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Evidence for redundancy in cysteine biosynthesis in Rhizobium leguminosarum RL3841: analysis of a cysE gene encoding serine acetyltransferase

Microbiology Division, School of Animal and Microbial Sciences, The University of Reading, Reading RG6 6AJ, UK¹

Author for correspondence: R. L. Robson. Tel: +44 118 9316639. Fax: +44 118 9316562. e-mail: r.l.robson{at}reading.ac.uk

A cysE gene encoding a serine acetyltransferase (SAT) potentially involved in the biosynthesis of cysteine was identified 4 kb upstream of the previously described aapJQMP gene cluster that encodes an amino acid permease in Rhizobium leguminosarum strain 3841. The gene exhibits >40% identity to the family of SATs containing N-terminal extensions that have been described for other bacteria and plants. The ORF has three possible translation initiation sites which potentially encode polypeptides of 311, 277 and/or 259 amino acid residues, respectively. All three ORFs complemented the cysE mutation in an Escherichia coli cysteine auxotroph, strain JM39. Insertion of Tn5-lacZ into cysE in the genome of R. leguminosarum (strain RU632) lowered SAT activity in crude extracts by >95%. However, RU632 was not a cysteine auxotroph, which suggests that R. leguminosarum possesses some redundancy in cysteine biosynthesis. Additional copies of cysE could not be detected in the genome when the R. leguminosarum cysE gene was used as a hybridization probe. Therefore it is possible that R. leguminosarum possesses an alternative pathway for cysteine biosynthesis which avoids O-acetylserine. Strain RU632 was unaffected in its ability to nodulate Pisum sativum, and the nodules were effective for N₂ fixation (measured by C₂H₂ reduction). Transcriptional activity of cysE was determined by measuring the ß-galactosidase arising from cysE::Tn5-lacZ fusions. Maximal levels of expression were observed during early exponential growth and were not influenced by the level of sulphur (supplied as sulphate). However, transcription was repressed by approximately twofold in ammonium-grown, as opposed to glutamate-grown, cultures. Repression by ammonium was not seen in a strain defective for ntrC.

Keywords: sulphur metabolism, symbiotic nitrogen fixation, SAT, nodulation

Abbreviations: DTNB, 5,5'-dithobis-(2-nitrobenzoic acid); SAT, serine acetyltransferase

The GenBank accession number for the sequence reported in this paper is AJ271648.