New Target Genes Controlled by the Bradyrhizobium japonicum Two-Component Regulatory System RegSR

Abstract

RegSR-like proteins, members of the family of two-component regulatory systems, are present in a large number of proteobacteria in which they globally control gene expression mostly in a redox-responsive manner. The controlled target genes feature an enormous functional diversity. In Bradyrhizobium japonicum, the facultative root nodule symbiont of soybean, RegSR activate the transcription of the nitrogen fixation regulatory gene nifA, thus forming a RegSR-NifA cascade which is part of a complex regulatory network for gene regulation in response to changing oxygen concentrations. Whole-genome transcription profiling was performed here in order to assess the full regulatory scope of RegSR. The comparative analysis of wild-type and ⌬regR cells grown under oxic and microoxic conditions revealed that expression of almost 250 genes is dependent on RegR, a result that underscores the important contribution of RegR to oxygen-or redoxregulated gene expression in B. japonicum. Furthermore, transcription profiling of ⌬regR bacteroids compared with wild-type bacteroids revealed expression changes for about 1,200 genes in young and mature bacteroids. Incidentally, many of these were found to be induced in symbiosis when wild-type bacteroids were compared with free-living, culture-grown wild-type cells, and they appeared to encode diverse functions possibly related to symbiosis and nitrogen fixation. We demonstrated direct RegR-mediated control at promoter regions of several selected target genes by means of DNA binding experiments and in vitro transcription assays, which revealed six novel direct RegR target promoters.

A Two-Component Regulatory System Integrates Redox State and Population Density Sensing in Pseudomonas putida

“…The sequence CGCTGCCAGCC was identified as a potential RoxR box, with the underlined hexamer highly conserved. This sequence differs from sequences proposed in Bradyrhizobium japonicum and Rhodobacter sphaeroides, where a degenerate hexamer followed by a pentamer located at a variable distance has been described as a binding site for the RoxR orthologs RegR and PrrA (10,19). Sequences resembling the consensus can be found both in the cioA and the ddcA promoters (CCC TGCGATCG and CGTGCCATGCC, respectively; nucleotides identical to the consensus sequence are underlined).…”

Comprehensive Assessment of the Regulons Controlled by the FixLJ-FixK2-FixK1Cascade inBradyrhizobium japonicum

“…For the identification of potential FixK 2 binding sites, we used a position-specific frequency matrix (PSFM) consisting of experimentally verified FixK 2 binding sites (see Table S1 in the supplemental material) in combination with a motif prediction algorithm (28). A similar strategy has previously been applied successfully for the identification of NifAϩ 54 -and RegRdependent targets (37,46). Putative promoter regions of 500 bp in length were searched for the PSFM motif.…”

“…B. japonicum cultures were grown to mid-exponential phase, which corresponded to an optical density at 600 nm of 0.4 to 0.5 in microoxic cultures (peptone-salts-yeast extractarabinose medium) and an optical density at 600 nm of 0.175 to 0.225 in anoxic cultures (yeast extract-mannitol-nitrate medium). Cell harvest, isolation of total RNA, cDNA synthesis, fragmentation, labeling, and conditions for microarray hybridization were done as described recently (37,46,56). A description of the custom-designed B. japonicum gene chip BJAPETHa520090 (Affymetrix, Santa Clara, CA) is given elsewhere (37).…”

A Link between Arabinose Utilization and Oxalotrophy in Bradyrhizobium japonicum

“…RNA was extracted from B. japonicum 110spc4 cultures grown in minimal medium, with 20 mM arabinose or 20 mM succinate as the C source, to mid-exponential phase. Cell harvest, RNA extraction, cDNA synthesis, and quantitative PCR (qPCR) analysis were done as described previously (30,31).…”