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Bradyrhizobium japonicum has two differentially regulated, functional homologs of the sigma 54 gene (rpoN).

ABSTRACT: Recognition of -24/-12-type promoters by RNA polymerase requires a special sigma factor, sigma 54 (RpoN NtrA GlnF). In the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum, two functional, highly conserved rpoN genes (rpoN1 and rpoN2) were identified and sequenced. The two predicted B. japonicum RpoN protein sequences were 87% identical, and both showed different levels of homology to the RpoN proteins of other bacteria. Downstream of rpoN2 (but not of rpoN1), two additional open reading frames were identified that corresponded to open reading frames located at similar positions in Klebsiella pneumoniae and Pseudomonas putida. Both B. japonicum rpoN genes complemented the succinate- and nitrate-negative phenotypes of a Rhizobium meliloti rpoN mutant. B. japonicum strains carrying single or double rpoN mutations were still able to utilize C4-dicarboxylates as a carbon source and histidine, proline, or arginine as a nitrogen source, whereas the ability to assimilate nitrate required expression of at least one of the two rpN genes. In symbiosis both rpoN genes could replace each other functionally. The rpoN1/2 double mutant induced about twice as many nodules on soybeans as did the wild type, and these nodules lacked nitrogen fixation activity completely. Transcription of a nifH'-'lacZ fusion was not activated in the rpoN1/2 mutant background, whereas expression of a fixR'-'lacZ fusion in this mutant was affected only marginally. By using rpoN'-'lacZ fusions, rpoN1 expression was shown to be activated at least sevenfold in microaerobiosis as compared with that in aerobiosis, and this type of regulation involved fixLJ. Expression of rpoN2 was observed under all conditions tested and was increased fivefold in an rpoN2 mutant. The data suggested that the rpoN1 gene was regulated in response to oxygen, whereas the rpoN2 gene was negatively autoregulated.

SUBMITTER: Kullik I

PROVIDER: S-EPMC207233 | biostudies-other | 1991 Feb

REPOSITORIES: biostudies-other

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Bradyrhizobium japonicum has two differentially regulated, functional homologs of the sigma 54 gene (rpoN).

Kullik I I Fritsche S S Knobel H H Sanjuan J J Hennecke H H Fischer H M HM

Journal of bacteriology 19910201 3

Recognition of -24/-12-type promoters by RNA polymerase requires a special sigma factor, sigma 54 (RpoN NtrA GlnF). In the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum, two functional, highly conserved rpoN genes (rpoN1 and rpoN2) were identified and sequenced. The two predicted B. japonicum RpoN protein sequences were 87% identical, and both showed different levels of homology to the RpoN proteins of other bacteria. Downstream of rpoN2 (but not of rpoN1), two additional open readin ...[more]

PMID: 1991712

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Project description:Copper-limiting growth conditions were thought to cause an induction of genes possibly involved in copper uptake and sorting. This rationale in mind, we performed microarray analyses on B. japonicum cells grown in three variations of the BVM minimal medium. Variant 1 contained 2 M-NM-<M CuSO4 (copper excess). Variant 2 was prepared in HCl-treated glassware without any copper added (copper starvation). The residual copper concentration in this copper-starvation medium was analyzed by GF-AAS and determined to be 5 nM. Variant 3 (extreme copper limitation) was prepared like variant 2 but with the addition of 10 M-NM-<M BCS and 1 mM ascorbic acid where BCS chelates Cu(I) selectively, and ascorbic acid reduces any Cu(II) to Cu(I). Changes in the transcription profiles were recorded by the pairwise comparison of cells grown in variant 2 vs. 1, and variant 3 vs. 2. Only a small set of genes were differentially up- or down-regulated when copper-starved cells were compared with cells grown in copper excess. Most notably, five genes located adjacent to each other on the B. japonicum genome displayed an increased expression: bll4882 to bll4878. The five genes were named pcuA, pcuB, pcuC, pcuD, and pcuE (mnemonic of proteins for Cu trafficking). The genes with decreased expression are either of unknown function or M-bM-^@M-^S not surprisingly M-bM-^@M-^S play a role in copper resistance. Extreme copper limitation (variant 3 vs. 2) did not further enhance the expression of the five pcu genes. Instead, another cluster of adjacent genes was strongly up-regulated: bll0889 to bll0883, which code for unidentified transport functions. Incidentally, the list also includes the copper chaperone ScoI. Taken together, copper-limiting growth conditions have led to the de-repression of genes potentially involved in copper acquisition. Microarray-based transcriptome analysis of B. japonicum 110spc4 wild-type cells grown under normal, copper-limiting and copper excess conditions

Project description: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 (> or = 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.

Dataset Information

Bradyrhizobium japonicum has two differentially regulated, functional homologs of the sigma 54 gene (rpoN).

Publications

Bradyrhizobium japonicum has two differentially regulated, functional homologs of the sigma 54 gene (rpoN).

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