Overlapping promoters for two different RNA polymerase holoenzymes control Bradyrhizobium japonicum nifA expression

Abstract

The Bradyrhizobium japonicum NifA protein, the central regulator for nitrogen fixation gene expression, is encoded in the fixRnifA operon. This operon is activated during free-living anaerobic growth and in the symbiotic root nodule bacteroid state. In addition, it is expressed in aerobic conditions, albeit at a low level. Here, we report that this pattern of expression is due to the presence of two overlapping promoters: fixRp 1 , which is of the ؊24/؊12 class recognized by the RNA polymerase 54 , and fixRp 2 , which shares homology with the ؊35 and ؊10 regions found in other putative B. japonicum housekeeping promoters. Primer extension analyses showed that fixRp 1 directed the synthesis of a transcript, P1, that starts 12 nucleotides downstream of the ؊12 region. In addition to 54 , P1 was dependent on NifA and low oxygen tension. Transcripts originating from fixRp 2 started at two sites: one coincided with P1, while the most abundant, P2, initiated just two nucleotides further downstream of P1. Expression from fixRp 2 was dependent on the upstream ؊68 promoter region, a region known to bind a putative activator protein, but it was independent of 54 and NifA. This promoter was expressed in aerobic and anaerobic conditions but was not expressed in 30-day-old bacteroids. Mutations in the conserved ؊12 region for the 54 promoter did not show any transcript, because these mutations also disrupted the overlapping ؊10 region of the fixRp 2 promoter. Conversely, mutations at the ؊24 region only affected the 54 -dependent P1 transcript, having no effect on the expression of P2. In the absence of 54 , anaerobic expression from the fixRp 2 promoter was enhanced threefold, suggesting that in the wild-type strain, the two RNA polymerase holoenzymes must compete for binding to the same promoter region.In the soybean root nodule endosymbiont Bradyrhizobium japonicum, as in most of the nitrogen-fixing bacteria, expression of the genes involved in free-living and symbiotic nitrogen fixation (nif and fix) is controlled by the oxygen status of the cell. This control is exerted by regulating both the activity and the synthesis of the central regulatory protein NifA (13,24,29). NifA belongs to the bacterial enhancer-binding protein family of transcriptional regulators that activate promoters recognized by the RNA polymerase holoenzyme containing the alternative sigma factor 54 (E 54 ) (15,21). The 54 promoters are unique among the prokaryotic promoters in having two closely spaced conserved sequences, centered at Ϫ12 and Ϫ24 bp from the transcription start, instead of the more common Ϫ10 and Ϫ35 boxes found in most of the prokaryotic promoters (23,27

Characterization of Azorhizobium caulinodans glnB and glnA genes: involvement of the P(II) protein in symbiotic nitrogen fixation

“…In A. caulinodans, only one glnBA transcriptional start site was detected in primer extension experiments, suggesting that the same site is shared by both promoters. This is relatively unusual but has been reported for the nifA gene of Bradyrhizobium japonicum (7), where the fixRnifA operon is transcribed from two overlapping promoters, recognized by different RNA polymerase holoenzymes, E 54 and E…”

Nucleotide sequence and predicted functions of the entire Sinorhizobium meliloti pSymA megaplasmid

“…We identified an ORF 58% identical to B. japonicum FixR (SMA1757). In B. japonicum, FixR function remains elusive, but its gene is cotranscribed with nifA, is activated during free-living anaerobic growth and during symbiosis, and may encode a 3-oxoacyl-(acyl carrier protein) reductase (39,40).…”

Dissection of the transcription machinery for housekeeping genes of Bradyrhizobium japonicum

“…The fbcFH genes encode the cytochrome bc 1 complex (32). The glnA (6) and glnB (21) genes are involved in nitrogen assimilation, and groESL 2 , groESL 4 , and groESL 5 are members of a multigene family encoding chaperonin-like proteins (2, 10). The etfSL genes code for subunits of the electron transfer flavoprotein complex (34).…”

“…This was mainly based on an alignment of the upstream sequences of the glnA, fbcF, and fixR genes. However, fixR, whose function is unknown, is aerobically expressed only in the presence of a sofar-unidentified activator (4,30). Therefore, that gene may not be preceded by a normal housekeeping promoter sequence.…”

“…Plasmids pRJ9503 and pRJ8516 were used for the determination of the in vitro transcription start sites of the rrn and groESL 4 genes. RNA subjected to primer extension was synthesized as described above for the specific transcription assay with the exception that no radiolabeled nucleoside triphosphate was used.…”