Citrate synthase mutants of Sinorhizobium meliloti are ineffective and have altered cell surface polysaccharides

The gltA gene, encoding Sinorhizobium meliloti 104A14 citrate synthase, was isolated by complementing an Escherichia coli gltA mutant, The S. meliloti gltA gene was mutated by inserting a kanamycin resistance gene and then us ing humologous recombination to replace the wild-type gltA with the gltA::k an allele. The resulting strain, CSDX1, was a glutamate auxotroph, and enzy me assays confirmed the absence of a requirement for glutamate, CSDX1 did n ot grow on succinate, malate, aspartate, pyruvate, or glucose. CSDX1 produc ed an unusual blue fluorescence on medium containing Calcofluor, which is d ifferent from the green fluorescence found with 104A14. High concentrations of arabinose (0.4%) or succinate (0.2%) restored the green fluorescence to CSDX1, High-performance liquid chromatography analyses showed that CSDX1 p roduced partially succinylated succinoglycan. CSDX1 was able to form nodule s on alfalfa, but these nodules were not able to fix nitrogen, The symbioti c defect of a citrate synthase mutant could thus be due to disruption of th e infection process or to the lack of energy generated by the tricarboxylic acid cycle.