Identification of Two New Genes Involved in Diazotrophic Growth via the Alternative Fe-Only Nitrogenase in the Phototrophic Purple Bacterium Rhodobacter capsulatus

Abstract

Growth of Rhodobacter capsulatus with molecular dinitrogen as the sole N source via the alternative Fe-only nitrogenase requires all seven gene products of the anfHDGK-1-2-3 operon. In contrast to mutant strains carrying lesions in the structural genes of nitrogenase (anfH, anfD, anfG, and anfK), strains defective for either anf1, anf2, or anf3 are still able to reduce the artificial substrate acetylene, although with diminished activity. To obtain further information on the role of Anf1, we screened an R. capsulatus genomic library designed for use in yeast two-hybrid studies with Anf1 as bait. Two genes, which we propose to call ranR and ranT (for genes related to alternative nitrogenase), coding for products that interact with Anf1 were identified. A ranR mutant exhibited a phenotype similar to that of an anf1 mutant strain (no growth with N 2 in the absence of molybdenum, but significant reduction of acetylene via the Fe-only nitrogenase), whereas a ranT mutant retained the ability to grow diazotrophically, but growth was clearly delayed compared to the parental strain. In contrast to the situation for anf1, expression of neither ranR nor ranT was regulated by ammonium or molybdenum. A putative role for Anf1, RanR, and RanT in the acquisition and/or processing of iron in connection with the Fe-only nitrogenase system is discussed.

Coordinated Expression of fdxD and Molybdenum Nitrogenase Genes Promotes Nitrogen Fixation by Rhodobacter capsulatus in the Presence of Oxygen

“…The bacterial strains and plasmids used in this study are listed in Table 1. Methods for conjugational plasmid transfer between Escherichia coli S17-1 and R. capsulatus, the selection of mutants, complex growth media, and antibiotic concentrations were previously described (22)(23)(24). R. capsulatus strains were grown in RCV (Rhodobacter capsulatus V) minimal medium as described previously (25), except that we omitted molybdate and a fixed-nitrogen source.…”

“…Disruption of R. capsulatus genes was achieved by insertion of gentamicin (Gm), kanamycin (Km), and spectinomycin (Sp) cassettes, as described previously (20,24,26). Briefly, the genes of interest were cloned into mobilizable suicide vectors.…”

NifA- and CooA-Coordinated cowN Expression Sustains Nitrogen Fixation by Rhodobacter capsulatus in the Presence of Carbon Monoxide

“…The bacterial strains and plasmids used in this study are listed in Table 1. Methods for conjugational plasmid transfer from Escherichia coli S17-1 to R. capsulatus and for mutant selection were previously described (22)(23)(24). R. capsulatus strains were grown in modified Rhodobacter capsulatus V (RCV) minimal medium as described previously (25).…”

Bacteroidales ectosymbionts of gut flagellates shape the nitrogen-fixing community in dry-wood termites

“…Using the presence of nifH mRNA as an indicator of nifH gene expression and diazotrophic activity, Noda et al (1999) demonstrated that the most highly expressed nitrogenases in the dry-wood termite Neotermes koshunensis (Kalotermitidae) were so-called alternative nitrogenases (anf) in Cluster II, paralogs of classical nitrogenases (nif) that contain neither molybdenum nor vanadium cofactors (Eady, 1996;Sicking et al, 2005). Closely related anfH genes are present also in Cryptotermes domesticus and Glyptotermes fuscus Yamada et al, 2007), but the general pattern of anfH expression in Kalotermitidae and the identity of the bacterium carrying these genes remain unknown.…”