Mutations in rpsL that confer streptomycin resistance show pleiotropic effects on virulence and the production of a carbapenem antibiotic in Erwinia carotovora.

Spontaneous streptomycin-resistant derivatives of Erwinia carotovora subsp. carotovora strain ATTn10 were isolated. Sequencing of the rpsL locus (encoding the ribosomal protein S12) showed that each mutant was missense, with a single base change, resulting in the substitution of the wild-type lysine by arginine, threonine or asparagine at codon 43. Phenotypic analyses showed that the rpsL mutants could be segregated into two groups: K43R mutants showed reduced production of the beta-lactam secondary metabolite 1-carbapen-2-em-3 carboxylic acid (Car), but little effect on exoenzyme production or virulence in potato tuber tests. By contrast, the K43N and K43T mutations were pleiotropic, resulting in reduced exoenzyme production and virulence, as well as diminished Car production. The effect on Car production was due to reduced transcription of the quorum-sensing-dependent car biosynthetic genes. The effects of K43N and K43T mutations on Car production were partially alleviated by provision of an excess of the quorum-sensing signalling molecule N-(3-oxohexanoyl)-L-homoserine lactone. Finally, a proteomic analysis of the K43T mutant indicated that the abundance of a subset of intracellular proteins was affected by this rpsL mutation.

Identification of the central quorum sensing regulator of virulence in the enteric phytopathogen, Erwinia carotovora: the VirR repressor.

In the Gram-negative phytopathogen, Erwinia carotovora ssp. atroseptica (Eca) virulence depends on the production of a N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) quorum sensing (QS) signal. This work identifies the elusive 'missing link' between QS and virulence in Erwinia. We have identified and characterized a novel regulator of virulence, VirR, in Eca and show that a virR mutation completely restores virulence factor production to an Eca mutant unable to synthesize OHHL. This effect of the virR mutation translates to a restoration of virulence to wild-type levels and thus provides evidence that VirR acts to prevent the production of virulence factors at low cell density. We also show that, in Eca, transcription of virulence genes is controlled by OHHL and that this control is effected through the action of VirR. We also demonstrate that the VirR regulatory pathway is present and functional in both blackleg and soft rotting species of Erwinia.

Carbapenem antibiotic biosynthesis in Erwinia carotovora is regulated by physiological and genetic factors modulating the quorum sensing-dependent control pathway.

Erwinia carotovora produces the beta-lactam antibiotic, carbapenem, in response to a quorum sensing signalling molecule, N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). We have mapped the OHHL-dependent promoter upstream of the first of the biosynthetic genes, carA. We have also analysed the effect on this promoter of the known genetic regulators of carbapenem expression, carR, carI (encoding homologues of LuxR and LuxI respectively) and hor (encoding a SlyA/MarR-like transcriptional regulator). We describe a previously unknown promoter located within the carA-H operon. This promoter does not respond to CarR and is required for quorum sensing-independent expression of the carbapenem resistance determinants encoded by the carFG genes. We have mapped the carR, carI and hor transcription start points, shown that CarR is positively autoregulated in the presence of OHHL, and have demonstrated negative feedback affecting transcription of carI. In addition, various environmental and physiological factors were shown to impinge on the transcription of the car biosynthetic genes. The nature of the carbon source and the temperature of growth influence carbapenem production by modulating the level of the OHHL signalling molecule, and thereby physiologically fine-tune the quorum sensing regulatory system.

The regulation of virulence in phytopathogenic Erwinia species: quorum sensing, antibiotics and ecological considerations.

Erwinia carotovora is a Gram-negative bacterial phytopathogen that causes soft-rot disease and potato blackleg. The organism is environmentally widespread and exhibits an opportunistic plant pathogenesis. The ability to secrete multiple plant cell wall-degrading enzymes is a key virulence trait and exoenzyme production is responsive to multiple environmental and physiological cues. One important cue is the cell population density of the pathogen. Cell density is monitored via an acylated homoserine lactone (acyl HSL) signalling molecule, which is thought to diffuse between Erwinia cells in a process now commonly known as 'quorum sensing'. This molecule also acts as the chemical communication signal controlling production of a broad-spectrum beta-lactam antibiotic (1-carbapen-2-em-3-carboxylic acid; carbapenem) synthesised in concert with exoenzyme elaboration, possibly for niche defence. In antibiotic production control, quorum sensing acts at the level of transcriptional activation of the antibiotic biosynthetic cluster. This is achieved via a dedicated LuxR-type protein, CarR that is bound to the signalling molecule. The molecular relay connecting acyl HSL production and exoenzyme induction is not clear, despite the identification of a multitude of global regulatory genes, including those of the RsmA/rsmB system, impinging on enzyme synthesis. Quorum sensing control mediated by acyl HSLs is widespread in Gram-negative bacteria and is responsible for the regulation of diverse phenotypes. Although there is still a paucity of meaningful information on acyl HSL availability and in-situ biological function, there is growing evidence that such molecules play significant roles in microbial ecology.