Altering substrate chain length specificity of an acylhomoserine lactone synthase in bacterial communication

Quorum sensing mediated by specific signal compounds (autoinducers) allows bacteria to monitor their cell density and enables a synchronized regulation of target gene sets. The best studied group of autoinducers are the acylhomoserine lactones (AHSLs), which are central to the regulation of virulence in many plant and animal pathogens. Variation of the acyl side chain of the AHSLs underlies the observed species specificity of this communication system. Here we show that even different strains of the plant pathogen Erwinia carotovora employ different dialects of this language and demonstrate the molecular basis for the acyl chain length specificity of distinct AHSL synthases. Under physiological concentrations, only the cognate AHSL with the "right" acyl chain is recognized as a signal that will switch on virulence genes. Mutagenesis of the AHSL synthase gene expI(SCC1) identified the changes M127T and F69L as sufficient to effectively alter ExpI(SCC1) (an N-3-oxohexanoyl-l-homoserine lactone producer) substrate specificity to that of an N-3-oxooctanoyl-l-homoserine lactone producer. Our data identify critical residues that define the size of the substrate-binding pocket of the AHSL synthase and will help in understanding and manipulating this bacterial language.     

Identification and characterization of Nip, necrosis-inducing virulence protein of Erwinia carotovora subsp. carotovora

Erwinia carotovora subsp. carotovora is a gram-negative bacterium that causes soft rot disease of many cultivated crops. When a collection of E. carotovora subsp. carotovora isolates was analyzed on a Southern blot using the harpin-encoding gene hrpN as probe, several harpinless isolates were found. Regulation of virulence determinants in one of these, strain SCC3193, has been characterized extensively. It is fully virulent on potato and in Arabidopsis thaliana. An RpoS (SigmaS) mutant of SCC3193, producing elevated levels of secreted proteins, was found to cause lesions resembling the hypersensitive response when infiltrated into tobacco leaf tissue. This phenotype was evident only when bacterial cells had been cultivated on solid minimal medium at low pH and temperature. The protein causing'the cell death was purified and sequenced, and the corresponding gene was cloned. The deduced sequence of the necrosis-inducing protein (Nip) showed homology to necrosis- and ethylene-inducing elicitors of fungi and oomycetes. A mutant strain of E. carotovora subsp. carotovora lacking the nip gene showed reduced virulence in potato tuber assay but was unaffected in virulence in potato stem or on other tested host plants.     

A novel plant ferredoxin-like protein and the regulator Hor are quorum-sensing targets in the plant pathogen Erwinia carotovora

Quorum sensing (QS), a population-density-sensing mechanism, controls the production of the main virulence determinants, the plant cell-wall-degrading enzymes (PCWDEs) of the soft-rot phytopathogen Erwinia carotovora subsp. carotovora. In this study, we used random transposon mutagenesis with a gusA reporter construct to identify two new QS-controlled genes encoding the regulator Hor and a plant ferredoxin-like protein, FerE. The QS control of the identified genes was executed by the QS regulators ExpR1 and ExpR2 and mediated by the global repressor RsmA. Hor was shown to contribute to bacterial virulence at least partly through its control of PCWDE production. Our results showed that FerE contributes to oxidative stress tolerance and in planta fitness of the bacteria and suggest that QS could be central to control of oxidative stress tolerance. The presence of the FerE protein appears to be rather unique in heterotrophic bacteria and suggests an acquisition of the corresponding gene from plant host by horizontal gene transfer.     

Transgenic plants producing the bacterial pheromone N-acyl-homoserine lactone exhibit enhanced resistance to the bacterial phytopathogen Erwinia carotovora

Bacterial pheromones, mainly different homoserine lactones, are central to a number of bacterial signaling processes, including those involved in plant pathogenicity. We previously demonstrated that N-oxoacyl-homoserine lactone (OHL) is essential for quorum sensing in the soft-rot phytopathogen Erwinia carotovora. In this pathogen, OHL controls the coordinate activation of genes encoding the main virulence determinants, extracellular plant cell wall degrading enzymes (PCWDEs), in a cell density-dependent manner. We suggest that E. carotovora employ quorum sensing to avoid the premature production of PCWDEs and subsequent activation of plant defense responses. To test whether modulating this sensory system would affect the outcome of a plant-pathogen interaction, we generated transgenic tobacco, producing OHL. This was accomplished by ectopic expression in tobacco of the E. carotovora gene expI, which is responsible for OHL biosynthesis. We show that expI-positive transgenic tobacco lines produced the active pheromone and partially complemented the avirulent phenotype of expI mutants. The OHL-producing tobacco lines exhibited enhanced resistance to infection by wild-type E. carotovora. The results were confirmed by exogenous addition of OHL to wild-type plants, which also resulted in increased resistance to E. carotovora.     

Type II quorum sensing regulates virulence in Erwinia carotovora ssp. carotovora

Quorum sensing is a process by which bacteria communicate using secreted chemical signaling molecules called autoinducers. In this study, the opportunistic plant pathogen Erwinia carotovora ssp. carotovora was observed to secrete type II signaling molecules. A homolog of luxS, the gene required for AI-2 synthesis in Vibrio harveyi, was isolated from the genome of the pathogen. To determine the potential role of AI-2 in virulence, an isogenic luxS- (ECC) mutant was constructed and tested for its ability to cause tissue maceration. The findings reported here demonstrate that the LuxS-dependent signaling affects the progression of disease symptoms during the early stages of infection by modulating the expression of pectinolytic enzymes.     

Potato plants genetically modified to produce N-acylhomoserine lactones increase susceptibility to soft rot erwiniae

Many gram-negative bacteria employ N-acylhomoserine lactones (AHL) to regulate diverse physiological processes in concert with cell population density (quorum sensing [QS]). In the plant pathogen Erwinia carotovora, the AHL synthesized via the carI/expI genes are responsible for regulating the production of secreted plant cell wall-degrading exoenzymes and the antibiotic carbapen-3-em carboxylic acid. We have previously shown that targeting the product of an AHL synthase gene (yenI) from Yersinia enterocolitica to the chloroplasts of transgenic tobacco plants caused the synthesis in planta of the cognate AHL signaling molecules N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL), which in turn, were able to complement a carI-QS mutant. In the present study, we demonstrate that transgenic potato plants containing the yenI gene are also able to express AHL and that the presence and level of these AHL in the plant increases susceptibility to infection by E. carotovora. Susceptibility is further affected by both the bacterial level and the plant tissue under investigation.