Nickel availability and hupSL activation by heterologous regulators limit symbiotic expression of the Rhizobium leguminosarum bv. viciae hydrogenase system in Hup(-) rhizobia

Abstract

A limited number of Rhizobium and Bradyrhizobium strains possess a hydrogen uptake (Hup) system that recycles the hydrogen released from the nitrogen fixation process in legume nodules. To extend this ability to rhizobia that nodulate agronomically important crops, we investigated factors that affect the expression of a cosmid-borne Hup system from Rhizobium leguminosarum bv. viciae UPM791 in R. leguminosarum bv. viciae, Rhizobium etli, Mesorhizobium loti, and Sinorhizobium meliloti Hup(-) strains. After cosmid pAL618 carrying the entire hup system of strain UPM791 was introduced, all recipient strains acquired the ability to oxidize H(2) in symbioses with their hosts, although the levels of hydrogenase activity were found to be strain and species dependent. The levels of hydrogenase activity were correlated with the levels of nickel-dependent processing of the hydrogenase structural polypeptides and with transcription of structural genes. Expression of the NifA-dependent hupSL promoter varied depending on the genetic background, while the hyp operon, which is controlled by the FnrN transcriptional regulator, was expressed at similar levels in all recipient strains. With the exception of the R. etli-bean symbiosis, the availability of nickel to bacteroids strongly affected hydrogenase processing and activity in the systems tested. Our results indicate that efficient transcriptional activation by heterologous regulators and processing of the hydrogenase as a function of the availability of nickel to the bacteroid are relevant factors that affect hydrogenase expression in heterologous rhizobia.

FIG. 1

Physical and genetic map of the hydrogenase gene cluster from R. leguminosarum bv. viciae UPM791 cloned in cosmid pAL618. The horizontal arrows below the pAL618 restriction map indicate the locations and orientations of the hup and hyp genes. The shaded arrows indicate genes whose encoded products were detected by an immunoblot analysis in this study. The thin arrows indicate promoters that control symbiotic expression of hydrogenase structural genes (P₁) (19) or microaerobic expression of the hypBFCDEX operon (P₅) (18). The vertical lines with black triangles indicate the sites of insertion and the orientations of the lacZ gene in pAL618-derived fusion constructs pHL55, pHL14, and pHL12 (31). Restriction sites: E, EcoRI; H, HindIII; X, XhoI.

FIG. 2

Immunological detection of HupL and HypB proteins in heterologous rhizobia carrying hup cosmid pAL618. Immunoreactive bands were detected by immunoblotting after bacteroid crude cell extracts were resolved in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Blots were developed with antisera raised against HupL (A) or HypB (B). The bacteroids used to prepare cell extracts were obtained from nodules of plants grown in standard nutrient solutions (lanes 1, 3, 5, 7, 9, 11, and 13) or in solutions supplemented with 170 μM NiCl₂ (lanes 2, 4, 6, 8, 10, 12, and 14). The numbers on the left indicate the molecular masses (in kilodaltons) of the different bands, as deduced from comparisons with standard molecular weight markers. The strains used were R. leguminosarum bv. viciae UPM791(pAL618) (lanes 1 and 2), UML2(pAL618) (lanes 3 and 4), and PRE(pAL618) (lanes 5 and 6); R. etli CFN42(pAL618) (lanes 7 and 8); S. meliloti 102F34(pAL618) (lanes 9 and 10); and M. loti Y3(pAL618) (lanes 11 and 12) and U226(pAL618) (lanes 13 and 14).