Dissection of the Bradyrhizobium japonicum NifA+σ54 regulon, and identification of a ferredoxin gene (fdxN) for symbiotic nitrogen fixation

Abstract

Hierarchically organized regulatory proteins form a complex network for expression control of symbiotic and accessory genes in the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum. A genome-wide survey of regulatory interactions was made possible with the design of a custom-made gene chip. Here, we report the first use of the microarray in a comprehensive and complete characterization of the B. japonicum NifA+sigma(54) regulon which forms an important node in the entire network. Comparative transcript profiles of anaerobically grown wild-type, nifA, and rpoN (1/2) mutant cells were complemented with a position-specific frequency matrix-based search for NifA- and sigma(54)-binding sites plus a simple operon definition. One of the newly identified NifA+sigma(54)-dependent genes, fdxN, encodes a ferredoxin required for efficient symbiotic nitrogen fixation, which makes it a candidate for being a direct electron donor to nitrogenase. The fdxN gene has an unconventional, albeit functional sigma(54 )promoter with the dinucleotide GA instead of the consensus GC motif at position -12. A GC-containing mutant promoter and the atypical GA-containing promoter of the wild type were disparately activated. Expression analyses were also carried out with two other NifA+sigma(54) targets (ectC; ahpC). Incidentally, the tiling-like design of the microarray has helped to arrive at completely revised annotations of the ectC- and ahpC-upstream DNA regions, which are now compatible with promoter locations. Taken together, the approaches used here led to a substantial expansion of the NifA+sigma(54 )regulon size, culminating in a total of 65 genes for nitrogen fixation and diverse other processes.

Characterization of the Flavonoid-Responsive Regulator FrrA and Its Binding Sites

“…B. japonicum cells were harvested 8 h after induction with 1 M genistein. Total RNA was isolated as described previously (8,41). Reverse transcription was carried out with Superscript II reverse transcriptase (Invitrogen) at 42°C for 50 min using the gene-specific primer SP1 ( Table 1).…”

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

“…B. japonicum was grown aerobically and microaerobically (0.5% O 2 in the gas phase) in a modified peptone-salts-yeast extract medium (59) (17) supplemented with 10 mM KNO 3 was used for B. japonicum growth under anoxic conditions (N 2 in the gas phase). Further details relevant for the growth of microaerobic and anaerobic cultures in transcriptome analyses have been described elsewhere (37,56). The concentrations of antibiotics in B. japonicum cultures were as follows (in g per ml): chloramphenicol, 20; spectinomycin, 100; kanamycin, 100; streptomycin, 50; and tetracycline, 50 (solid medium) or 25 (liquid medium).…”

“…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).…”

Posttranslational control of transcription factor FixK 2 , a key regulator for the Bradyrhizobium japonicum –soybean symbiosis

“…Harvest and storage of cells and subsequent RNA extraction were done as described in ref. 44. RNA was then purified, reverse-transcribed to cDNA, processed to fragments, and hybridized to a whole-genome B. japonicum Affymetrix GeneChip (44).…”

“…44. RNA was then purified, reverse-transcribed to cDNA, processed to fragments, and hybridized to a whole-genome B. japonicum Affymetrix GeneChip (44). For each strain, a minimum of three biological replicates was prepared.…”