Hydrogenovibrio Marinus Research Articles

Improved production of biohydrogen in light-powered Escherichia coli by co-expression of proteorhodopsin and heterologous hydrogenase

BackgroundSolar energy is the ultimate energy source on the Earth. The conversion of solar energy into fuels and energy sources can be an ideal solution to address energy problems. The recent discovery of proteorhodopsin in uncultured marine γ-proteobacteria has made it possible to construct recombinant Escherichia coli with the function of light-driven proton pumps. Protons that translocate across membranes by proteorhodopsin generate a proton motive force for ATP synthesis by ATPase. Excess protons can also be substrates for hydrogen (H2) production by hydrogenase in the periplasmic space. In the present work, we investigated the effect of the co-expression of proteorhodopsin and hydrogenase on H2 production yield under light conditions.ResultsRecombinant E. coli BL21(DE3) co-expressing proteorhodopsin and [NiFe]-hydrogenase from Hydrogenovibrio marinus produced ~1.3-fold more H2 in the presence of exogenous retinal than in the absence of retinal under light conditions (70 μmole photon/(m2·s)). We also observed the synergistic effect of proteorhodopsin with endogenous retinal on H2 production (~1.3-fold more) with a dual plasmid system compared to the strain with a single plasmid for the sole expression of hydrogenase. The increase of light intensity from 70 to 130 μmole photon/(m2·s) led to an increase (~1.8-fold) in H2 production from 287.3 to 525.7 mL H2/L-culture in the culture of recombinant E. coli co-expressing hydrogenase and proteorhodopsin in conjunction with endogenous retinal. The conversion efficiency of light energy to H2 achieved in this study was ~3.4%.ConclusionHere, we report for the first time the potential application of proteorhodopsin for the production of biohydrogen, a promising alternative fuel. We showed that H2 production was enhanced by the co-expression of proteorhodopsin and [NiFe]-hydrogenase in recombinant E. coli BL21(DE3) in a light intensity-dependent manner. These results demonstrate that E. coli can be applied as light-powered cell factories for biohydrogen production by introducing proteorhodopsin.

Structural basis for a [4Fe-3S] cluster in the oxygen-tolerant membrane-bound [NiFe]-hydrogenase

Membrane-bound respiratory [NiFe]-hydrogenase (MBH), a H(2)-uptake enzyme found in the periplasmic space of bacteria, catalyses the oxidation of dihydrogen: H(2) → 2H(+) + 2e(-) (ref. 1). In contrast to the well-studied O(2)-sensitive [NiFe]-hydrogenases (referred to as the standard enzymes), MBH has an O(2)-tolerant H(2) oxidation activity; however, the mechanism of O(2) tolerance is unclear. Here we report the crystal structures of Hydrogenovibrio marinus MBH in three different redox conditions at resolutions between 1.18 and 1.32 Å. We find that the proximal iron-sulphur (Fe-S) cluster of MBH has a [4Fe-3S] structure coordinated by six cysteine residues--in contrast to the [4Fe-4S] cubane structure coordinated by four cysteine residues found in the proximal Fe-S cluster of the standard enzymes--and that an amide nitrogen of the polypeptide backbone is deprotonated and additionally coordinates the cluster when chemically oxidized, thus stabilizing the superoxidized state of the cluster. The structure of MBH is very similar to that of the O(2)-sensitive standard enzymes except for the proximal Fe-S cluster. Our results give a reasonable explanation why the O(2) tolerance of MBH is attributable to the unique proximal Fe-S cluster; we propose that the cluster is not only a component of the electron transfer for the catalytic cycle, but that it also donates two electrons and one proton crucial for the appropriate reduction of O(2) in preventing the formation of an unready, inactive state of the enzyme.

