A leucine biosynthesis mutant of Rhizobium tropici CIAT899 which survives at pH 3.5.

Abstract

To investigate whether leucine biosynthesis plays a role in survival and growth under acidic conditions, leucine auxotrophs were produced from acid-tolerant Rhizobium tropici CIAT899. An internal fragment of the R. tropici leuA gene was PCR amplified using degenerate primers and cloned into an integrative plasmid for mutagenesis of the locus. Sequence and homology analysis confirmed the identity of the 550-bp gene fragment, with highest similarity (86% identity) to leuA from Sinorhizobium meliloti. The resultant mutants were strict leucine auxotrophs when tested on solid and liquid media. The mutants could be divided into two groups dependent on their ability to grow in the presence of leucine at both pH 6.8 and pH 5.5, or only at pH 5.5. One mutant, HS20, which could only grow properly in the presence of leucine at pH 5.5, was tested for its acid tolerance. When inoculated into medium at pH 3.5, the mutant was able to survive and to alter the pH from 3.5 to 3.8 whereas the wildtype could not. The wildtype was able to grow at pH 4.0, 4.5 and 5.0 without raising the extracellular pH, indicating that alteration of extracellular pH is not one of the strategies used by the wildtype for growth under acidic conditions. The ability of the mutant to raise the extracellular pH from 3.5 to 3.8 may enable it to survive at the extreme of its pH range of growth.