Three cosmids previously shown to contain information necessary for the expression of uptake of hydrogenase in Rhodobacter capsulatus were found to be present in a cluster on the chromosome. Earlier genetic experiments suggested the presence of at least six genes essential for hydrogenase activity that are now shown to be in a region of approximately 18 kb that includes the structural genes for the enzyme. A potential response regulator gene was sequenced as a part of the hup gene region.Certain microbial enzymes, hydrogenases, catalyze H2 evolution for the removal of excess reductant in anaerobes or H2 oxidation as the first step in the use of H2 as an energy source. In the photosynthetic bacterium Rhodobacter capsulatus, H2 evolution appears to occur only as an obligate by-product of nitrogenase activity (21). In contrast, H2 consumption is mediated by a membrane-bound hydrogenase (5).R. capsulatus appears to express a single hydrogenase (19), with subunits of 67 and 31 kDa, that is immunologically related to the dimeric hydrogenases from several aerobic diazotrophs (15). Physiological studies of R. capsulatus suggested that the enzyme was a nickel-metallo protein (20), and nickel was later shown to be present in the purified enzyme (5).The genes encoding the two subunits of hydrogenase have been identified by homology with a probe containing the Bradyrhizobium japonicum structural genes (9) and by complementation of photoautotrophic mutants lacking hydrogenase activity (23). Information concerning the number and location of other genes essential for hydrogen oxidation by R. capsulatus is still fragmentary. Three cosmids were isolated by Vignais and coworkers that appear to complement most of their 9). Recently, these cosmids have been shown to contain sequences nearly contiguous on the chromosome (4), and it was suggested that a stretch of about 15 kb of DNA is necessary for the expression of hydrogenase genes in R. capsulatus.Independently, we isolated three small cosmids that had unique restriction patterns and complemented different sets of . To confirm the arrangement of hup genes on the chromosome and to relate our cosmids to those reported by others, we sought to determine the chromosomal arrangement of the DNA found in our cosmids. We found these cosmids to be nearly contiguous on the chromosome, forming a cluster of approximately 18 kb. In contrast to the conclusion drawn by Colbeau et al. (4) All analyses were of DNA from R. capsulatus B100, grown as described previously (23). The cosmids pRHP4, pRHP8, and pRHP20 were isolated by complementation of hup mutations (23) and are pLAFR1 with R. capsulatus Hup-specific DNA inserts. The cosmids were maintained in and prepared from Escherichia coli HB101. Individual EcoRI fragments used as hybridization probes were subcloned in pUC18 (24) and were maintained and prepared from E. coli DH5a (8). Cosmid A163 was prepared from a partial Sau3A genomic digest of R. capsulatus DNA in a Lorist 2 vector (7) and was a generous gift from J. Williams. Chromosomal and p...

Mutants of Rhodobacter capsulatus unable to grow photoautotrophically with H2 and CO2 were isolated.Those lacking uptake hydrogenase activity as measured by H2-dependent methylene blue reduction were analyzed genetically and used in complementation studies for the isolation of the wild-type genes. Results of further subcloning and transposon TnS mutagenesis suggest the involvement of a minimum of five genes. Hybridization to the 2.2-kilobase-pair SstI fragment that lies within the coding region for the large and small subunits of Bradyrhizobium japonicum uptake hydrogenase showed one region of strong homology among the R. capsulatus fragments isolated, which we interpret to mean that one or both structural genes were among the genes isolated.The nonsulfur purple photosynthetic bacterium Rhodobacter capsulatus produces and consumes molecular hydrogen. In this diazotroph, H2 production occurs primarily as a function of the nitrogenase enzyme complex (26, 43) and its consumption via an uptake hydrogenase (6, 40). Although hydrogenase catalyzes the reversible oxidation of H2 to protons and electrons, the enzyme found in R. capsulatus appears to be physiologically important only in an H2 uptake capacity (5, 40). The directionality of the enzyme underscores its essential role in photoautotrophic (19) and chemoautotrophic (31) growth and in recycling H2 generated during N2 reduction (40).Because H2 metabolism influences the efficiency of nitrogen fixation (14, 15), emphasis has been placed on gaining a clearer understanding of the regulation and function of hydrogenases in the diazotrophs (7,14,21). In addition, the potential application of the phototrophs for H2 production from biomass at the expense of light energy (44) provides a second impetus for analysis of this enzyme system. The enzyme has been purified from R. capsulatus and was initially reported to be a monomer (6). Later purification yielded a preparation with higher specific activity and showed the hydrogenase to be a dimer with subunits of 67,000 and 31,000 daltons (Da) (37). As yet, the number of genes essential for uptake hydrogenase biosynthesis and function has not been determined, although at least two have been isolated (7). Recently, the hydrogenase control system in Alcaligenes eutrophus has been implicated in the regulation of five polypeptides in addition to the hydrogenase peptides (28). Thus, it is possible that a regulon may be necessary for the formation, maturation, and metal metabolism of uptake hydrogenase and electron flux from this enzyme.To obtain a more complete picture of the hydrogenase genetic system in R. capsulatus, we isolated mutants unable to grow photoautotrophically and unable to reduce methylene blue with H2. After transductional analysis, complementing fragments were obtained from a pLAFR1 cosmid library. Subcloning and complementation revealed the presence of a minimum of five genes essential for restoration of * Corresponding author. t Missouri Agricultural Experiment Station journal series no. 10644.the mutant phenotype...