The Bradyrhizobium japonicum hsfA gene exhibits a unique developmental expression pattern in cowpea nodules

The Bradyrhizobium japonicum host-specific fixation gene hsfA was identifie d as essential for nitrogen fixation on cowpea, but not required for nitrog en fixation on soybean or siratro. The DNA sequence of the hsfA promoter co ntains a consensus RpoN, -24/-12 binding site, suggesting the involvement o f a regulatory protein that binds to an upstream activating sequence (UAS). To further explore the regulation of this interesting gene, serial deletio ns of the hsfA promoter were made and fused with the beta -glucuronidase (G US) gene. The HsfA3 deletion, containing 60 bp 5' of the -24/-12 sequence, showed a similar level of GUS expression to that shown by the longest fusio n construct (HsfA1), containing 464 bp of upstream sequence. In contrast, t he HsfA4-GUS fusion, containing only 20 bp 5' of the -24/-12 region, showed no GUS activity, delimiting the location of a putative UAS to a 40-bp regi on. During nodule development, GUS expression first appeared in nodules 12 days postinoculation (dpi) and reached a maximum level of expression in app roximately 17-day-old nodules. By 28 dpi, HsfA-GUS expression had returned to a low, basal level. These data were consistent with the detection of hsf A mRNA by in situ hybridization in 17-day-old nodules, but not in 28-day-ol d nodules. In contrast to the stage-specific expression in cowpea, HsfA-GUS expression increased with nodule development in HsfA3-inoculated soybean. These data indicate that HsfA expression is regulated in cowpea in a unique developmental manner and that the DNA regulatory regions that control this expression are confined to a short, promoter-proximal region.