Genetic disruption of both Chlamydomonas reinhardtii [FeFe]-hydrogenases: Insight into the role of HYDA2 in H₂ production

Chlamydomonas reinhardtii (Chlamydomonas throughout) encodes two [FeFe]-hydrogenases, designated HYDA1 and HYDA2. While HYDA1 is considered the dominant hydrogenase, the role of HYDA2 is unclear. To study the individual functions of each hydrogenase and provide a platform for future bioengineering, we isolated the Chlamydomonas hydA1-1, hydA2-1 single mutants and the hydA1-1 hydA2-1 double mutant. A reverse genetic screen was used to identify a mutant with an insertion in HYDA2, followed by mutagenesis of the hydA2-1 strain coupled with a H(2) chemosensor phenotypic screen to isolate the hydA1-1 hydA2-1 mutant. Genetic crosses of the hydA1-1 hydA2-1 mutant to wild-type cells allowed us to also isolate the single hydA1-1 mutant. Fermentative, photosynthetic, and in vitro hydrogenase activities were assayed in each of the mutant genotypes. Surprisingly, analyses of the hydA1-1 and hydA2-1 single mutants, as well as the HYDA1 and HYDA2 rescued hydA1-1 hydA2-1 mutant demonstrated that both hydrogenases are able to catalyze H(2) production from either fermentative or photosynthetic pathways. The physiology of both mutant and complemented strains indicate that the contribution of HYDA2 to H(2) photoproduction is approximately 25% that of HYDA1, which corresponds to similarly low levels of in vitro hydrogenase activity measured in the hydA1-1 mutant. Interestingly, enhanced in vitro and fermentative H(2) production activities were observed in the hydA1-1 hydA2-1 strain complemented with HYDA1, while maximal H(2)-photoproduction rates did not exceed those of wild-type cells.     

Cloning of hydrogenase genes and fine structure analysis of an operon essential for H2 metabolism in Escherichia coli.

Escherichia coli has two unlinked genes that code for hydrogenase synthesis and activity. The DNA fragments containing the two genes (hydA and hydB) were cloned into a plasmid vector, pBR322. The plasmids containing the hyd genes (pSE-290 and pSE-111 carrying the hydA and hydB genes, respectively) were used to genetically map a total of 51 mutant strains with defects in hydrogenase activity. A total of 37 mutants carried a mutation in the hydB gene, whereas the remaining 14 hyd were hydA. This complementation analysis also established the presence of two new genes, so far unidentified, one coding for formate dehydrogenase-2 (fdv) and another producing an electron transport protein (fhl) coupling formate dehydrogenase-2 to hydrogenase. Three of the four genes, hydB, fhl, and fdv, may constitute a single operon, and all three genes are carried by a 5.6-kilobase-pair chromosomal DNA insert in plasmid pSE-128. Plasmids carrying a part of this 5.6-kilobase-pair DNA (pSE-130) or fragments derived from this DNA in different orientations (pSE-126 and pSE-129) inhibited the production of active formate hydrogenlyase. This inhibition occurred even in a prototrophic E. coli, strain K-10, but only during an early induction period. These results, based on complementation analysis with cloned DNA fragments, show that both hydA and hydB genes are essential for the production of active hydrogenase. For the expression of active formate hydrogenlyase, two other gene products, fhl and fdv are also needed. All four genes map between 58 and 59 min in the E. coli chromosome.     

Heterologous expression of a hydrogenase gene in Enterobacter aerogenes to enhance hydrogen gas production

The hydrogenase gene from Enterobacter cloacae (IIT-BT 08) was amplified and inserted into a prokaryotic expression vector to create a recombinant plasmid (pGEX-4T-2-Cat/hydA). The recombinant plasmid was transformed into a hydrogen-producing strain of Enterobacter aerogenes (ATCC13408). SDS–PAGE and western blot analysis confirmed the successful expression of the GST-tagged hydA protein. Anaerobic fermentation for the production of hydrogen from glucose was investigated using E. aerogenes ATCC13408 and the recombinant strain. The results showed that the hydrogen yield markedly increased, from 442.82 ± 22.61 ml/g glucose in the ATCC13408 strain to 864.02 ± 36.8 ml/g glucose in the recombinant. The maximum rate of hydrogen production was found to be 53.49 ± 3.34 ml l⁻¹ h⁻¹ using 1% (w/v) glucose as the substrate at pH 6.0 and a reaction temperature of 37°C.     

