Molecular analysis of a microaerobically induced operon required for hydrogenase synthesis in Rhizobium leguminosarum biovar viciae

Abstract

The nucleotide sequence (6138 bp) of a microaerobically inducible region (hupV/VI) from the Rhizobium leguminosarum bv. viciae hydrogenase gene cluster has been determined. Six genes, arranged as a single operon, were identified, and designated hypA, B, F, C, D and E based on the sequence similarities of all of them, except hypF, to genes from the hydrogenase pleiotropic operon (hyp) from Escherichia coli. The gene products from hypBFCDE were identified by in vivo expression analysis in E. coli, and their molecular sizes were consistent with those predicted from the nucleotide sequence. Transposon Tn5 insertions into hypB, hypF, hypD and hypE resulted in R. leguminosarum mutants that lacked any hydrogenase activity in symbiosis with peas, but still were able to synthesize the polypeptide for the hydrogenase large subunit. The gene products HypA, HypB, HypF and HypD contained CX2C motifs characteristic of metal-binding proteins. In addition, HypB bore a long histidine-rich stretch of amino acids near the N-terminus, suggesting a possible role in nickel binding for this protein. The gene product HypF, which was translationally coupled to HypB, presented two cysteine motifs (CX2CX18CX2C) with a capacity to form zinc finger-like structures in the N-terminal third of the protein. A role in nickel metabolism in relation to hydrogenase synthesis is postulated for proteins HypB and HypF.

Purification of Rhizobium leguminosarum HypB, a nickel-binding protein required for hydrogenase synthesis

“…E. coli BL21(DE3) was used as the host for T7 polymerase-dependent gene expression (43). Plasmids pBluescript SK+ (41), pRLH60 (pBluescript carrying hypB under control of the T7 promoter [39]), and pRLH47 (pBluescript carrying hypBF in the opposite orientation with respect to the T7 promoter [39]) have been described. Bacterial strains were grown in the media and conditions previously described (22).…”

“…This cluster contains six genes, hypABFCDE (39), which show homology to genes from the Escherichia coli hydrogenase pleiotropic operon (26) and are conserved in a number of uptake hydrogenase-containing bacteria (9,47). These genes are absolutely required for hydrogenase activity in R. leguminosarum (39) and have been shown to be required for maturation of hydrogenase in E. coli (17). The hypB gene is particularly interesting.…”

“…However, no nickel binding to the purified protein could be demonstrated. The sequence of the R leguminosarum hypB product contains a histidine-rich region (39) which is absent from the E. coli hypB product. Taking advantage of this feature, we have developed a one-step purification procedure for the R leguminosarum HypB protein expressed in E. coli and have been able to show that R leguminosarum HypB binds Ni2+ in solution.…”

Nickel availability to pea (Pisum sativum L.) plants limits hydrogenase activity of Rhizobium leguminosarum bv. viciae bacteroids by affecting the processing of the hydrogenase structural subunits

“…viciae and Rhizobium etli in symbiosis with peas and beans, respectively (25). The DNA region spanning the H2 uptake gene cluster has been sequenced, and 17 genes, closely linked and oriented in the same direction, were identified (15,38,40). The first two genes, hupS and hupL, encode the hydrogenase structural polypeptides (14).…”

“…The precise molecular functions of the remaining 15 genes, named hupCDEFGHIJK and hypABFCDE, are unknown, although analysis of TnS insertion mutants indicates that they are required for the synthesis of an active hydrogenase (14,38,40). Besides the hydrogenase structural genes, accessory genes homologous to the R leguminosarum hup and hyp genes have also been identified in other bacteria (see references 9 and 46 for reviews).…”