In-frame deletions were introduced into each of the eight genes of the hyc operon coding for products required for the formation of the formate hydrogenlyase (FHL) system. The deletions were transferred to the chromosome and the resulting mutants were analysed for development of formate dehydrogenase H and hydrogenase 1, 2 and 3 activity. It was found that hycA, the promoter-proximal gene, is a regulatory gene and that it codes for a product counteracting transcriptional activation by FhlA. Deletions within the hycB to hycH genes specifically affected formate dehydrogenase H activity or hydrogenase 3 activity, or both. None of the mutations affected hydrogenase 1 or 2 activity. A model is proposed for the functional interaction of the different hyc operon gene products in the formate hydrogenlyase complex, which is based on the results of the mutational analysis, on the determination of the subcellular localization of the FdhF, HycE, HycF and HycG polypeptides and on the similarity of hyc gene product sequences with those from other hydrogenase systems. HycH, the product of the most promoter-distal gene, does not seem to form part of the functional FHL complex but rather is required for the conversion of a precursor form of the large subunit of hydrogenase 3 into the mature form.
An 8kb segment of DNA from the 58/59 min region of the E. coli chromosome, which complements the defect of a mutant devoid of hydrogenase 3 activity, has been sequenced. Eight open reading frames were identified which are arranged in a transcriptional unit; all open reading frames were transcribed and translated in vivo in a T7 promoter/polymerase system. Analysis of the amino acid sequences derived from the nucleic acid sequences revealed that one of them, open reading frame 5 (ORF5), exhibits significant sequence similarity to conserved regions of the large subunit from Ni/Fe hydrogenases. Two of the open reading frames (orf2, orf6) code for proteins apparently carrying iron-sulphur clusters of the 4Fe/4S ferredoxin type. The product of one of the open reading frames, orf7, displays extensive sequence similarity with protein G from the chloroplast electron transport chain. ORF3 and ORF4, on the other hand, are extremely hydrophobic proteins with nine and six putative transmembrane helices, respectively. Over a limited hydrophilic sequence stretch, bordered by putative transmembrane areas, ORF3 and ORF4 exhibit homology with subunits 4 and 1 of mitochondrial and plastid NADH-ubiquinol oxidoreductases, respectively. The operon described, therefore, appears to comprise genes for redox carriers linking formate oxidation to proton reduction and for a hydrogenase of hitherto unique composition.
The 58/59 min region of the Escherichia coli chromosome contains two divergently oriented gene clusters coding for proteins with a function in hydrogenase formation. One cluster (the hyc operon), transcribed counterclockwise with respect to the E. coli chromosome, codes for gene products with a structural role in hydrogenase 3 formation (Böhm et al., 1990). The nucleotide sequence of the divergently transcribed operon (hyp) has been determined. It contains five genes, all of which are expressed in vivo in a T7 promoter/polymerase system, and the sizes of the synthesized products correspond with those predicted from the amino acid sequence. Complementation analysis of previously characterized mutants showed that the hypB, hypC and hypD genes have a function in the formation of all three hydrogenase isoenzymes, lesions in hypB being complemented by high nickel ion concentration in the medium. Prevention of hypBCDE gene expression led to an altered electrophoretic pattern of hydrogenase 1 and 2 constituent subunits, indicating increased chemical or proteolytic subunits, Under fermentative growth conditions, operon expression was governed by an NtrA-dependent promoter lying upstream of hypA working together with an fnr gene product-dependent promoter which was localized within the hypA gene. The latter (operon-internal) promoter is responsible for hypBCDE transcription under non-fermentative conditions when the -24/-12 NtrA-dependent promoter upstream of hypA is silent.
The regulatory region of two divergently oriented transcriptional units involved in the formation of the gas-evolving hydrogenase (isoenzyme 3) of Escherichia coli was investigated. DNA sequence analysis revealed the existence of a 210 bp non-coding region containing two sequences showing homology to -24/-12 NtrA-dependent promoters. These sequences were arranged in a divergent orientation entirely consistent with their being involved in transcribing the divergent operons. Through S1 protection experiments it could be shown that transcription of both promoters was NtrA-dependent and that it was regulated in an identical manner: oxygen repressed expression, as did anaerobic growth in the presence of nitrate; transcription was induced in cells grown anaerobically in the absence of exogenous electron acceptors and formate was found to be obligately required for this anaerobic induction. Lying at an approximately equal distance between both promoters was a short stretch of DNA which showed similarity to the sequence previously identified (Birkmann and Böck, 1989a) as being necessary for formate induction of the fdhF gene.
We analyzed the involvement of chaperonins GroES and GroEL in the biosynthesis of the three hydrogenase isoenzymes, HYD1, HYD2, and HYD3, of Escherichia coli. These hydrogenases are NiFe-containing, membranebound enzymes composed of small and large subunits, each of which is proteolytically processed during biosynthesis. Total hydrogenase activity was found to be reduced by up to 60% in groES and groEL thermosensitive mutant strains. This effect was specific because it was not seen for another oligomeric, membranebound metalloenzyme, i.e., nitrate reductase. Analyses of the single hydrogenase isoenzymes revealed that a temperature shift during the growth of groE mutants led to an absence of HYD1 activity and to an accumulation of the precursor of the large subunit of HYD3, whereas only marginal effects on the processing of HYD2 and its activity were observed under these conditions. A decrease in total hydrogenase activity, together with accumulation of the precursors of the large subunits of HYD2 and HYD3, was also found to occur in a nickel uptake mutant (nik). The phenotype of this nik mutant was suppressed by supplementation of the growth medium with nickel ions. On the contrary, Ni 2؉ no longer restored hydrogenase activity and processing of the large subunit of HYD3 when the nik and groE mutations were combined in one strain. This finding suggests the involvement of these chaperonins in the biosynthesis of a functional HYD3 isoenzyme via the incorporation of nickel. In agreement with these in vivo results, we demonstrated a specific binding of GroEL to the precursor of the large subunit of HYD3 in vitro. Collectively, our results are consistent with chaperonin-dependent incorporation of nickel into the precursor of the large subunit of HYD3 as a prerequisite of its proteolytic processing and the acquisition of enzymatic activity.The enterobacterium Escherichia coli synthesizes three NiFe-hydrogenase isoenzymes under anaerobic conditions (25). Hydrogenases 1 and 2 (HYD1 and HYD2, respectively) catalyze the anaerobic oxidation of hydrogen linked to the ultimate reduction of a terminal electron acceptor. These energy-conserving respiratory pathways, which enable the bacterium to use hydrogen as an energy source, allow the organism to grow on nonfermentable carbon sources such as fumarate in the presence of hydrogen. HYD3 is part of the formate hydrogenlyase complex and is responsible for formate-dependent dihydrogen evolution. This system catalyzes the oxidation of endogenously produced formate to carbon dioxide and passes the electrons so generated to protons to evolve gaseous hydrogen. The overall reaction is scalar and non-energy conserving and functions both to remove redox equivalents exchangeable with formate and to help offset acidification of the growth medium during fermentative growth. The structural genes of these hydrogenases lie in three operons, hya, hyb, and hyc, each of which contains four, five, and seven additional accessory genes specifically required for HYD1, HYD2, and HYD3 activities, res...
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