INCREASED NITROGENASE-DEPENDENT H-2 PHOTOPRODUCTION BY HUP MUTANTS OFRHODOSPIRILLUM-RUBRUM

Transposon Tn5 mutagenesis was used to isolate mutants of Rhodospirill um rubrum which lack uptake hydrogenase (Hup) activity. Three Tn5 inse rtions mapped at different positions within the same 13-kb EcoRI fragm ent (fragment E1). Hybridization experiments revealed homology to the structural hydrogenase genes hupSLM from Rhodobacter capsulatus and hu pSL from Bradyrhizobium japonicum in a 3.8-kb EcoRI-ClaI subfragment o f fragment E1. It is suggested that this region contains at least some of the structural genes encoding the nickel-dependent uptake hydrogen ase of R. rubrum. At a distance of about 4.5 kb from the fragment homo logous to hupSLM, a region with homology to a DNA fragment carrying hy pDE and hoxXA from B. japonicum was identified. Stable insertion and d eletion mutations were generated in vitro and introduced into R. rubru m by homogenotization. In comparison with the wild type, the resulting hup mutants showed increased nitrogenase-dependent H-2 photoproductio n. However, a mutation in a structural hup gene did not result in maxi mum H-2 production rates, indicating that the capacity to recycle H-2 was not completely lost. Highest H-2 production rates were obtained wi th a mutant carrying an insertion in a nonstructural hup-specific sequ ence and with a deletion mutant affected in both structural and nonstr uctural hup genes. Thus, besides the known Hup activity, a second, pre viously unknown Hup activity seems to be involved in H-2 recycling. A single regulatory or accessory gene might be responsible for both enzy mes. In contrast to the nickel-dependent uptake hydrogenase, the secon d Hup activity seems to be resistant to the metal chelator EDTA.