Function and regulation of the nolA gene of Bradyrhizobium japonicum

Two Bradyrhizobium japonicum nolA mutants were constructed and used to test the functional role of NolA in nodulation. Contrary to the previous hypothesis that NolA acts as a repressor of nod gene transcription, the expression of a nodD1-lacZ or nodY-lacZ fusion in the nolA mutant strains was similar to that found in the wild type. However, NolA does appear to act as a transcriptional regulatory protein since it is required for its own expression, as well as that of nodD2. Expression of NodD2 from a constitutive promoter led to a significant reduction in nodC-lacZ activity. Therefore, the repression of nod gene expression by NolA is likely an indirect effect, perhaps mediated by other genes (e.g., nodD2) that are regulated by NolA. When inoculated onto soybean (Glycine max cultivar Essex) roots, the nolA mutant strains showed only a slight delay in nodulation as compared to the wild type. However, the mutant strains were grossly defective in nodulation and nitrogen fixation on cowpea (Vigna unguiculata cultivar Caloona) plants. Microscopic examination of soybean nodules induced by the nolA mutant strains showed similar developmental and morphological characteristics to nodules formed by the wild type with only a slight delay in bacteroid maturation. In contrast, cowpea nodules induced by the nolA mutant strains contained fewer infected cells and bacteroids were not found in a typical symbiosome structure. These results indicate that NolA is a transcriptional activator required for the expression of genes that play a role not only in the early stages of infection, but also during the later stages of bacteroid development and maintenance.

Examination of the nolA gene sequence of Bradyrhizobium japonicum shows the presence of two putative ATG start codons. Translation for the first ATG (ATG1) would predict a protein (NolA_L) having an N-terminal, helix-turn-helix DNA-binding motif which shares sequence similarity to the DNA-binding domains of the MerR-type regulatory proteins. Translation from the second ATG (ATG2) would give an N-terminally truncated protein (NolA_s) lacking the DNA-binding domain. To investigate the possibility that nolA encodes two proteins, plasmids encoding N-terminal or C-terminal nolA-lacZ translational fusions were constructed. Expression of lacZ fused to NolA_L prior to ATG2 was inducible by soybean seed extract but not by genistein, a normal isoflavone inducer of nod gene expression. In contrast, β-galactosidase activity resulting from a C-terminal fusion of lacZ to nolA was not inducible by soybean seed extract but did require the presence of a functional NolA. These results indicate that nolA can be translated from two ATG start codons and that the expression of NolA_L and NolA_s is differentially regulated. Consistent with these results, western blot analysis of B. japonicum cell lysates revealed two immunoreactive bands of 29kd and 27 kd that were absent in lysates prepared from a nolA mutant strain. Primer extension experiments revealed that nolA is transcribed from two promoters, one of which is immediately downstream of a putative NolA_L DNA-binding site. Similarly, nodD2, which requires NolA for expression, is also transcribed from a site immediately downstream of a putative NolA_L DNA-binding site. Since only NolA_L would contain a helix-turn-helix DNA-binding domain, these results suggest that nolA and nodD2 require NolA_L for transcription.