Unusual organization of the genes coding for HydSL, the stable [NiFe]hydrogenase in the photosynthetic bacterium Thiocapsa roseopersicina BBS

Abstract

The characterization of a hyd gene cluster encoding the stable, bidirectional [NiFe]hydrogenase 1 enzyme in Thiocapsa roseopersicina BBS, a purple sulfur photosynthetic bacterium belonging to the family Chromatiaceae, is presented. The heterodimeric hydrogenase 1 had been purified to homogeneity and thoroughly characterized (K. L. Kovacs et al., J. Biol. Chem. 266:947-951, 1991; C. Bagyinka et al., J. Am. Chem. Soc. 115:3567-3585, 1993). As an unusual feature, a 1,979-bp intergenic sequence (IS) separates the structural genes hydS and hydL, which encode the small and the large subunits, respectively. This IS harbors two sequential open reading frames (ORFs) which may code for electron transfer proteins ISP1 and ISP2. ISP1 and ISP2 are homologous to ORF5 and ORF6 in the hmc operon, coding for a transmembrane electron transfer complex in Desulfovibrio vulgaris. Other accessory proteins are not found immediately downstream or upstream of hydSL. A hup gene cluster coding for a typical hydrogen uptake [NiFe]hydrogenase in T. roseopersicina was reported earlier (A. Colbeau et al. Gene 140:25-31, 1994). The deduced amino acid sequences of the two small (hupS and hydS) and large subunit (hupL and hydL) sequences share 46 and 58% identity, respectively. The hup and hyd genes differ in the arrangement of accessory genes, and the genes encoding the two enzymes are located at least 15 kb apart on the chromosome. Both hydrogenases are associated with the photosynthetic membrane. A stable and an unstable hydrogenase activity can be detected in cells grown under nitrogen-fixing conditions; the latter activity is missing in cells supplied with ammonia as the nitrogen source. The apparently constitutive and stable activity corresponds to hydrogenase 1, coded by hydSL, and the inducible and unstable second hydrogenase may be the product of the hup gene cluster.

FIG. 1

Cloning, restriction map, and arrangement of hyd genes. The locations of PCR primer sequences, designed on the basis of the N-terminal and internal peptide sequences, are indicated. The principal PCR products have been cloned as pTSUP and pACRG4 for sequencing. The positive cosmid was identified by hybridization with the PCR product ACRG4. This cosmid insert was digested with BamHI and PstI, and the 5-kb BamHI and 4.5-kb PstI fragments, giving positive hybridization, were subcloned into the corresponding sites of pBluescribe vector (Stratagene), yielding clones pTSH2/8 and pTSH4/5, respectively. The overlapping clones were further subcloned into pBluescribe and pBluescriptKS and -SK and then sequenced by using specific synthetic primers and/or T3 and T7 primers. The hydS and hydL genes code for the small and large subunits, respectively, of hydrogenase 1. IS is the 2-kb IS harboring two ORFs, isp1 and isp2. Abbreviations for restriction endonucleases: BHI, BamHI; BHII, BssHII; BII, BglII; BXI, BstXI; E, EcoRI; H, HincII; P, PstI; S, SphI; X, XhoI.

FIG. 2

Alignment of translated ORFs ISP1 and ISP2 (TrISP1 and TrISP2) from the DNA region intercalated between hydS and hydL of T. roseopersicina with the predicted products of ORF5 and ORF6 of the hmc operon of D. vulgaris (DvHMC5). The multiple alignment was done by using the CLUSTALW and PRETTYPLOT programs of the Genetics Computer Group program package. Homologous amino acids are boxed, the putative transmembrane hydrophobic domains in the T. roseopersicina sequence are marked with continuous lines between arrows, the heme (ORF5)-binding conserved histidines (at positions 83, 96, 180, and 198) are shown marked with asterisks, and the iron-sulfur cluster (ORF6)-binding domains are indicated with dashed lines between arrows. As evident from the homologies, ORF5 and ORF6 contain the corresponding sequences and therefore are not marked separately.

FIG. 3

DEAE chromatogram of total cell extract prepared from ammonia-grown cells (a; nitrogenase repressed) and cells grown with glutamate as the nitrogen source (b; nitrogenase active). The hydrogen evolution activity of each fraction was determined after elution (•) and following incubation at 4°C under air for 24 h (□). Activities are expressed as micromoles of H₂ produced per hour per milligram of protein.