing Dehalococcoides.

Hydrogen production by using Rhodobacter capsulatus mutants with genetically modified electron transfer chains

In Rhodobacter capsulatus excess reducing equivalents generated by organic acid oxidation is consumed to reduce protons into hydrogen by the activity of nitrogenase. Nitrogenase serves as a redox-balancing tool and is activated by the RegB/RegA global regulatory system during photosynthetic growth. The terminal cytochrome cbb 3 oxidase and the redox state of the cyclic photosynthetic electron transfer chain serve redox signaling to the RegB/RegA regulatory systems in Rhodobacter. In this study, hydrogen production of various R. capsulatus strains harboring the genetically modified electron carrier cytochromes or lacking the cyt cbb 3 oxidase or the quinol oxidase were compared with the wild type. The results indicated that hydrogen production of mutant strains with modified electron carrier cytochromes decreased 3- to 4-fold, but the rate of hydrogen production increased significantly in a cbb 3 - mutant. Moreover, hydrogen production efficiency of various R. capsulatus strains further increased by inactivation of uptake hydrogenase genes.

Characterization of Fe-hydrogenase genes diversity and hydrogen-producing population in an acidophilic sludge

According to the DNA sequences of six Fe-hydrogenase genes (FHG) of Clostridium species retrieved from the GenBank, a set of primers specific for Fe-hydrogenase genes were identified from their common conserved regions. The length of DNA fragments amplified using these two primers averaged 313 bps. This primer set was then used to investigate the FHG diversity in an acidophilic rice-degrading sludge by methods based on polymerase chain reaction (PCR). Eight new Fe-hydrogenase gene fragments were identified from the sludge, as a result. Similarity based on amino acids among the 14 hydrogenase genes (8 newly found plus 6 known ones) was 39-97%, which is comparable to the similarity of 41-82% among the 6 known hydrogenase genes alone. The low similarity indicates a great diversity on Fe-hydrogenase among the Clostridium species. The primer set was then used to monitor the change of hydrogen-producing microbial population in a batch reaction using the technique of quantitative real-time polymerase chain reaction (qRT-PCR) with SYBR Green I as the fluorescent reagent. Results showed that the hydrogen producers had an average generation time of 4.2h, and a production rate of 7.0 x 10(16) H2-molecule cell(-1)h(-1).

Detection in soil of aerobic hydrogen-oxidizing bacteria related to Alcaligenes eutrophus by PCR and hybridization assays targeting the gene of the membrane-bound (NiFe) hydrogenase

The presence and abundance of the aerobic, chemolithoautotrophic Alcaligenes eutrophus, and related hydrogen-oxidizing bacteria, in soil samples were determined by using a hydrogenase gene probe and two different PCR primer sets that were derived from the alignment of (NiFe) hydrogenase gene sequences available in the EMBL data bank. The specificity of both the gene probe and the primer sets was tested on the genomic DNAs of 19 hydrogenase-containing, or hydrogenase-lacking, bacteria. The hydrogenase gene probe detected hydrogenase-containing Knallgas bacteria, N2-fixing bacteria, and enterobacteria, belonging to the alpha, beta, and gamma subclasses of the Proteobacteria. Hydrogenase-containing sulfate reducers (delta subclass) were not detected. One primer set was degenerated and resulted in the amplification of gene fragments of Knallgas bacteria and N2-fixing bacteria. The other primer set was specifically designed for the gene of the small subunit of the membrane-bound hydrogenase of A. eutrophus. In addition to the target organism, only Variovorax (Alcaligenes) paradoxus gave a gene product of the expected length. A. eutrophus-related bacteria were detected in soil by using two different strategies. The first strategy required cultivation and isolation of soil bacteria on complex media. Colonies were isolated from six different soils and tested by colony hybridization with the hydrogenase gene probe. Fifteen of the positive colonies were chosen for further characterization with the PCR primers. Two of the isolates gave amplification products with the A. eutrophus-specific PCR primers, which were sequenced and found to be 89–90% identical on a nucleotide level to the hydrogenase gene of A. eutrophus H16. The second strategy avoided a cultivation step. Total DNA was isolated from the rhizosphere of rice plants and tested in PCR amplification experiments with the A. eutrophus-specific PCR primers. Two different types of hydrogenase fragments were cloned and partially sequenced. The gene fragment sequences showed 73 and 75% identity to the A. eutrophus hydrogenase sequence. In quantitative PCR amplification assays, the number (about 2.6×107 g−1 fresh roots) of these two species of hydrogenase-containing bacteria was determined in the rice rhizosphere and was found to be similar to the number (about 7.4×106 g−1 fresh roots) of Knallgas bacteria that were determined by enumeration on selective media. Thus, it was apparently possible to detect and enumerate A. eutrophus-related bacteria in soil samples by molecular techniques.

