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Journal of Bacteriology, December 1999, p. 7608-7613, Vol. 181, No. 24
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Citrate Synthase Mutants of Sinorhizobium meliloti Are Ineffective and Have Altered Cell Surface Polysaccharides

Michael W. Mortimer,1 Timothy R. McDermott,1,dagger Gregory M. York,2 Graham C. Walker,2 and Michael L. Kahn1,3,*

Institute of Biological Chemistry1 and the Department of Microbiology,3 Washington State University, Pullman, Washington 99164-6340, and Biology Department, Massachusetts Institute of Technology, Cambridge, Massachusetts 021392

Received 5 January 1999/Accepted 22 September 1999

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 using homologous recombination to replace the wild-type gltA with the gltA::kan allele. The resulting strain, CSDX1, was a glutamate auxotroph, and enzyme assays confirmed the absence of a requirement for glutamate. CSDX1 did not grow on succinate, malate, aspartate, pyruvate, or glucose. CSDX1 produced an unusual blue fluorescence on medium containing Calcofluor, which is different 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 produced partially succinylated succinoglycan. CSDX1 was able to form nodules on alfalfa, but these nodules were not able to fix nitrogen. The symbiotic defect of a citrate synthase mutant could thus be due to disruption of the infection process or to the lack of energy generated by the tricarboxylic acid cycle.

* Corresponding author. Mailing address: Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340. Phone: (509) 335-8327. Fax: (509) 335-7643. E-mail: kahn{at}wsu.edu.

dagger Present address: Department of Land Resources and Environmental Sciences, Montana State University, Bozeman, MT 59717.

Journal of Bacteriology, December 1999, p. 7608-7613, Vol. 181, No. 24
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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