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J. Bacteriol. doi:10.1128/JB.00920-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

RelA-dependent (p)ppGpp production controls exoenzyme synthesis in Erwinia carotovora subsp. atroseptica

Department of Biochemistry, Tennis Court Road, University of Cambridge, Cambridge CB2 1QW, UK

^* To whom correspondence should be addressed. Email: mw240{at}mole.bio.cam.ac.uk.

	Abstract

In this report, we investigate the link between nutrient limitation, RelA-mediated (p)ppGpp production, and virulence in the phytopathogen, Erwinia carotovora subsp. atrospectica (Eca). A relA null mutant (JWC7) was constructed by allelic exchange, and we confirmed that, unlike the wild type progenitor, this mutant did not produce elevated levels of (p)ppGpp upon nutrient downshift. However, (p)ppGpp production could be restored in JWC7 during nutrient limitation by supplying relA in trans. During growth on exoenzyme-inducing minimal medium, the relA mutant showed a diminution in secreted pectate lyase and protease activities and a severe defect in motility. The relA mutant was also impaired in its ability to cause rot in potato tubers. In the presence of serine hydroxamate (a competitive inhibitor of seryl tRNA synthase and a potent inducer of the stringent response in wild-type Eca) exoenzyme production was essentially abolished in JWC7, but could be restored in the presence of plasmid-borne relA. The inhibition of exoenzyme production in JWC7 caused by serine hydroxamate could not be overcome by addition of the quorum sensing signal molecule, N-3-oxohexanoyl-L-homoserine lactone. Quantitative RT-PCR analysis of selected RNA species confirmed that the effects of relA on secreted pectate lyase activity and motility could be attributed to a reduction in transcription of the corresponding genes. We conclude that nutrient limitation is a potent environmental cue that triggers (p)ppGpp-dependent exoenzyme production in Eca. Furthermore, our data suggest that nutrient limitation (or rather, (p)ppGpp accumulation) is a pre-requisite for effective quorum sensing-dependent activation of exoenzyme production.