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The hya operon of Escherichia coli is composed of the genes which synthesize uptake hydrogenase isoenzyme 1 (Hyd1). Although hya expression and Hyd1 synthesis occur only under anaerobic conditions, Hyd1 is not essential for growth. In this study we used a hya′-′lacZ fusion to characterize parameters of anaerobic growth that maximize hya expression in an attempt to further elucidate Hyd1 function. We found that the expression pattern of hya followed a decline of external pH. In buffered media where the pH value was set, the onset of hya expression initiated earlier in growth and reached a greater peak level in acidic than in alkaline medium. When cultures expressing hya were shifted from acidic to alkaline conditions, hya expression was arrested; shifting from alkaline to acidic conditions stimulated hya expression. Maximal expression of hya under all growth conditions required the sigma factor RpoS and transcriptional regulators AppY and ArcA. In the absence of RpoS or AppY, the response of hya expression onset to external pH was evident and maximal hya levels remained greater in acidic than in alkaline medium. However, the absence of ArcA led to a diminished response of expression onset to external pH and the loss of elevated expression at an acidic external pH. The fermentation end product formate slightly altered hya expression levels but was not required for hya to respond to external pH. In contrast to hya expression, the onset of hyb operon expression, encoding uptake hydrogenase isoenzyme 2, was constitutive with respect to external pH. However, external pH did affect hyb expression levels, which, in contrast to hya , were maximal in alkaline rather than acidic medium.

Hydrogenase-1 (HYD1), overexpressed by twofold, has been purified to homogeneity and to a high specific activity from a mutant strain (AP6) of Escherichia coli which lacks hydrogenase-2. Plasma emission spectroscopy indicated that 0.93 atom of nickel and 11.4 iron atoms were present in HYD1. EPR studies on the as isolated HYD1 detected a complex 3Fe-4S signal and a Ni(III) species. Reduction with hydrogen gas caused disappearance of both the 3Fe–4S cluster and initial Ni(III) signals. At the same time the EPR signature (small g=2.19 signal) of the activated hydrogenase appeared. The detection of a 4Fe–4S cluster signal was noted. Reduction of HYD1 with sodium dithionite caused all nickel signals to disappear. The 4Fe–4S complex intensity was slightly increased. The EPR responses in the three oxidation-reduction states are consistent with other known (NiFe)-hydrogenases.

The genes encoding the two structural subunits of Escherichia coli hydrogenase 2 (HYD2) have been cloned and sequenced. They occur in an operon (hyb) which contains seven open reading frames. An hyb deletion mutant (strain AP3) failed to grown on dihydrogen-fumarate medium and also produced very low levels of HYD1. All seven open reading frames are required for restoration of wild-type levels of active HYD2 in AP3. The hyb operon was mapped at 65 min on the E. coli chromosome.

We have cloned the gene encoding Desulfovibrio gigas ferredoxin using a photodigoxigenin-labelled probe synthesized with the polymerase chain reaction. The DNA sequence of the gene predicts a polypeptide of 58 residues after removal of the initial formyl methionine (polypeptide Mr = 6,276). The ferredoxin gene was expressed in aerobically grown E. coli behind the lac promoter of pUC18 resulting in a high level of ferredoxin expression which comprises about 10% of the total cell protein. EPR analysis of recombinant ferredoxin revealed the presence of a [3Fe-4S] cluster which is characteristic of native D. gigas ferredoxin II.

Deletion mutants of Escherichia coli specific for hydrogenase isoenzyme 1 (HYD1) have been constructed and characterized. The hya operon, which contains genes for the two HYD1 structural subunits and four additional genes, was mapped at 22 min on the E. coli chromosome. The total hydrogenase activities of the HYD1-negative mutant and wild-type strains were similar. However, the formate dehydrogenase activity associated with the formate hydrogen lyase pathway was lower in the mutant. The hya mutant (strain AP1), complemented with only the hydrogenase structural genes (hyaAB), produced antigenically identifiable but inactive HYD1 protein. The first five genes of hya (hyaA to hyaE) were required for the synthesis of active HYD1, but wild-type levels of HYD1 activity were restored only when mutant cells were transformed with all six genes of the operon. When AP1 was complemented with hya carried on a high-copy-number plasmid, the HYD1 structural subunits were overexpressed, but the excess protein was unprocessed and localized in the soluble fraction of the cell. The products of hyaDEF are postulated to be involved in the processing of nascent structural subunits (HYAA and HYAB). This processing takes place only after the subunits are inserted into the cell membrane. It is concluded that the biosynthesis of active HYD1 is a complex biochemical process involving the cellular localization and processing of nascent structural subunits, which are in turn dependent on the insertion of nickel into the nascent HYD1 large subunit.

