Identification and isolation of genes essential for H2 oxidation in Rhodobacter capsulatus

Abstract

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...

A self-transmissible, narrow-host-range endogenous plasmid of Rhodobacter sphaeroides 2.4.1: physical structure, incompatibility determinants, origin of replication, and transfer functions

“…The remarkable metabolic diversity of this organism and its ability to synthesize photosynthetic membrane invaginations (intracytoplasmic membranes) when grown anaerobically have made it and related bacteria, such as R. capsulatus, excellent model systems for the study of complex biological and biophysical phenomena. For example, photosynthetic membrane biogenesis (18), carbon dioxide fixation (42), nitrogen fixation (20), biophysical and structural studies of the light reactions of photosynthesis (5,19,35), pigment biosynthesis (1,31), and hydrogen metabolism (49), to mention only a few, are fertile areas of research employing these facultative photosynthetic bacteria.…”

Nucleotide sequences and genetic analysis of hydrogen oxidation (hox) genes in Azotobacter vinelandii

“…eutrophus (20). The predicted amino acid sequence of ORF8' is 80% identical to the N terminus of an ORF immediately downstream of the putative hoxQ gene equivalent in A. eutrophus (20 (30), and R. capsulatus (11,67,68). In all these organisms, the arrangement of the genes for the ao and L subunits and a third ORF corresponding to hoxZ is conserved (18,29,48,56).…”

Hup-Type Hydrogenases of Purple Bacteria: Homology Modeling and Computational Assessment of Biotechnological Potential

“…However, there is another group of hydrogenases in purple bacteria, namely HupSL-hydrogenases [20][21][22][23][24][25]. They haven't drawn much attention as in vitro catalysts for a while, but recently discovered 4Fe3S clusters led to hypothesis that they should be treated as possible oxygen-tolerant hydrogen oxidation catalysts like the mentioned membrane-bound hydrogenases, which are their close homologues.…”