Nucleic Acids Research: VOLUME 36 ISSUE 22 2008

The Bradyrhizobium japonicum N2 fixation regulatory gene, nifA, was sequenced and Its transcription start site determined. Between the start of transcription and the nifA gene an open reading frame of 278 codons was found and named fixR. A deletion in fixR which allowed transcription into nifA resulted in a 50% reduced Fix activity. The fixRnifA operon was expressed 1n soybean root nodules, in cultures grown anaerobically with nitrate as terminal electron acceptor, in microaerobic cultures, and in aerobic cultures. The transcription start site (+1) was preceded by a characteristic nif(-24/-12)-type promoter consensus sequence. Double base-pair exchanges in the -12 but not in the -24 region resulted in a 'promoter-down' phenotype. A promoter-upstream DNA region between -50 and -148 was essential for maximal promoter activity. Expression from the promoter was not dependent on nifA. We conclude that the fixRnifA promoter is positively controlled, and that it requires a newly postulated transcriptional factor in order to become activated. INTRODUCTION Expression of nitrogen fixation genes is subject to positive control. It has first been shown in the enterobacterium Klebsiella pneumoniae that the product of the nifA gene is required to activate other nif genes (for review see Refs. 1, 2). The presence of a nifA gene has also been demonstrated in several species of the symbiotic, nitrogen-fixing root and stem nodule bacteria belonging to the genera Rhizobium, Bradyrhizobium and Azorhizobium (3, 4, 5, 6). The NifA protein is believed to bind to a characteristic activator sequence (5'-TGT-N1Q-ACA-3'; 7, 8) at a position more than 100 bp upstream of the typical jivf(jvt£,-24/-12) consensus promoter sequence (5'-CTGGCAC-NgTTGCA-3'; 9, 10, 11) thereby activating the initiation of nif gene transcription by RNA polymerase, which in turn is dependent on a specific sigma factor (the product of ntrA [rpoN, glnF]; see Ref. 2 for review). [For reasons outlined 1n the Discussion section we use the term "-24/-12" promoter throughout this paper.] In K. pneumoniae the function of the NifA protein 1s adversely affected by oxygen and intermediate concentrations of C IR L Press Limited, Oxford, England. 8479 Nucleic Acids Research ammonia via the product of the nifL gene (12, 13) which 1s located upstream of nifA and forms part of the nifLA operon (1). Evidence for a similar antagonistic effect of a NifL-like protein in other diazotrophs is lacking. In Bradyrhizobium japonicum a nifL-independent, direct response of nifAmediated nif gene regulation to oxygen has been demonstrated (14). Of particular interest 1s the question how the expression of the nifA gene itself is regulated. In K. pneumoniae the nifLA operon 1s regulated by the global nitrogen control circuitry, which includes that it is activated by the active form of the NtrC protein, whereas 1t is not expressed under conditions of ammonia excess (2). In Rhizobium meiiloti the nifA gene 1s transcribed from two separate promoters (15, 16). One of them (a -24/-12 promoter) is responsible for the expression of a fixABCXnifA transcript and appears to be activatable by the NtrC and N1fA proteins (17). Nevertheless, ntrC mutants of R. meliloti are Fix (17), and this has been explained by the activity of a second promoter located in front of nifA (15, 16). It is not known how transcription from this second promoter is controlled. The present work deals with the analysis of the transcription of the B^ japonicum nifA gene. Since 1t has recently been shown that the expression of B. japonicum nif and fix genes is controlled by oxygen via a mechanism that Involves the NifA protein (5, 14), we wished to know whether or not the nifA gene itself was subject to control by oxygen. We have determined the transcriptional start and the complete nucleotide sequence of the nifA-contain1ng operon and show that it is also expressed aerobically. Expression of this operon does not appear to be dependent on (auto)activation by NifA; however, its promoter carries a -24/-12 consensus sequence and requires an upstream DNA region (between nucleotides -50 and -148 from the transcription start site) for Its maximal activity. The presence, 1n B. japonicum, of an activator protein other than NifA is postulated. MATERIALS AND METHODS Bacterial strains, plasmids and M13 phages These are listed in Table 1. Plasmids constructed in this work are described 1n the text. Media, and growth of E. coll and B. japonicum Growth of E. coli was done routinely in LB medium (18). Selective precultures of E. coll strains used as recipients for M13 phages were grown 1n minimal-medium as described (19). Antibiotics were added at the following concentrations (ug/ml): ampicillin 200, kanamycin 50, streptomycin 50, tetra-