Clustering of genes necessary for hydrogen oxidation in Rhodobacter capsulatus

Abstract

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...

In vivo nickel insertion into the carbon monoxide dehydrogenase of Rhodospirillum rubrum: molecular and physiological characterization of cooCTJ

“…The histidine-rich portion of CooJ is similar to motifs of HypB and UreE proteins, insofar as most contain several His residues, though their arrangement is inconstant (18,50,61) and is not evident in the hyp system of E. coli (39) or in the urease accessory systems of a Bacillus sp. (38) and Helicobacter pylori (10).…”

Purification and characterization of Klebsiella aerogenes UreE protein: A nickel‐binding protein that functions in urease metallocenter assembly

“…Potential candidates for nickel-binding proteins that could substitute for UreE include auxiliary peptides that are essential for hydrogenase biosynthesis (hydrogenase is another nickel-containing enzyme [Haus-inger, 19871). For example, ORF5 in the Azotobacter vinelandii hox gene cluster possesses a 13-residue region containing 10 histidines (Chen & Mortenson, 1992) and ORF4 in the Rhodobacter capsulatus hup gene cluster possesses a region that contains 23 histidines in 62 residues (Xu &Wall, 1991). The roles of these hydrogenaserelated genes have not been elucidated but they are related in sequence to hypB, an E. coli gene that is known to function in hydrogenase activation involving nickel incorporation (Lutz et al, 1991).…”

Analysis of a Caulobacter crescentus gene cluster involved in attachment of the holdfast to the cell

“…HfaB showed similarity to SoxR and LasR, small transcriptional activators from E. coli and Pseudomonas aeruginosa, respectively, and to RCORF2, a protein encoded by open reading frame 2 of the hup-related gene cluster of Rhodobacter capsulatus and implicated in transcriptional activation (10,46,47) (Table 2).…”