Rhizobial FixK-like proteins play essential roles in activating genes for endosymbiotic life in legume root nodules, such as genes for micro-oxic respiration. In the facultative soybean symbiont, Bradyrhizobium japonicum, the FixK 2 protein is the key player in a complex regulatory network. The fixK 2 gene itself is activated by the 2-component regulatory system FixLJ in response to a moderate decrease of the oxygen tension, and the FixK 2 protein distributes and amplifies this response to the level of approximately 200 target genes. Unlike other members of the cAMP receptor protein family, to which FixK 2 belongs, the FixK2 protein does not appear to be modulated by small effector molecules. Here, we show that a critical, single cysteine residue (C183) near the DNA-binding domain of FixK 2 confers sensitivity to oxidizing agents and reactive oxygen species. Oxidation-dependent inactivation occurs not only in vitro, as shown with cell-free transcription assays, but also in vivo, as shown by microarray-assisted transcriptome analysis of the FixK 2 regulon. The oxidation mechanism may involve a reversible dimerization by intermolecular disulfide-bridge formation and a direct, irreversible oxidation at the cysteine thiol, depending on the oxidizing agent. Mutational exchange of C183 to alanine renders FixK 2 resistant to oxidation, yet allows full activity, shown again both in vitro and in vivo. We hypothesize that posttranslational modification by reactive oxygen species is a means to counterbalance the cellular pool of active FixK 2, which would otherwise fill unrestrictedly through FixLJ-dependent synthesis.CPR/FNR ͉ gene regulation ͉ nitrogen fixation ͉ nodules ͉ rhizobia T he cAMP receptor protein (CRP)/fumarate-nitrate reductase regulator (FNR) family comprises transcription factors that mainly act as activators in a wide range of bacteria (reviewed in ref. 1). In all cases studied, the active form consists of homodimeric proteins in which each monomer contains an N-terminal sensor domain linked to the C-terminal DNA binding domain via a long ␣-helix that causes dimerization. These regulators control expression of specific sets of genes implicated in a broad spectrum of processes such as oxidative stress response, micro-oxic and anoxic metabolism, carbon catabolism, and stationary phase survival. The response to the respective environmental or intracellular stimuli is usually transduced through an interaction between a signaling molecule and the sensory domain, whereby a conformational change is induced that leads to the binding of the active dimer to operators near the promoters of the target genes (reviewed in ref.2). Three modes of signal perception have been described: (i) direct perception of a stressor, as in the Lactobacillus casei FLP protein (3); (ii) dependency on a prosthetic group such as a [4Fe-4S] 2ϩ cluster or heme in Escherichia coli FNR (4) or Rhodospirillum rubrum CooA (5), respectively; and (iii) binding of a small effector molecule like cAMP for E. coli CRP (6) or 2-oxoglutarate for cyanob...
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 (H2O2) and singlet oxygen (1O2). 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 H2O2-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 H2O2-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 H2O2 stress while cysteine 129 is possibly needed for EcfF-OsrA interaction.
DnaB and DnaI proteins conserved in low-GC content Gram-positive bacteria are apparently involved in helicase loading at the replication initiation site and during the restarting of stalled replication forks. In this study, we found five novel dnaB mutants and three novel dnaI mutants by screening 750 temperature-sensitive Gram-positive Staphylococcus aureus mutants. All of the mutants had a single amino acid substitution in either DnaB or DnaI that controlled temperature-sensitive growth, as confirmed by transduction experiments using phage 80a. DNA synthesis as measured by [ 3 H]thymine incorporation, origin-to-terminus ratios and flow cytometric analysis revealed that the dnaB and dnaI mutants were unable to initiate DNA replication at restrictive temperatures, which is similar to previous findings in Bacillus subtilis. Furthermore, some of the mutants were found to exhibit asynchrony in the initiation of DNA replication. Also, a fraction of the dnaI mutant cells showed arrested replication, and the dnaI mutant tested was sensitive to mitomycin C, which causes DNA lesions. These results suggest that DnaB and DnaI are required not only for replication initiation and but also for regulation of its synchrony, and they provide support for the involvement of DnaI activity in the restart of arrested replication forks in vivo.
Alternative sigma (σ) factors, proteins that recruit RNA polymerase core enzyme to target promoters, are one mechanism by which bacteria transcriptionally regulate groups of genes in response to environmental stimuli. A class of σ(70) proteins, termed extracytoplasmic function (ECF) σ factors, are involved in cellular processes such as bacterial stress responses and virulence. Here, we describe an ECF16 σ factor, EcfS (Blr4928) from the gram-negative soil bacterium Bradyrhizobium japonicum USDA110, that plays a critical role in the establishment of a functional symbiosis with soybean. Nonpolar insertional mutants of ecfS form immature nodules that do not fix nitrogen, a defect that can be successfully complemented by expression of ecfS. Overexpression of the cocistronic gene, tmrS (blr4929), phenocopies the ecfS mutant in planta and, therefore, we propose that TmrS is a negative regulator of EcfS, a determination consistent with the prediction that it encodes an anti-σ factor. Microarray analysis of the ecfS mutant and tmrS overexpressor was used to identify 40 transcripts misregulated in both strains. These transcripts primarily encode proteins of unknown and transport-related functions and may provide insights into the symbiotic defect in these strains.
Several essential Bradyrhizobium japonicum genes for a symbiotic, nitrogen-fixing root-nodule symbiosis are positively controlled under micro-oxic conditions by the FixLJ-FixK(2) regulatory cascade. Negative control is exerted by reactive oxygen species at the level of the FixK(2) protein. Furthermore, we noticed that fixK (2) gene expression is increased in a fixK (2) mutant, suggesting that FixK(2) in the wild type has a negative effect, directly or indirectly, on its own expression. To possibly understand this effect, the transcription pattern of the fixLJ-bll2758-fixK (2) gene region was examined more closely. While fixK (2) gene transcription is activated by FixJ, the bll2758 gene is transcribed from its own promoter in a FixK(2)-dependent manner, and there is no read-through transcription from bll2758 into fixK (2). The bll2758-encoded protein is predicted to be a stand-alone receiver domain of a response regulator, making it a prime candidate for exerting an inhibitory role on the expression of fixK (2). Transcriptome profiling of a bll2758 knock-out mutant revealed, however, that neither fixK (2) itself nor any of the known FixJ- and FixK(2)-dependent target genes is significantly affected in their expression. This precludes a role of the bll2758 product as a so-called FixT-like protein in the inhibition of FixLJ function, as was proposed for Sinorhizobium meliloti and Caulobacter crescentus. Instead, we rationalize that other transcription factors, whose genes are activated by FixK(2), might be involved in the negative autoregulation of fixK (2) gene expression.
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