Enhanced nitrogenase activity in strains of Rhodobacter capsulatus that overexpress the rnf genes

Abstract

In the photosynthetic bacterium Rhodobacter capsulatus, a putative membrane-bound complex encoded by the rnfABCDGEH operon is thought to be dedicated to electron transport to nitrogenase. In this study, the whole rnf operon was cloned under the control of the nifH promoter in plasmid pNR117 and expressed in several rnf mutants. Complementation analysis demonstrated that transconjugants which integrated plasmid pNR117 directed effective biosynthesis of a functionally competent complex in R. capsulatus. Moreover, it was found that strains carrying pNR117 displayed nitrogenase activities 50 to 100% higher than the wild-type level. The results of radioactive labeling experiments indicated that the intracellular content of nitrogenase polypeptides was marginally altered in strains containing pNR117, whereas the levels of the RnfB and RnfC proteins present in the membrane were four- and twofold, respectively, higher than the wild-type level. Hence, the enhancement of in vivo nitrogenase activity was correlated with a commensurate overproduction of the Rnf polypeptides. In vitro nitrogenase assays performed in the presence of an artificial electron donor indicated that the catalytic activity of the enzyme was not increased in strains overproducing the Rnf polypeptides. It is proposed that the supply of reductants through the Rnf complex might be rate limiting for nitrogenase activity in vivo. Immunoprecipitation experiments performed on solubilized membrane proteins revealed that RnfB and RnfC are associated with each other and with additional polypeptides which may be components of the membrane-bound complex.

FIG. 1

pNR117-mediated complementation of rnf mutant strains. (A) Western blot analysis of whole-cell protein extracts using anti-RnfC antibodies. Odd numbers refer to strains B10S, R363B, R386I, R368I, KS92I, and R219BBII carrying pNF3 in this order; even numbers refer to the same strains carrying pNR117. (B) Nitrogen fixation abilities of the complemented strains. +, Nif⁺ phenotype; −, Nif⁻ phenotype.

FIG. 2

Southern blot analysis of genomic DNA from pNR117-containing transconjugants. Genomic DNA from each transconjugant was digested by HindIII and NdeI, electrophoresed on a 0.8% agarose gel, and transferred onto a Hybond-N⁺ membrane. (A) Southern blot with a DIG-labeled rnfB probe. Lane 1, B10S(pNR117); lane 2, R363B(pNR117); lane 3, R386I(pNR117); lane 4, R368I(pNR117); lane 5, KS92I(pNR117); lane 6, R219BBII(pNR117). Sizes of DNA markers are indicated at the right. (B) Predicted physical map of the region containing the rnf genes in each transconjugant, as deducted from Southern blot analysis. The orientation of the interposon cassette in each strain is shown as a boxed arrow. The nifH promoter from the pNR117 plasmid is indicated as a closed triangle. H, HindIII; N, NdeI; Gm, gentamicin.

FIG. 3

SDS-PAGE analysis of ³⁵S-labeled cytoplasmic proteins from pNR117-carrying strains. Resting cells of strains containing or lacking pNR117 were subjected to 3 h of derepression in the presence of a mixture of ³⁵S-labeled Met and Cys. Protein extracts were prepared and analyzed by SDS-PAGE as described in Materials and Methods. Lanes 1 to 4 compare the radioactive band patterns of the cytoplasmic proteins from strains B10S(pNF3), B10S(pNR117), R368I(pNF3), and R368I(pNR117), respectively. Arrows indicate the nitrogenase polypeptides, the α and β subunits of Rc1 (55 and 59.5 kDa), and the Rc2 subunit (33.5 kDa). Sizes of marker proteins are indicated in kilodaltons.

FIG. 4

³⁵S-labeled membrane polypeptides from pNR117-containing strains immunoprecipitated with anti-RnfB or anti-RnfC antibodies. Four strains were subjected to in vivo radioactive labeling as defined in the legend to Fig. 3. Chromatophores were isolated; proteins were solubilized and then incubated with immobilized anti-RnfB or anti-RnfC antibodies as described in Materials and Methods. Membrane proteins captured by immobilized anti-RnfB antibodies or anti-RnfC antibodies were analyzed by SDS-PAGE on a 12.5% polyacrylamide gel and visualized using a PhosphorImager system. Lanes 1 to 4 were loaded with proteins from strains B10S(pNF3), B10S(pNR117), R368I(pNF3), and R368I(pNR117), respectively. RnfB and RnfC polypeptides are marked with arrows. Triangles indicate coimmunoprecipitated polypeptides. Sizes of marker proteins are indicated in kilodaltons.

FIG. 5

Levels of ³⁵S-labeled RnfB and RnfC polypeptides recovered from strains containing or lacking pNR117. In vivo-labeled membrane proteins from four strains were prepared and then subjected to immunoprecipitation with immobilized anti-RnfC and anti-RnfB antibodies as defined in the legend to Fig. 4. After separation by SDS-PAGE, the radioactivity incorporated in the RnfB and RnfC bands was quantified by integration of the PhosphorImager signals. Standard errors calculated from three independent experiments are shown.

FIG. 6

In vivo and in vitro nitrogenase activity of pNR117-containing strains. Transconjugants of strains B10S, R386I (rnfB mutant), and R368I (rnfC mutant) carrying pNR117 were derepressed for 4 h, at which time nitrogenase was assayed both in vivo (white bars) and in vitro (gray bars). Strain B10S(pNF3) was used as a control. For in vitro assays, protein extracts were anaerobically prepared (see Materials and Methods), and sufficient purified Rc2 (1.5 nmol) was added to titrate the Rc1 component present in each extract. Activities are expressed as nanomoles of C₂H₂ reduced per minute per milligram of dry weight (in vivo assays) or per milligram of protein (in vitro assays). Standard errors calculated from at least five different experiments are shown.