Identification of a putative LPS-associated cation exporter from Rhizobium leguminosarum bv. viciae

A gene, cpaA, with similarity to calcium proton antiporters has been identified adjacent to lpcAB in Rhizobium leguminosarum. LpcA is a galactosyl transfererase while LpcB is a 2-keto-3-deoxyoctonate transfererase, both of which are required to form the lipopolysaccharide (LPS) core in R. leguminosarum. Mutations in lpcAB result in a rough LPS phenotype with a requirement for elevated calcium concentrations to allow growth, suggesting that truncation of the LPS core exposes a highly negatively charged molecule. This is consistent with the LPS core being one of the main sites for binding calcium in the Gram-negative outer membrane. Strain RU1109 (cpa::Tn5-lacZ) has a normal LPS layer, as measured by silver staining and Western blotting. This indicates that cpaA mutants are not grossly affected in their LPS layer. LacZ fusion analysis indicates that cpaA is constitutively expressed and is not directly regulated by the calcium concentration. Over-expression of cpaA increased the concentration of calcium required for growth, consistent with CpaA mediating calcium export from the cytosol. The location of lpcA, lpcB and cpaA as well as the phenotype of lpcB mutants suggests that CpaA might provide a specific export pathway for calcium to the LPS core.