DNA encompassing the structural genes of an Escherichia coli [NiFe] hydrogenase has been cloned and sequenced. The genes were identified as those encoding the large and small subunits of hydrogenase isozyme 1 based on NH2-terminal sequences of purified subunits (kindly provided by K. Francis and K. T. Shanmugam). The structural genes formed part of a putative operon that contained four additional open reading frames. We have designated the operon hya and the six open reading frames hyaA through F. hyaA and hyaB encode the small and large structural subunits, respectively. The nucleotide-derived amino acid sequence of hyaC has a calculated molecular mass of 27.6 kilodaltons, contains 20% aromatic residues, and has four potential membrane-spanning regions. Open reading frames hyaD through F could encode polypeptides of 21.5, 14.9, and 31.5 kilodaltons, respectively. These putative peptides have no homology to other reported protein sequences, and their functions are unknown.

The structural genes for 3'-phosphoadenylyl sulfate (PAPS) reductase (cysH) and sulfite reductase (alpha and beta subunits; EC 1.8.1.2)(cysI and cysJ) of Escherichia coli K-12 have been cloned by complementation. pCYSI contains two PstI fragments (18.3 and 2.9 kb) which complement cysH-, cysI-, and cysJ- mutants. Subcloning showed that the cysH gene is located on a 1.6-kb ClaI subfragment (pCYSI-3) whereas cysI and most of cysJ are carried on a 3.7-kb ClaI subfragment (pCYSI-5). The PAPS reductase gene is closely linked to the sulfite reductase genes, but its expression is regulated by a unique promoter. The cysI and cysJ genes, on the other hand, are transcribed as an operon and the promoter precedes the cysI gene. Maxicell analysis demonstrated that pCYSI encodes three polypeptides of Mr 27,000, 57,000, and 60,000, in addition to the tetracycline-resistance determinant. The 60- and 57-kDa proteins are most likely the alpha and beta subunits, respectively, of E. coli sulfite reductase while the 27-kDa protein is putatively identified as PAPS reductase. Preliminary data suggest that the alpha and beta subunits of sulfite reductase are encoded by cysI and cysJ, respectively.

The structural genes for the large and small subunits of Desulfovibrio gigas periplasmic [NiFe]hydrogenase were identified and isolated by immunological and oligonucleotide screening. The gene for the small subunit codes for a 266-amino-acid, 28,724-dalton polypeptide which is separated by 63 nucleotides from the large subunit gene that codes for a 560-amino-acid, 61,707-dalton polypeptide. A putative signal peptide precedes the small subunit coding region, which may direct transport of the enzyme into the periplasmic compartment. Comparison of the amino acid sequence of this enzyme with those of two other classes of hydrogenase found in Desulfovibrio revealed that the D. gigas periplasmic hydrogenase has some homologies to the periplasmic [NiFeSe]hydrogenase of D. baculatus but none to the periplasmic [Fe]hydrogenase of D. vulgaris. The genes for the large and small subunits of the D. gigas hydrogenase hybridize strongly to genomic DNAs from several species of Desulfovibrio, indicating molecular similarity of the [NiFe]hydrogenase among sulfate reducers.

Two electrophoretic forms of the large subunit of the soluble periplasmic [NiFe] hydrogenase from Desulfovibrio gigas have been detected by Western analysis. The faster moving form co-migrates with the large subunit from purified, active enzyme. Amino acid sequence and composition of the C-terminal tryptic peptide of the large subunit from purified hydrogenase revealed that it is 15 amino acids shorter than that predicted by the nucleotide sequence. Processing of the nascent large subunit occurs by C-terminal cleavage between His536 and Val537, residues which are highly conserved among [NiFe] hydrogenases. Mutagenesis of the analogous residues, His582 and Val583, in the E. coli hydrogenase-1 (HYD1) large subunit resulted in significant decrease in processing and HYD1 activity.

A PyrF- mutant of Escherichia coli (SK1108, pyrF::Tn5 Kanr) was complemented with the Desulfovibrio vulgaris (Hildenborough) structural gene for orotidine-5'-phosphate decarboxylase (EC 4.1.1.23). Either orientation of a 1.6-kilobase-pair D. vulgaris DNA fragment (pLP3B or pLP3A) complemented the PyrF- strain suggesting that the D. vulgaris pyrF promoter was functional. The apparent product of the D. vulgaris pyrF gene was a single 26-kilodalton polypeptide. These results demonstrate the utility of E. coli cloning systems in studying metabolic and energetic pathways in sulfate-reducing bacteria.

Formyltetrahydrofolate synthetase (FTHFS) (EC 6.3.4.3), a thermostable protein of four identical subunits from Clostridium thermoaceticum was cloned into Escherichia coli SK1592. The clone (CRL47) contained a 9.5 kb EcoRI fragment of C. thermoaceticum DNA ligated into pBR322. It produced catalytically active, thermostable FTHFS, that was not found in E. coli SK1592 containing native pBR322. The identity of the expressed enzyme was confirmed by specific binding of rabbit polyclonal anti-FTHFS serum produced against C. thermoaceticum FTHFS. The specific activities (mumol.min-1.mg-1) of FTHFS in cell free extracts of CRL47 were 28-89 when assayed at 50 degrees C and pH 8. This was from 3-10-fold higher than in C. thermoaceticum extracts. FTHFS was purified to homogeneity from CRL47. The purified enzyme behaved during electrophoresis and gel chromatography and it had similar specific activity and thermostability as the enzyme purified from C. thermoaceticum.