The hupC gene product is a component of the electron transport system for hydrogen oxidation in Pseudomonas hydrogenovora.

The hydrogenase gene cluster containing nine genes (hupSLCDFGHIJ) was identified by sequencing of an 8.8-kb DNA region from Pseudomonas hydrogenovora. To investigate the function of the hupC gene product, we isolated a hupC-null mutant (HID3) of P. hydrogenovora by introducing an in-frame deletion into the hupC. The mutant, HID3, could not grow autotrophically but retained half the level of hydrogenase activity of the wild-type strain. Results of the oxygen consumption test and Western blot analysis revealed that the hupC gene product is a b-type cytochrome but not involved in the hydrogenase maturation process.

Anaerobic bacterial degradation for the effective utilization of biomass

Biomass is originally photosynthesized from inorgainic compounds such as CO2, minerals, water and solar energy. Recent studies have shown that anaerobic bacteria have the ability to convert recalcitrant biomass such as cellulosic or chitinoic materials to useful compounds. The biomass containing agricultural waste, unutilized wood and other garbage is expected to utilize as feed, food and fuel by microbial degradation and other metabolic functions. In this study we isolated several anaerobic, cellulolytic and chitinolytic bacteria from rumen fluid, compost and soil to study their related enzymes and genes. The anaerobic and cellulolytic bacteria,Clostridium thermocellum, Clostridium stercorarium, andClostridium josui, were isolated from compost and the chitinolyticClostridium paraputrificum from beach soil andRuminococcus albus was isolated from cow rumen. After isolation, novel cellulase and xylanase genes from these anaerobes were cloned and expressed inEscherichia coli. The properties of the cloned enzymes showed that some of them were the components of the enzyme (cellulase) complex,i.e., cellulosome which is known to form complexes by binding cohesin domains on the cellulase integrating protein (Cip: or core protein) and dockerin domains on the enzymes. Several dockerin and cohesin polypeptides were independently produced byE. coli and their binding properties were specified with BIAcore by measuring surface plasmon resonance. Three pairs of cohesin-dockerin with differing binding specificities were selected. Two of their genes encoding their respective cohesin polypeptides were combined to one gene and expressed inE. coli as a chimeric core protein, on which two dockerin-dehydrogenase chimeras, the dockerin-formaldehyde dehydrogenase and the dockerin-NADH dehydrogenase are planning to bind for catalyzing CO2 reduction to formic acid by feeding NADH. This reaction may represent a novel strategy for the reduction of the green house gases. Enzymes from the anaerobes were also expressed in tobacco and rice plants. The activity of a xylanase fromC. stercorarium was detected in leaves, stems, and rice grain under the control of CaMV35S promoter. The digestibility of transgenic rice leaves in goat rumen was slightly accelerated.C. paraputrificum was found to solubilize shrimp shells and chitin to generate hydrogen gas. Hydrogen productivity (1.7 mol H2/mol glucose) of the organism was improved up to 1.8 times by additional expression of the own hydrogenase gene inC. paraputrficum using a modified vector ofClostridium perfringens. The hydrogen producing microflora from soil, garbage and dried pelletted garbage, known as refuse derived fuel (RDF), were also found to be effective in converting biomass waste to hydrogen gas.