Affiliation:
1. Institut für Mikrobiologie, Eidgenössische Technische Hochschule, CH-8092 Zürich, Switzerland1;
2. Departamento de Microbiologia del Suelo y Sistemas Simbioticos, Estacion Experimental del Zaidin, CSIC, E-18080-Granada, Spain2; and
3. Institut für Genetik, Technische Universität Dresden, D-01062 Dresden, Germany3
Abstract
ABSTRACT
Previously, we screened the symbiotic gene region of the
Bradyrhizobium japonicum
chromosome for new NifA-dependent genes by competitive DNA-RNA hybridization (A. Nienaber, A. Huber, M. Göttfert, H. Hennecke, and H. M. Fischer, J. Bacteriol. 182:1472–1480, 2000). Here we report more details on one of the genes identified, a
hemN
-like gene (now called
hemN
1
) whose product exhibits significant similarity to oxygen-independent coproporphyrinogen III dehydrogenases involved in heme biosynthesis in facultatively anaerobic bacteria. In the course of these studies, we discovered that
B. japonicum
possesses 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 region; it is located 1.5 kb upstream of
nirK
, the gene for a Cu-containing nitrite 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% identical (68% similar) amino acids. Expression of both
hemN
genes was monitored with the help of chromosomally integrated translational
lacZ
fusions. No significant expression of either gene was detected in aerobically grown cells, whereas both genes were strongly induced (≥20-fold) under microaerobic or anaerobic conditions. Induction was in both cases dependent on the transcriptional activator protein FixK
2
. In addition, maximal anaerobic
hemN
1
expression was partially dependent on NifA, which explains why this gene had been identified by the competitive DNA-RNA hybridization approach. Strains were constructed carrying null mutations either in individual
hemN
genes or simultaneously in both genes. All mutants showed normal growth in rich medium under aerobic conditions. Unlike the
hemN
1
mutant, strains lacking a functional
hemN
2
gene were unable to grow anaerobically under nitrate-respiring conditions and largely failed to fix nitrogen in symbiosis with the soybean host plant. Moreover, these mutants lacked several
c
-type cytochromes which are normally detectable by heme staining of proteins from anaerobically grown wild-type cells. Taken together, our results revealed that
B. japonicum hemN
2
, but not
hemN
1
, encodes a protein that is functional under the conditions tested, and this conclusion was further corroborated by the successful complementation of a
Salmonella enterica
serovar Typhimurium
hemF hemN
mutant with
hemN
2
only.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology