Cloning, sequencing, and mutational analysis of the hyb operon encoding Escherichia coli hydrogenase 2.

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.     

Cloning and sequencing of a putative Escherichia coli [NiFe] hydrogenase-1 operon containing six open reading frames.

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.     

Involvement of the GroE chaperonins in the nickel-dependent anaerobic biosynthesis of NiFe-hydrogenases of Escherichia coli.

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, membrane-bound 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, membrane-bound 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, Ni2+ 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.     

A genetic region downstream of the hydrogenase structural genes of Bradyrhizobium japonicum that is required for hydrogenase processing.

Deletion of a 2.9-kb chromosomal EcoRI fragment of DNA located 2.2 kb downstream from the end of the hydrogenase structural genes resulted in the complete loss of hydrogenase activity. The normal 65- and 35-kDa hydrogenase subunits were absent in the deletion mutants. Instead, two peptides of 66.5 and 41 kDa were identified in the mutants by use of anti-hydrogenase subunit-specific antibody. A hydrogenase structural gene mutant did not synthesize either the normal hydrogenase subunits or the larger peptides. Hydrogenase activity in the deletion mutants was complemented to near wild-type levels by plasmid pCF1, containing a 6.5-kb BglII fragment, and the 65- and 35-kDa hydrogenase subunits were also recovered in the mutants containing pCF1.     

Purification and characterization of two forms of hydrogenase isoenzyme 1 from Escherichia coli.

A hydrogenase associated with dihydrogen uptake (HUP hydrogenase) was purified from an Escherichia coli mutant (strain SE1100) defective in utilization of molybdate and thus fermentative dihydrogen production. This protein had two subunits with apparent molecular weights of 59,000 and 28,000 (form 1). An immunologically cross-reactive hydrogenase was also purified from E. coli K10 grown in glucose-minimal medium and harvested at the mid-exponential phase of growth. Upon purification to homogeneity, this hydrogenase contained only one subunit with an apparent molecular weight of 59,000 (form 2). The two forms of the HUP hydrogenase exhibited similar kinetic characteristics. The electrophoretic properties of the enzyme and its response to pH suggest that this HUP hydrogenase is the HYD1 isoenzyme. The HYD1 isoenzyme was the only hydrogenase detectable during the stationary phase of growth in E. coli grown in Mo-deficient medium.     

Pleiotropic hydrogenase mutants of Escherichia coli K12: growth in the presence of nickel can restore hydrogenase activity

Anaerobic growth in the presence of 0.6 mM NiCl2 was able to restore hydrogenase and benzyl-viologen-linked formate dehydrogenase activities to a mutant (FD12), which is normally defective in these activities. This mutant carries a mutation located near minute 58 in the genome. Hydrogenase isoenzyme I and II activities were restored along with the hydrogenase activity that forms part of the formate hydrogen lyase system. A plasmid (pRW1) was constructed, containing a 4.8 kb chromosomal DNA insert, which was able to complement the lesion in mutant FD12. Further mutants with mutations near 58 minutes on the chromosome, and which lacked hydrogenase and formate dehydrogenase activities were isolated. These mutants were divided into three groups. Class I mutants were restored to the wild-type phenotype either by growth with 0.6 mM NiCl2 or following transformation with pRW1. Class II mutants were also complemented by pRW1 but were unaffected by growth with NiCl2. Class III mutants were unaffected by both pRW1 and growth with NiCl2. The cloned 4.8 kb fragment of chromosomal DNA therefore encodes two genes essential for hydrogenase activity. Restriction analysis indicates that the cloned DNA is the same as a fragment that has previously been cloned and which complements the hydB locus (Sankar et al. (1985) J. Bacteriol., 162, 353-360). None of the three classes of mutants possess mutations in hydrogenase structural genes.     

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.     

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.