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Characterization of a fixLJ-regulated Bradyrhizobium japonicum gene sharing similarity with the Escherichia coli fnr and Rhizobium meliloti fixK genes.

ABSTRACT: We describe the cloning, sequencing, regulation, and mutational analysis of a Bradyrhizobium japonicum fixK-like gene whose product belongs to the family of Fnr-Crp-related regulatory proteins. The predicted 237-amino-acid FixK protein was found to share between 28 and 38% sequence identity with the Escherichia coli Fnr protein, other bacterial Fnr-like proteins (FnrN, Anr, and HlyX), and two rhizobial FixK proteins. The B. japonicum fixK-like gene, when expressed from a lac promoter, could functionally complement an fnr mutant strain of E. coli and activate transcription from an fnr-dependent promoter in the E. coli background; this activation was sixfold higher in anaerobic cultures than in aerobically grown cells, a finding that suggested oxygen sensitivity of the FixK protein and was consistent with the presence of a cysteine-rich, putatively oxygen-responsive domain at its N-terminal end. Similar to the situation in Rhizobium meliloti, expression of the fixK gene in B. japonicum was shown to be induced at low O2 tension and this induction was dependent on the two-component regulatory system FixLJ. Despite this dependency, however, a B. japonicum fixK mutant did not have the phenotypic characteristics of B. japonicum fixL and fixJ mutants: the fixK mutant was neither Fix- in symbiosis with soybean plants nor defective in anaerobic respiration with nitrate as the terminal electron acceptor. Also, the fixK mutant was unaffected in the expression of one of the two B. japonicum sigma 54 genes, rpoN1, which was previously shown to be controlled by the fixLJ genes. When fixK was introduced into the B. japonicum fixJ mutant and expressed therein from a constitutive promoter (i.e., uncoupling it from regulation by FixJ), the FixK protein thus synthesized fully restored anaerobic nitrate respiration in that strain. We interpret this to mean that the B. japonicum wild type has two homologs of fixLJ-regulated fixK genes which can functionally substitute for each other.

SUBMITTER: Anthamatten D

PROVIDER: S-EPMC205827 | biostudies-other | 1992 Apr

REPOSITORIES: biostudies-other

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Characterization of a fixLJ-regulated Bradyrhizobium japonicum gene sharing similarity with the Escherichia coli fnr and Rhizobium meliloti fixK genes.

Anthamatten D D Scherb B B Hennecke H H

Journal of bacteriology 19920401 7

We describe the cloning, sequencing, regulation, and mutational analysis of a Bradyrhizobium japonicum fixK-like gene whose product belongs to the family of Fnr-Crp-related regulatory proteins. The predicted 237-amino-acid FixK protein was found to share between 28 and 38% sequence identity with the Escherichia coli Fnr protein, other bacterial Fnr-like proteins (FnrN, Anr, and HlyX), and two rhizobial FixK proteins. The B. japonicum fixK-like gene, when expressed from a lac promoter, could func ...[more]

PMID: 1551834

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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

Dataset Information

Characterization of a fixLJ-regulated Bradyrhizobium japonicum gene sharing similarity with the Escherichia coli fnr and Rhizobium meliloti fixK genes.

Publications

Characterization of a fixLJ-regulated Bradyrhizobium japonicum gene sharing similarity with the Escherichia coli fnr and Rhizobium meliloti fixK genes.

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