Characterisation of the cysQ locus of Escherichia coli : cysQ is activated by entry into stationary phase

Abstract

Cysteine biosynthesis is an essential biosynthetic activity of Escherichia coli, as it is the sole pathway for synthesis of reduced sulphur. In this study a mutant, AM77, was found to be a cysteine requiring bradytroph. AM77 had previously been thought to be defective in osmoregulatory trehalose synthesis because of a mini Tn10 insertion in a gene designated otsC. The mini Tn10 insertion causing AM77's cysteine requirement was shown by complementation and sequencing to be within cysQ, a gene required for cysteine biosynthesis and which is immediately to 5' of the putative otsC insertion site. Another eight independently isolated osmosensitive mutants were also found to contain lesions in cysQ, but their phenotypes differed depending on the position of the mini Tn10 insertion and on whether they contained secondary suppressor mutations. No evidence was obtained to support a role for AM77 in osmoregulatory trehalose synthesis, with all mutant phenotypes reversed by the addition of cysteine or other forms of reduced sulphur. The cysQ gene product is a putative phosphatase, and a member of a family of phosphatases which are inhibited by monovalent cations, specifically lithium. In this study it was shown that AM77 had a partially active CysQ protein, the activity of which was inhibited by the addition of NaCl. These results suggested that inhibition of the cysQ product might be a major cause of halosensitivity in E. coli. The transcriptional regulation of cysQ was characterised, and it was found that cysQ was activated by entry into stationary phase. Activation was at least partially modulated by the stationary phase σ factor, σs. Transcription of cysQ was also found to be affected by anaerobiosis, osmolarity and sulphur source. The observations in this study support the previously hypothesised role for cysQ function as a mediator of PAPS, a toxic intermediate in cysteine biosynthesis. In combination with the evidence for stationary phase activation of the cysQ gene, the hypothesis is presented that as well as being required for modulation of cysteine biosynthesis during growth, cysQ has a second role as a "switch", enabling rapid suppression of cysteine biosynthesis during stationary phase, possibly in response to carbon availability.