Production of biohydrogen by heterologous expression of oxygen-tolerant Hydrogenovibrio marinus [NiFe]-hydrogenase in Escherichia coli

Oxygen sensitivity of hydrogenase is a critical issue in efficient biological hydrogen production. In the present study, oxygen-tolerant [NiFe]-hydrogenase from the marine bacterium, Hydrogenovibrio marinus, was heterologously expressed in Escherichia coli, for the first time. Recombinant E. coli BL21 expressing H. marinus [NiFe]-hydrogenase actively produced hydrogen, but the parent strain did not. Recombinant H. marinus hydrogenase required both nickel and iron for biological activity. Compared to the recombinant E. coli [NiFe]-hydrogenase 1 described in our previous report, recombinant H. marinus [NiFe]-hydrogenase displayed 1.6- to 1.7-fold higher hydrogen production activity in vitro. Importantly, H. marinus [NiFe]-hydrogenase exhibited relatively good oxygen tolerance in analyses involving changes of surface aeration and oxygen proportion within a gas mixture. Specifically, recombinant H. marinus [NiFe]-hydrogenase produced ∼7- to 9-fold more hydrogen than did E. coli [NiFe]-hydrogenase 1 in a gaseous environment containing 5-10% (v/v) oxygen. In addition, purified H. marinus [NiFe]-hydrogenase displayed a hydrogen evolution activity of ∼28.8 nmol H₂/(minmg protein) under normal aerobic purification conditions. Based on these results, we suggest that oxygen-tolerant H. marinus [NiFe]-hydrogenase can be employed for in vivo and in vitro biohydrogen production without requirement for strictly anaerobic facilities.

Crystallization and preliminary X-ray diffraction analysis of membrane-bound respiratory [NiFe] hydrogenase fromHydrogenovibrio marinus

Membrane-bound respiratory [NiFe] hydrogenase is an H2-uptake enzyme found in the periplasmic space of bacteria that plays a crucial role in energy-conservation processes. The heterodimeric unit of the enzyme from Hydrogenovibrio marinus was purified to homogeneity using chromatographic procedures. Crystals were grown using the sitting-drop vapour-diffusion method at room temperature. Preliminary crystallographic analysis revealed that the crystals belonged to space group P2(1), with unit-cell parameters a=75.72, b=116.59, c=113.40 Å, β=91.3°, indicating that two heterodimers were present in the asymmetric unit.

Purification and characterization of a highly thermostable, oxygen-resistant, respiratory [NiFe]-hydrogenase from a marine, aerobic hydrogen-oxidizing bacterium Hydrogenovibrio marinus

The membrane-bound [NiFe]-hydrogenase from Hydrogenovibrio marinus ( HmMBH) was purified homogeneously under anaerobic conditions. Its molecular weight was estimated as 110 kDa, consisting of a heterodimeric structure of 66 kDa and 37 kDa subunits. The purified enzyme exhibited high activity in a wide temperature range: 185 U/mg at 30 °C and 615 U/mg at 85 °C (the optimum temperature). The K m and k cat/ K m values for H 2 were, respectively, 12 μM and 8.58 × 10 7 M −1 s −1. The optimum reaction pH was 7.8, but its stability was particularly high at pH 4.0–7.0. Results show that HmMBH was remarkably thermostable and oxygen-resistant: its half-life was 75 h at 80 °C under H 2, and more than 72 h at 4 °C under air. The air-oxidized HmMBH for 72 h showed only weak EPR signals of Ni–B, suggesting a structural feature in which the active center is not easily oxidized.

Universal degenerate oligonucleotide-primed-polymerase chain reaction for detection and amplification of NiFe-hydrogenase genes

Hydrogenase plays an important role in biological hydrogen production. Many researchers have paid attention to this enzyme in attempts to enhance the efficiency of biological hydrogen production. In contrast to the well-characterized Fe-hydrogenase, NiFe-hydrogenase, a heterodimeric metalloenzyme composed of two subunits, has not been examined in detail for biotechnological application. It has been reported that NiFe-hydrogenases have relatively higher tolerance to oxygen than Fe-hydrogenases and particularly, some enzymes are highly oxygen-tolerant. Because inhibition by oxygen is one of the serious problems in biological hydrogen production, the development of techniques for the effective detection of oxygen-tolerant NiFe-hydrogenase genes is important. In this study, we designed a universal degenerate primer pair based on highly conserved motifs at the N-terminus of the small subunit and C-terminus of the large subunit. We successfully demonstrated that the universal degenerate oligonucleotide-primed-polymerase chain reaction (DOP-PCR) amplified 2.9–3.0 kb NiFe-hydrogenase genes from various microorganisms. This technique was applied to detect and reveal unknown sequences from the large subunit of oxygen-tolerant NiFe-hydrogenase in Hydrogenovibrio marinus. Thus, this universal DOP-PCR procedure can be successfully employed to identify or amplify novel/known oxygen-tolerant NiFe-hydrogenase genes for engineering biological hydrogen production.

Diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes in the MgCl2-dominated deep hypersaline anoxic basin discovery

Partial sequences of the form I (cbbL) and form II (cbbM) of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) large subunit genes were obtained from the brine and interface of the MgCl2-dominated deep hypersaline anoxic basin Discovery. CbbL and cbbM genes were found in both brine and interface of the Discovery Basin but were absent in the overlying seawater. The diversity of both genes in the brine and interface was low, which might caused by the extreme saline conditions in Discovery of approximately 5 M MgCl2. None of the retrieved sequences were closely related to sequences deposited in the GenBank database. A phylogenetic analysis demonstrated that the cbbL sequences were affiliated with a Thiobacillus sp. or with one of the RuBisCO genes from Hydrogenovibrio marinus. The cbbM sequences clustered with thiobacilli or formed a new group with no close relatives. The results implicate that bacteria with the potential for carbon dioxide fixation and chemoautotrophy are present in the Discovery Basin. This is the first report demonstrating that RuBisCO genes are present under hypersaline conditions of 5 M MgCl2.

The role of two CbbRs in the transcriptional regulation of three ribulose-1,5-bisphosphate carboxylase/oxygenase genes in Hydrogenovibrio marinus strain MH-110

Hydrogenovibrio marinus MH-110 possesses three different sets of genes for ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO): two form I (cbbLS-1 and cbbLS-2) and one form II (cbbM). We have previously shown that the expression of these RubisCO genes is dependent on the ambient CO2 concentration. LysR-type transcriptional regulators, designated CbbR1 and CbbRm, are encoded upstream of the cbbLS-1 and cbbM genes, respectively. In this study, we revealed by gel shift assay that CbbR1 and CbbRm bind with higher affinity to the promoter regions of cbbLS-1 and cbbM, respectively, and with lower affinity to the other RubisCO gene promoters. The expression patterns of the three RubisCOs in the cbbR1 and the cbbRm gene mutants showed that CbbR1 and CbbRm were required to activate the expression of cbbLS-1 and cbbM, respectively, and that neither CbbR1 nor CbbRm was required for the expression of cbbLS-2. The expression of cbbLS-1 was significantly enhanced under high-CO2 conditions in the cbbRm mutant, in which the expression of cbbM was decreased. Although cbbLS-2 was not expressed under high-CO2 conditions in the wild-type strain or the single cbbR mutants, the expression of cbbLS-2 was observed in the cbbR1 cbbRm double mutant, in which the expression of both cbbLS-1 and cbbM was decreased. These results indicate that there is an interactive regulation among the three RubisCO genes.

CO2-responsive expression and gene organization of three ribulose-1,5-bisphosphate carboxylase/oxygenase enzymes and carboxysomes in Hydrogenovibrio marinus strain MH-110.

