A Gene Region Conferring Stress Tolerance to Rhizobium leguminosarum bv viciae and Sinorhizobium meliloti

Root nodule bacteria differ in their response to acidity. The inability of the prokaryotic symbiont to survive in acid conditions will be manifested as a lack of nodulation of the host leading to decreased legume productivity. Sinorhizobium meliloti is one of the most acid-sensitive root nodule bacteria, growing only down to pH 5.5, whereas Rhizobium leguminosarum bv viciae is far more acid-tolerant and can grow down to pH 5.0. An understanding of these differences is being investigated at the physiological and genetic level (Glenn et al., 1999). One of the basic questions is whether acid-tolerant strains possess unique genes conferring acid-tolerance or whether they carry adapted versions of genes held in common with acid-sensitive strains. Rhizobial mutants with acid-sensitive phenotypes have been identified using insertional inactivation of genes in S. meliloti and R. leguminosarum. The acid-sensitive mutants R. leguminosarum bv. viciae WR1-14 and Sinorhizobium meliloti RT3-27 were constructed by Tn5 mutagenesis of the wild-type strains WSM710 and WSM419, respectively. These two mutants belong to a “calcium-irrepairable” subclass of acid-sensitive mutants whose acid tolerance cannot be restored by high concentrations of calcium. Each mutant contains a single copy of Tn5 inserted within a gene (actP) encoding a P-type ATPase that belongs to the CPx heavy metal transporting subfamily. Both WR1-14 and RT3-27 show sensitivity specifically to the heavy metal copper; omission of copper from low pH buffered media restores acid tolerance to these strains. The acidor copper -sensitive defect in both R. leguminosarum and S. meliloti can be complemented by a plasmid clone containing the actP gene from S. meliloti. A R. leguminosarum actP-gusA fusion was transcriptionally regulated in a copper-dependent manner in R. leguminosarum but not in S. meliloti. The fusion is activated specifically in response to copper and not to any other heavy metal. In both organisms, downstream to the site of Tn5 insertion is a gene (hmrR) encoding a putative heavy metal regulator. The precise function of the HmrR protein in either Sinorhizobium or Rhizobium has not been determined. Two more partial open reading frames were identified in the R. leguminosarum DNA sequence, one upstream to actP encoded an enzyme (TreY) possibly required for trehalose biosynthesis and another downstream to hmrR encoded an extracytoplasmic sigma factor (EcfR). Additional studies using EcfR mutants R. leguminosarum and Sinorhizobium should provide an indication of the importance of this protein in cell response to stress. Furthermore, the role of EcfR in the regulation of low pH-inducible genes such as phrR (Reeve et al., 1998) and lpiA (Reeve et al., 1999) can now be examined. The significance of actP and its adjacent genes in the stress response of root nodule bacteria will be presented.