One of two hemN genes in Bradyrhizobium japonicum is functional during anaerobic growth and in symbiosis

Previously, we screened the symbiotic gene region of the Bradyrhizobium jap onicum chromosome for new NifA-dependent genes by competitive DNA-RNA hybri dization (A. Nienaber, A. Huber, M. Gottfert, B. Hennecke, and H. M. Fische r, J. Bacteriol. 182:1472-1480, 2000). Here we report more details on one o f the genes identified, a hemN-like gene (now called hemN(1)) whose product exhibits significant similarity to oxygen-independent coproporphyrinogen I II dehydrogenases involved in heme biosynthesis in facultatively anaerobic bacteria. In the course of these studies, we discovered that B. japonicum p ossesses a second hemN-like gene (hemN(2)), which was then cloned by using hemN(1) as a probe. The hemN(2) gene maps outside of the symbiotic gene reg ion; it is located 1.5 kb upstream of nirK, the gene for a Cu-containing ni trite reductase. The two deduced HemN proteins are similar in size (445 and 450 amino acids for HemN(1) and HemN(2), respectively) and share 53% ident ical (68% similar) amino acids. Expression of both hemN genes was monitored with the help of chromosomally integrated translational lacZ fusions. No s ignificant expression of either gene was detected in aerobically grown cell s, whereas both genes were strongly induced (greater than or equal to 20-fo ld) under microaerobic or anaerobic conditions. Induction was in both cases dependent on the transcriptional activator protein FixK(2). In addition, m aximal anaerobic hemN(1) expression was partially dependent on NifA, which explains why this gene had been identified by the competitive DNA-RNA hybri dization approach. Strains were constructed carrying null mutations either in individual hemN genes or simultaneously in both genes. All mutants showe d normal growth in rich medium under aerobic conditions. Unlike the hemN(1) mutant, strains lacking a functional hemN(2) gene were unable to grow anae robically under nitrate-respiring conditions and largely failed to fix nitr ogen in symbiosis with the soybean host plant. Moreover, these mutants lack ed several c-type cytochromes which are normally detectable by heme stainin g of proteins from anaerobically grown wild-type cells. Taken together, our results revealed that B. japonicum hemN(2), but not hemN(1), encodes a pro tein that is functional under the conditions tested, and this conclusion wa s further corroborated by the successful complementation of a Salmonella en terica serovar Typhimurium hemF hemN mutant with hemN(2) only.