Hydrogenovibrio marinus strain MH-110, an obligately lithoautotrophic hydrogen-oxidizing bacterium, fixes CO2 by the Calvin-Benson-Bassham cycle. Strain MH-110 possesses three different sets of genes for ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO): CbbLS-1 and CbbLS-2, which belong to form I (L8S8), and CbbM, which belongs to form II (Lx). In this paper, we report that the genes for CbbLS-1 (cbbLS-1) and CbbM (cbbM) are both followed by the cbbQO genes and preceded by the cbbR genes encoding LysR-type regulators. In contrast, the gene for CbbLS-2 (cbbLS-2) is followed by genes encoding carboxysome shell peptides. We also characterized the three RubisCOs in vivo by examining their expression profiles in environments with different CO2 availabilities. Immunoblot analyses revealed that when strain MH-110 was cultivated in 15% CO2, only the form II RubisCO, CbbM, was expressed. When strain MH-110 was cultivated in 2% CO2, CbbLS-1 was expressed in addition to CbbM. In the 0.15% CO2 culture, the expression of CbbM decreased and that of CbbLS-1 disappeared, and CbbLS-2 was expressed. In the atmospheric CO2 concentration of approximately 0.03%, all three RubisCOs were expressed. Transcriptional analyses of mRNA by reverse transcription-PCR showed that the regulation was at the transcriptional level. Electron microscopic observation of MH-110 cells revealed the formation of carboxysomes in the 0.15% CO2 concentration. The results obtained here indicate that strain MH-110 adapts well to various CO2 concentrations by using different types of RubisCO enzymes.

Analysis of the molecular species of hydrogenase in the cells of an obligately chemolithoautotrophic, marine hydrogen-oxidizing bacterium, Hydrogenovibrio marinus.

Hydrogenovibrio marinus was suggested to have only membrane-bound hydrogenase (MBH). The change of cultivation pO2 did not affect the molecular species of hydrogenase expressed. We propose the MBH is grouped in class I [NiFe] MBH according to the subunit composition, size (Mw 38,000 and Mw 74,000 subunits) and N-terminal sequences of the subunits, and arrangement of the structural genes. Ni-requirement for the autotrophic growth on H2 also suggested the MBH is the Ni-containing type. Southern hybridization analysis using a part of the MBH gene showed a possibility of the presence of two highly homologous MBHs which were not separated by SDS-PAGE.

Isolation and Characterization of CO2-Fixing Hydrogen-Oxidizing Marine Bacteria.

A CO2-fixing bacterium, strain YN-1, that can fix CO2 under chemoautotrophic conditions but not photoautotrophic conditions was isolated from seawater. Identification of the isolate was carried out using biochemical tests and 16S rDNA sequence analysis, and its characteristics were investigated. From the results of partial 16S rDNA sequence analysis, strain YN-1 showed low identity with previously reported hydrogen-oxidizing bacteria, Hydrogenovibrio marinus MH-110 and Hydrogenophilus thermoluteolus. This result indicates that strain YN-1 may be a new hydrogen-oxidizing marine bacterium. Strain YN-1 showed considerable CO2 fixation ability during continuous cultivation even at high CO2 concentration. Strain YN-1 used H2 and CO2 as energy and carbon sources, respectively. Growth characteristics were examined in batch and continuous cultivation with a view to improving the CO2 fixation rate. The results showed that CO2 fixation occurred in the absence of a light source and that the strain exhibited good growth at high CO2 concentration (40%). On the other hand, the dry cell weight was 13.4 g/l following continuous cultivation for 76 h in 10% CO2 (0.1 l/min), and at that time the amount of fixed CO2 was 18.08 g CO2/l. This indicates that strain YN-1 can efficiently fix CO2 even at high CO2 concentrations, which would allow its application to the removal of industrially discharged CO2.

Isolation and characterization of CO 2-fixing hydrogen-oxidizing marine bacteria

A CO 2-fixing bacterium, strain YN-1, that can fix CO 2 under chemoautotrophic conditions but not photoautotrophic conditions was isolated from seawater. Identification of the isolate was carried out using biochemical tests and 16S rDNA sequence analysis, and its characteristics were investigated. From the results of partial 16S rDNA sequence analysis, strain YN-1 showed low identity with previously reported hydrogen-oxidizing bacteria, Hydrogenovibrio marinus MH-110 and Hydrogenophilus thermoluteolus. This result indicates that strain YN-1 may be a new hydrogen-oxidizing marine bacterium. Strain YN-1 showed considerable CO 2 fixation ability during continuous cultivation even at high CO 2 concentration. Strain YN-1 used H 2 and CO 2 as energy and carbon sources, respectively. Growth characteristics were examined in batch and continuous cultivation with a view to improving the CO 2 fixation rate. The results showed that CO 2 fixation occurred in the absence of a light source and that the strain exhibited good growth at high CO 2 concentration (40%). On the other hand, the dry cell weight was 13.4 g/ l following continuous cultivation for 76 h in 10% CO 2 (0.1 l/min), and at that time the amount of fixed CO 2 was 18.08 g CO 2/ l. This indicates that strain YN-1 can efficiently fix CO 2 even at high CO 2 concentrations, which would allow its application to the removal of industrially discharged CO 2.

