Three New NifA-Regulated Genes in the Bradyrhizobium japonicum Symbiotic Gene Region Discovered by Competitive DNA-RNA Hybridization

Abstract

The so-called symbiotic region of the Bradyrhizobium japonicum chromosome (C. Kündig, H. Hennecke, and M. Göttfert, J. Bacteriol. 175:613-622, 1993) was screened for the presence of genes controlled by the nitrogen fixation regulatory protein NifA. Southern blots of restriction enzyme-digested cosmids that represent an ordered, overlapping library of the symbiotic region were competitively hybridized with in vitro-labeled RNA from anaerobically grown wild-type cells and an excess of RNA isolated either from anaerobically grown nifA and rpoN mutant cells or from aerobically grown wild-type cells. In addition to the previously characterized nif and fix gene clusters, we identified three new NifA-regulated genes that were named nrgA, nrgB, and nrgC (nrg stands for NifA-regulated gene). The latter two probably form an operon, nrgBC. The proteins encoded by nrgC and nrgA exhibited amino acid sequence similarity to bacterial hydroxylases and N-acetyltransferases, respectively. The product of nrgB showed no significant similarity to any protein with a database entry. Primer extension experiments and expression studies with translational lacZ fusions revealed the presence of a functional ؊24/ ؊12-type promoter upstream of nrgA and nrgBC and proved the NifA-and RpoN ( 54 )-dependent transcription of the respective genes. Null mutations introduced into nrgA and nrgBC resulted in mutant strains that exhibited wild-type-like symbiotic properties, including nitrogen fixation, when tested on soybean, cowpea, or mung bean host plants. Thus, the discovery of nrgA and nrgBC further emphasizes the previously suggested role of NifA as an activator of anaerobically induced genes other than the classical nitrogen fixation genes.

Key Role of Bacterial NH 4 + Metabolism in Rhizobium-Plant Symbiosis

“…Instead, if these plants are infected with a nifA mutant strain, SBs are degraded at an early stage (25,58,209). This indicates that the nifA regulatory gene, as in the case of elongated nodules, is required for SB maintenance, while nifH, and thus nitrogenase activity, is not (60,129).…”

Potential Symbiosis-Specific Genes Uncovered by Sequencing a 410-Kilobase DNA Region of the Bradyrhizobium japonicum Chromosome

“…id102 (frxA; probability value, 97) encodes a ferredoxin-like protein (16). Expression was demonstrated for id774r2 (hsfA; probability value, 64 [11]) and id813r1 (nrgB; probability value, 1 [68]). For id725f2 (nolM; probability value, 5 [59]), no data are available that could prove its functionality.…”

The Iron Control Element, Acting in Positive and Negative Control of Iron-RegulatedBradyrhizobium japonicumGenes, Is a Target for the Irr Protein

“…B. japonicum wild-type cells were grown aerobically to mid-exponential phase in PSY medium either lacking iron or supplemented with 50 M FeSO 4 . RNA was isolated and used for reverse transcription as described previously (3,44), using primers 6680-7 and 6680-8 (for bll6680) and 7895-8 and 7895-11 (for blr7895). Extension products were analyzed on 6% denaturing polyacrylamide gels adjacent to sequencing ladders generated with the same oligonucleotides and plasmids pRJ2562 and pRJ2563.…”