Restoration of hydrogenase activity in hydrogenase-negative strains of Escherichia coli by cloned DNA fragments from Chromatium vinosum and Proteus vulgaris

DNA fragments from Proteus vulgaris and Chromatium vinosum were isolated which restored hydrogenase activities in both hydA and hydB mutant strains of Escherichia coli. The hydA and hydB genes, which map near minute 59 of the genome map, 17 kb distant from each other, are not structural hydrogenase genes, but mutation in either of these genes leads to failure to synthesize any of the hydrogenase isoenzymes. The smallest DNA fragments which restored hydrogenase activity to both E. coli mutant strains were 4.7 kb from C. vinosum and 2.3 kb from P. vulgaris. These fragments were cleaved into smaller fragments which did not complement either of the E. coli mutations. The cloned heterologous genes also restored formate hydrogenlyase activity but they did not restore activity in hydE, hupA or hupB mutant strains of E. coli. The cloned genes, on plasmids, did not lead to the synthesis of proteins of sufficient size to be the hydrogenase catalytic subunit. The hydrogenase proteins synthesized by hydA and hydB mutant strains of E. coli transformed by cloned genes from P. vulgaris and C. vinosum were shown by isoelectric and immunological methods to be E. coli hydrogenase. Thus, these genes are not hydrogenase structural genes.     

Molecular cloning, characterization, and overexpression of a novel [Fe]-hydrogenase isolated from a high rate of hydrogen producing Enterobacter cloacae IIT-BT 08

Degenerate primers were designed from the conserved zone of hydA structural gene encoding for catalytic subunit of [Fe]-hydrogenase of different hydrogen producing bacteria. A 750 bp of PCR product was amplified by using the above-mentioned degenerate primers and genomic DNA of Enterobacter cloacae IIT-BT 08 as template. The amplified PCR product was cloned and sequenced. The sequence showed the presence of an ORF of 450 bp with significant similarity (40%) with C-terminal end of the conserved zone (H-cluster) of [Fe]- hydrogenase. hydA ORF was then amplified and cloned in-frame with GST in pGEX4T-1 and overexpressed in a non-hydrogen producing Escherichia coli BL-21 to produce a GST-fusion protein of a calculated molecular mass of about 42.1 kDa. Recombinant protein was purified and specifically recognized by anti-GST monoclonal antibody through Western blot. Southern hybridization confirmed the presence of this gene in E. cloacae IIT-BT 08 genome. In vitro hydrogenase assay with the overexpressed hydrogenase enzyme showed that it is catalytically active upon anaerobic adaptation. In vivo hydrogenase assay confirmed the presence of H2 gas in the gas mixture obtained from the batch culture of recombinant E. coli BL-21. A tentative molecular mechanism has been proposed about the transfer of electron from electron donor to H-cluster without the mediation of the F-cluster.     

Identification of proteins encoded in Escherichia coli hydA, hydB and analysis of the hydA locus

The hydB gene of Escherichia coli, which is related with the expression of hydrogenase activity, was cloned into the plasmid (pES1). Using the maxicell protein-labeling method, the molecular weight of hydB gene product was estimated. Comparing between the gene products from the mutant strains and that of the hydB genes cloned strains, the molecular weight of the gene product was 35,000 Mr. Similarly, the molecular weight of the gene product of hydA, which had been previously cloned, was determined by maxicell analysis. The molecular weight of hydA gene product was estimated to be 80,000 Mr. Using deletion analysis and Tn1000 insertional inactivation of hydA's function, the hydA coding region was estimated between 2.2 kb and 2.8 kb in a 3.1 kb EcoRI-MluI fragment on the recombinant plasmid pEH3.     

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

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSION: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.     

Expression and nucleotide sequence of the Clostridium acetobutylicum beta-galactosidase gene cloned in Escherichia coli.

A gene library for Clostridium acetobutylicum NCIB 2951 was constructed in the broad-host-range cosmid pLAFR1, and cosmids containing the beta-galactosidase gene were isolated by direct selection for enzyme activity on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) plates after conjugal transfer of the library to a lac deletion derivative of Escherichia coli. Analysis of various pSUP202 subclones of the lac cosmids on X-Gal plates localized the beta-galactosidase gene to a 5.1-kb EcoRI fragment. Expression of the Clostridium beta-galactosidase gene in E. coli was not subject to glucose repression. By using transposon Tn5 mutagenesis, two gene loci, cbgA (locus I) and cbgR (locus II), were identified as necessary for beta-galactosidase expression in E. coli. DNA sequence analysis of the entire 5.1-kb fragment identified open reading frames of 2,691 and 303 bp, corresponding to locus I and locus II, respectively, and in addition a third truncated open reading frame of 825 bp. The predicted gene product of locus I, CbgA (molecular size, 105 kDa), showed extensive amino acid sequence homology with E. coli LacZ, E. coli EbgA, and Klebsiella pneumoniae LacZ and was in agreement with the size of a polypeptide synthesized in maxicells containing the cloned 5.1-kb fragment. The predicted gene product of locus II, CbgR (molecular size, 11 kDa) shares no significant homology with any other sequence in the current DNA and protein sequence data bases, but Tn5 insertions in this gene prevent the synthesis of CbgA. Complementation experiments indicate that the gene product of cbgR is required in cis with cbgA for expression of beta-galactosidase in E. coli.