The cbbQ Genes, Located Downstream of the Form I and Form II RubisCO Genes, Affect the Activity of Both RubisCOs

Hydrogenovibrio marinus strain MH-110, an obligately lithoautotrophic hydrogen-oxidizing bacterium, possesses three sets of the genes for ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO): namely, two form I type (cbbLS-1 and cbbLS-2) and one form II type (cbbM) enzymes. The cbbQ-m gene was located downstream of cbbM, and showed high similarity to other cbbQ genes and nirQ/norQ genes in denitrifying bacteria. Phylogenetic analysis of CbbQ and NirQ/NorQ indicated that CbbQ-m from Hv. marinus closely resembled CbbQ from Thiobacillus intermedius and Thiobacillus neapolitannus and less closely resembled NirQ and NorQ. The cbbQ-m gene has been shown to activate the form II RubisCO in E. coli cells, and the cbbQ-t from Hydrogenophilus thermoluteolus could also activate the form II RubisCO. Both cbbQ genes have also been shown to activate the form I RubisCO from Hp. thermoluteolus in E. coli cells. However, the activation levels of two form I RubisCOs from Hv. marinus were smaller than that of form I RubisCOs from Hp. thermoluteolus. Form II RubisCO activated by CbbQ-m (QM) was purified from E. coli cells. The result of the 8-anilino-1-naphthalenesulfonate binding assay and the circular dichroism spectra indicated that QM was conformationally different from Form II RubisCO that was not activated by CbbQ.

Distribution and diversity of sulfur-oxidizing Thiomicrospira spp. at a shallow-water hydrothermal vent in the Aegean Sea (Milos, Greece).

A shallow-water hydrothermal vent system in the Aegean Sea close to the island of Milos (Greece) was chosen to study the diversity and distribution of the chemolithoautotrophic sulfur-oxidizing bacterium Thiomicrospira. Cell numbers in samples from different regions around a solitary vent decreased toward the center of the vent (horizontal distribution), as well as with depth (vertical distribution), corresponding to an increase in temperature (from ca. 25 to 60 degrees C) and a decrease in pH (from ca. pH 7 to 5). Thiomicrospira was one of the most abundant culturable sulfur oxidizers and was even dominant in one region. Phylogenetic analysis of Thiomicrospira spp. present in the highest most-probable-number (MPN) dilutions revealed that most of the obtained sequences grouped in two new closely related clusters within the Thiomicrospira branch. Two different new isolates, i.e., Milos-T1 and Milos-T2, were obtained from high-dilution (10(-5)) enrichments. Phylogenetic analysis indicated that isolate Milos-T1 is related to the recently described Thiomicrospira kuenenii and Hydrogenovibrio marinus, whereas isolate Milos-T2 grouped with the MPN sequences of cluster 2. The predominance of strain Milos-T2 was indicated by its identification in several environmental samples by hybridization analysis of denaturing gradient gel electrophoresis (DGGE) patterns and by sequencing of one of the corresponding bands, i.e., ML-1, from the DGGE gel. The results shown in this paper support earlier indications that Thiomicrospira species are important members of hydrothermal vent communities.

Phylogenetic position of an obligately chemoautotrophic, marine hydrogen-oxidizing bacterium, Hydrogenovibrio marinus, on the basis of 16S rRNA gene sequences and two form I RuBisCO gene sequences.

