Use of a promoter-probe vector system in the cloning of a new NifA-dependent promoter ( ndp) from Bradyrhizobium japonicum

Abstract

Many of the symbiotic nitrogen-fixation genes in the soybean root nodule bacterium, Bradyrhizobium japonicum, are transcribed from -24/-12 promoters that are recognized by the sigma 54-RNA polymerase and activated by the transcriptional regulator protein, NifA. Several lines of evidence suggest that the B. japonicum genome has more than those seven NifA-regulated promoters which were characterized previously. Here, we present a strategy aimed at the cloning of new NifA-activated promoters. It makes use of (i) a promoter-probe vector into which random B. japonicum genomic fragments were cloned in front of a promoterless reporter gene and (ii) a screening procedure that allowed us to distinguish constitutive promoters from promoters that were specifically activated by NifA under microaerobic or anaerobic conditions. With certain modifications, the system may be generally applicable to clone positively regulated, anaerobically induced genes. A novel NifA-dependent promoter region (ndp) of B. japonicum was found by these means. The transcription start point was mapped, and its 5'-flanking DNA carried a -24/-12-type promoter sequence plus potential binding sites for NifA and integration host factor. Further transcript analyses confirmed that maximal transcription from this promoter occurred only in the presence of NifA and sigma 54 during anaerobic growth of B. japonicum. In Escherichia coli, expression of beta-galactosidase derived from a transcriptional ndp::lacZ fusion was activated 11-fold by B. japonicum NifA, and this activation also required sigma 54 but was independent of NtrC. The DNA around ndp shared no similarity with known sequences in databases.(ABSTRACT TRUNCATED AT 250 WORDS)