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Cytochrome aa3 gene regulation in members of the family Rhizobiaceae: comparison of copper and oxygen effects in Bradyrhizobium japonicum and Rhizobium tropici.

ABSTRACT: Dithionite-reduced minus ferricyanide-oxidized difference spectra on membranes from Rhizobium tropici (formerly Rhizobium leguminosarum bv. phaseoli) incubated at progressively lower O2 concentrations showed only a slight concomitant decrease in A603, the alpha-peak of cytochrome aa3. In contrast to previous results on Bradyrhizobium japonicum, R. tropici showed no significant O2-mediated reduction in the level of either coxA transcription or cytochrome aa3 activity (as measured by ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine [TMPD] oxidase) even in the cells incubated at 12.5 microM O2. Bean nodule R. tropici bacteroids contained 65% of the fully aerobic free-living levels of the coxA transcript. Primer extension analyses established the transcription initiation site of the R. tropici coxA genes. Sequence analyses of the regions upstream of the transcription initiation site revealed no homology with previously reported Rhizobiaceae family promoters, including the coxA promoter of B. japonicum. The R. tropici deduced CoxA sequence itself is highly homologous to the B. japonicum and Paracoccus denitrificans CoxA sequences. In both B. japonicum and R. tropici, coxA transcript levels were the same for cells grown with copper (0.02 microM) in the medium or in medium completely devoid of copper. However, a posttranscriptional effect of copper deprivation was observed for both bacteria; difference absorption spectra on membranes from cells grown without copper showed that B. japonicum lacked spectroscopically detectable cytochrome aa3, whereas R. tropici retained approximately 50% of normal cytochrome aa3 levels.

SUBMITTER: Gabel C

PROVIDER: S-EPMC201281 | biostudies-other | 1994 Jan

REPOSITORIES: biostudies-other

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Cytochrome aa3 gene regulation in members of the family Rhizobiaceae: comparison of copper and oxygen effects in Bradyrhizobium japonicum and Rhizobium tropici.

Gabel C C Bittinger M A MA Maier R J RJ

Applied and environmental microbiology 19940101 1

Dithionite-reduced minus ferricyanide-oxidized difference spectra on membranes from Rhizobium tropici (formerly Rhizobium leguminosarum bv. phaseoli) incubated at progressively lower O2 concentrations showed only a slight concomitant decrease in A603, the alpha-peak of cytochrome aa3. In contrast to previous results on Bradyrhizobium japonicum, R. tropici showed no significant O2-mediated reduction in the level of either coxA transcription or cytochrome aa3 activity (as measured by ascorbate-N,N ...[more]

PMID: 8117073

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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:Extracytoplasmic function (ECF) σ factors control the transcription of genes involved in different cellular functions, such as stress responses, metal homeostasis, virulence-related traits, and cell envelope structure. The genome of Bradyrhizobium japonicum, the nitrogen-fixing soybean endosymbiont, encodes 17 putative ECF σ factors belonging to nine different ECF σ factor families. The genes for two of them, ecfQ (bll1028) and ecfF (blr3038), are highly induced in response to the reactive oxygen species hydrogen peroxide (H(2)O(2)) and singlet oxygen ((1)O(2)). The ecfF gene is followed by the predicted anti-σ factor gene osrA (blr3039). Mutants lacking EcfQ, EcfF plus OsrA, OsrA alone, or both σ factors plus OsrA were phenotypically characterized. While the symbiotic properties of all mutants were indistinguishable from the wild type, they showed increased sensitivity to singlet oxygen under free-living conditions. Possible target genes of EcfQ and EcfF were determined by microarray analyses, and candidate genes were compared with the H(2)O(2)-responsive regulon. These experiments disclosed that the two σ factors control rather small and, for the most part, distinct sets of genes, with about half of the genes representing 13% of the members of H(2)O(2)-responsive regulon. To get more insight into transcriptional regulation of both σ factors, the 5' ends of ecfQ and ecfF mRNA were determined. The presence of conserved sequence motifs in the promoter region of ecfQ and genes encoding EcfQ-like σ factors in related α-proteobacteria suggests regulation via a yet unknown transcription factor. By contrast, we have evidence that ecfF is autoregulated by transcription from an EcfF-dependent consensus promoter, and its product is negatively regulated via protein-protein interaction with OsrA. Conserved cysteine residues 129 and 179 of OsrA are required for normal function of OsrA. Cysteine 179 is essential for release of EcfF from an EcfF-OsrA complex upon H(2)O(2) stress while cysteine 129 is possibly needed for EcfF-OsrA interaction.

Dataset Information

Cytochrome aa3 gene regulation in members of the family Rhizobiaceae: comparison of copper and oxygen effects in Bradyrhizobium japonicum and Rhizobium tropici.

Publications

Cytochrome aa3 gene regulation in members of the family Rhizobiaceae: comparison of copper and oxygen effects in Bradyrhizobium japonicum and Rhizobium tropici.

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