Two form ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes from the obligately autotrophic, marine hydrogen oxidizer Hydrogenovibrio marinus were sequenced. The deduced amino acid sequences of both RuBisCOs revealed that they are similar to those of sulfur oxidizers (Thiobacillus) and a purple sulfur bacterium (Chromatium vinosum). According to the 16S rRNA gene sequences, H. marinus is also affiliated with these microorganisms, members of Thiomicrospira being the closest relatives. Sequence similarities of the 16S rRNA genes and of the RuBisCO genes among these gamma-Proteobacteria suggest a common autotrophic ancestry. An ancestor of purple sulfur bacteria might be a common root of H. marinus and related sulfur oxidizers.

Different properties of gene products of three sets ribulose 1,5-bisphosphate carboxylase/oxygenase from a marine obligately autotrophic hydrogen-oxidizing bacterium, Hydrogenovibrio marinus strain MH-110

Hydrogenovibrio marinus strain MH-110 is an obligately lithoautotrophic hydrogen-oxidizing bacterium, possessing three sets of the genes for ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO); one form II type (L x) and two form I type (L 8S 8) enzymes. The genes for the form I type enzymes are named cbbLS-1 and cbbLS-2, and that for the form II type enzyme is named cbbM. These three sets of genes were cloned in plasmid vectors, and the expression of the genes in E. coli cells were studied. The three RubisCOs were purified through the same steps, and their specificity factors (τ values) were measured. The τ values of CbbLS-1 and CbbLS-2 were higher than that of CbbM, but lower than those of other form I RubisCOs. The specific activity of CbbM was very low and CbbM was inactivated easily during the process of purification. Immunochemical analyses revealed that the antibody against form I type reacted only with CbbL-1 and CbbL-2, and the antibody against form II type reacted only with CbbM. The antibody to neither form of RubisCO demonstrated cross-reactivity for the other form.

Cloning and sequence of the L2 form of RubisCO from a marine obligately autotrophic hydrogen-oxidizing bacterium, Hydrogenovibrio marinus strain MH-110.

A form II ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) gene was isolated and sequenced from a marine hydrogen-oxidizing bacterium, Hydrogenovibrio marinus strain MH-110. To our knowledge, this is the first report for the sequence of a form II (L2-form) RubisCO gene derived from a chemolithoautotrophic bacterium. The form II RubisCO gene coded for 463 amino acid residues (1389 bp, M(r) = 50,655). The deduced amino acid sequence had high homology with those of the L2-form RubisCO of Rhodospirillum rubrum (63%) and the L4-form RubisCO from Rhodobacter sphaeroides (62%), but low similarity to other L8S8 form RubisCOs (under 40%).

Purification of form L2 RubisCO from a marine obligately autotrophic hydrogen-oxidizing bacterium, Hydrogenovibrio marinus strain MH-110

Purification of form L2 RubisCO from a marine obligately autotrophic hydrogen-oxidizing bacterium, Hydrogenovibrio marinus strain MH-110

Purification of form L2RubisCO from a marine obligately autotrophic hydrogen-oxidizing bacterium,Hydrogenovibrio marinusstrain MH-110

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) was purified from an obligately autotrophic hydrogen-oxidizing bacterium, Hydrogenovibrio marinus MH-110. The protein has a M(r) value of approximately 110,000, and is composed of two identical subunits of 55,000. To our knowledge, the existence of L2-form RubisCO in a chemolithoautotrophic bacterium is first reported in this paper. The N-terminal amino acid sequence determination of the purified enzyme showed high homology with those of the L2-form RubisCO of Rhodospirillum rubrum and the Lx-form RubisCO from Rhodobacter sphaeroides.

Production and properties of glycogen in the marine obligate chemolithoautotroph, Hydrogenovibrio marinus

Production of an intracellular polysaccharide was observed during cultivation of a marine, obligately chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenovibrio marinus, under nitrogen or magnesium starvation or oxygen limitation in a continuous gas supply. The polysaccharide formed in oxygen-limited cells was isolated and shown to be composed of only glucose. Instrumental and enzymatic analyses revealed that the polysaccharide has a glycogen-like structure.