To isolate genes from Escherichia coli which regulate the labile hydrogenase activity, a plasmid library was used to transform hydL mutants lacking the labile hydrogenase. A single type of gene, designated hydG, was isolated. This gene also partially restored the hydrogenase activity in hydF mutants (which are defective in all hydrogenase isoenzymes), although the low hydrogenase 1 and 2 levels were not induced. Therefore, hydG apparently regulates, specifically, the labile hydrogenase activity. Restoration of this latter activity in hydF mutants was accompanied by a proportional increase of the H2 uptake activity, suggesting a functional relationship. H2:fumarate oxidoreductase activity was not restored in complemented hydL mutants. These latter strains may therefore lack, in addition to the labile hydrogenase, a second component (provisionally designated component R), possibly an electron carrier coupling H2 oxidation to the anerobic respiratory chain. Sequence analysis showed an open reading frame of 1,314 base pairs for hydG. It was preceded by a ribosome-binding site but apparently lacked a promoter. Minicell experiments revealed a single polypeptide of approximately 50 kilodaltons. Comparison of the predicted amino acid sequence with a protein sequence data base revealed strong homology to NtrC from Klebsiella pneumoniae, a DNA-binding transcriptional activator. The 411 base pairs upstream from pHG40 contained a second open reading frame overlapping hydG by four bases. The deduced amino acid sequence showed considerable homology with the C-terminal part of NtrB. This sequence was therefore assumed to be part of a second gene, encoding the NtrB-like component, and was designated hydH. The labile hydrogenase activity in E. coli is apparently regulated by a multicomponent system analogous to the NtrB-NtrC system. This conclusion is in agreement with the results of Birkmann et al. (A. Birkmann, R. G. Sawers, and A. Böck, Mol. Gen. Genet. 210:535-542, 1987), who demonstrated ntrA dependence for the labile hydrogenase activity.

A mutant of Escherichia coli K-12 is described that is specifically impaired in only one hydrogenase isoenzyme. By means of Tn5-mediated insertional mutagenesis, a class of mutants was isolated (class I) that had retained 20% of the overall hydrogenase activity. As determined by neutral polyacrylamide gel electrophoresis, the mutant contained normal amounts of the hydrogenase isoenzymes 1 and 2. Therefore, the hydrogenase activity affected seemed to be electrophoretically labile and was called hydrogenase L. The presence of such an activity was recently suggested in various papers and was called isoenzyme 3. Hydrogenase L might be identical or part of the latter isoenzyme. By DEAE ion-exchange chromatography it could be separated from hydrogenases 1 and 2. Hydrogenase activity in the parent strain HB101, determined manometrically with cell-free preparations and methylviologen as the electron acceptor, immediately showed maximal activity. However, class I mutants showed a lag phase which was dependent on the protein concentration utilized in the assay. This suggested that the fast initial activity of HB101 was due to hydrogenase L. The enzyme or enzyme complex showed an Mr around 300,000 and a pH optimum between 7 and 8. Strong indications about its physiological role were provided by the finding that in class I mutants H2 production by the formate-hydrogen lyase pathway was unimpaired, whereas fumarate-dependent H2 uptake was essentially zero. Complementation with F-prime factor F'116 but not with F'143 and coconjugation and cotransduction experiments localized the mutation (hydL) close to metC at approximately 64.8 min.

Systematic screening of 6.104 independent Tn5 insertion mutants of Escherichia coli yielded one new hydrogenase locus, hydF, mapping near 64.8 min, i.e., close to the hydL locus (K. Stoker, L. F. Oltmann, and A. H. Stouthamer, J. Bacteriol. 170:1220-1226, 1988. It regulated specifically the activity of the hydrogenase isoenzymes, formate dehydrogenase and lyase activities being unaffected. In hydF mutants, hydrogenase 1 and 2 activities were reduced to 1 % of the parental level, whereas the electrophoretically labile part was present at about 20% of the parental level. H2 uptake was also reduced to about 20%, which suggested a relationship between these two activities. Experiments with 63Ni indicated that hydrogenase isoenzymes 1 and 2 might be present in these strains but in an inactive form. The hydF product might therefore be a posttranslational activator. At least three other mutant classes were isolated. Additional data were obtained on coisolated, nickel-restorable hydC mutants (L. F. Wu and M.-A. Mandrand-Berthelot, Biochimie 68:167-179, 1986). These strains were found to suffer a general impairment of nickel uptake. Restoration of hydrogenase activities was specific for NiCl2 and inhibited by chloramphenicol, which indicated an effect either on the transcription of hydrogenase(-associated) genes or by cotranslational incorporation in nickel-containing enzymes (e.g., in hydrogenases). The hydC mutation could not be complemented in trans, evidence that the hydC product is not a nickel transport protein but rather a cis-acting regulatory gene. Parent HB101, hydF mutants, and the other mutants were further analyzed by monitoring the induction of hydrogenase and hydrogenase-associated activities upon transition of cells from aerobic to anaerobic growth. These experiments also revealed a correlation between the early-induced H2 uptake route and labile hydrogenase activity. The formate hydrogenlyase induction patterns followed quite well the slower induction patterns of hydrogenases 1 and 2. Expression of most of these components is inducible, depending on the redox state of the environment, the presence of substrates (e.g., formate), and the source of energy (27). The underlying genetic regulation mechanisms, however, are still poorly understood. Although more than 10 genetic loci have been described thus far, mapping near 58 min (11, 15, 22, 24, 30), 65 min (15, 28), and 77 min (31), none of these searches was exhaustive, and it is still unknown whether these studies revealed all loci involved in H, metabolism. We therefore undertook a systematic screening of a large number of Tn5 insertion mutants; this search yielded one new locus, which was further characterized.Another problem is how the products expressed by these loci carry out their functions. This too is largely unknown, although some suggestions have been made. For example, hydC (31) and the loci affected in mutants FD-12 (11) and AK23 (6) might be involved in nickel metabolism, the hyd-17 * Corresponding author. 831 locus might make up the structu...

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