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.     

Look who's talking: communication and quorum sensing in the bacterial world

For many years bacteria were considered primarily as autonomous unicellular organisms with little capacity for collective behaviour. However, we now appreciate that bacterial cells are in fact, highly communicative. The generic term 'quorum sensing' has been adopted to describe the bacterial cell-to-cell communication mechanisms which co-ordinate gene expression usually, but not always, when the population has reached a high cell density. Quorum sensing depends on the synthesis of small molecules (often referred to as pheromones or autoinducers) that diffuse in and out of bacterial cells. As the bacterial population density increases, so does the synthesis of quorum sensing signal molecules, and consequently, their concentration in the external environment rises. Once a critical threshold concentration has been reached, a target sensor kinase or response regulator is activated (or repressed) so facilitating the expression of quorum sensing-dependent genes. Quorum sensing enables a bacterial population to mount a co-operative response that improves access to nutrients or specific environmental niches, promotes collective defence against other competitor prokaryotes or eukaryotic defence mechanisms and facilitates survival through differentiation into morphological forms better able to combat environmental threats. Quorum sensing also crosses the prokaryotic-eukaryotic boundary since quorum sensing-dependent signalling can be exploited or inactivated by both plants and mammals.     

Quorum-sensing regulation in rhizobia and its role in symbiotic interactions with legumes

Legume-nodulating bacteria (rhizobia) usually produce N-acyl homoserine lactones, which regulate the induction of gene expression in a quorum-sensing (or population-density)-dependent manner. There is significant diversity in the types of quorum-sensing regulatory systems that are present in different rhizobia and no two independent isolates worked on in detail have the same complement of quorum-sensing genes. The genes regulated by quorum sensing appear to be rather diverse and many are associated with adaptive aspects of physiology that are probably important in the rhizosphere. It is evident that some aspects of rhizobial physiology related to the interaction between rhizobia and legumes are influenced by quorum sensing. However, it also appears that the legumes play an active role, both in terms of interfering with the rhizobial quorum-sensing systems and responding to the signalling molecules made by the bacteria. In this article, we review the diversity of quorum-sensing regulation in rhizobia and the potential role of legumes in influencing and responding to this signalling system.     

Inhibition of Quorum Sensing Mediated Virulence Factors Production in Urinary Pathogen Serratia marcescens PS1 by Marine Sponges

The focal intent of this study was to find out an alternative strategy for the antibiotic usage against bacterial infections. The quorum sensing inhibitory (QSI) activity of marine sponges collected from Palk Bay, India was evaluated against acyl homoserine lactone (AHL) mediated violacein production in Chromobacterium violaceum (ATCC 12472), CV026 and virulence gene expressions in clinical isolate Serratia marcescens PS1. Out of 29 marine sponges tested, the methanol extracts of Aphrocallistes bocagei (TS 8), Haliclona (Gellius) megastoma (TS 25) and Clathria atrasanguinea (TS 27) inhibited the AHL mediated violacein production in C. violaceum (ATCC 12472) and CV026. Further, these sponge extracts inhibited the AHL dependent prodigiosin pigment, virulence enzymes such as protease, hemolysin production and biofilm formation in S. marcescens PS1. However, these sponge extracts were not inhibitory to bacterial growth, which reveals the fact that the QSI activity of these extracts was not related to static or killing effects on bacteria. Based on the obtained results, it is envisaged that the marine sponges could pave the way to prevent quorum sensing (QS) mediated bacterial infections.     

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.     

Acyl-homoserine lactone-dependent eavesdropping promotes competition in a laboratory co-culture model

Many Proteobacteria use acyl-homoserine lactone (AHL)-mediated quorum sensing to activate the production of antibiotics at high cell density. Extracellular factors like antibiotics can be considered public goods shared by individuals within a group. Quorum-sensing control of antibiotic production may be important for protecting a niche or competing for limited resources in mixed bacterial communities. To begin to investigate the role of quorum sensing in interspecies competition, we developed a dual-species co-culture model using the soil saprophytes Burkholderia thailandensis (Bt) and Chromobacterium violaceum (Cv). These bacteria require quorum sensing to activate the production of antimicrobial factors that inhibit growth of the other species. We demonstrate that quorum-sensing-dependent antimicrobials can provide a competitive advantage to either Bt or Cv by inhibiting growth of the other species in co-culture. Although the quorum-sensing signals differ for each species, we show that the promiscuous signal receptor encoded by Cv can sense signals produced by Bt, and that this ability to eavesdrop on Bt can provide Cv an advantage in certain situations. We use an in silico approach to investigate the effect of eavesdropping in competition, and show conditions where early activation of antibiotic production resulting from eavesdropping can promote competitiveness. Our work supports the idea that quorum sensing is important for interspecies competition and that promiscuous signal receptors allow eavesdropping on competitors in mixed microbial habitats.     

Protist predation can favour cooperation within bacterial species

Here, we studied how protist predation affects cooperation in the opportunistic pathogen bacterium Pseudomonas aeruginosa, which uses quorum sensing (QS) cell-to-cell signalling to regulate the production of public goods. By competing wild-type bacteria with QS mutants (cheats), we show that a functioning QS system confers an elevated resistance to predation. Surprisingly, cheats were unable to exploit this resistance in the presence of cooperators, which suggests that resistance does not appear to result from activation of QS-regulated public goods. Instead, elevated resistance of wild-type bacteria was related to the ability to form more predation-resistant biofilms. This could be explained by the expression of QS-regulated resistance traits in densely populated biofilms and floating cell aggregations, or alternatively, by a pleiotropic cost of cheating where less resistant cheats are selectively removed from biofilms. These results show that trophic interactions among species can maintain cooperation within species, and have further implications for P. aeruginosa virulence in environmental reservoirs by potentially enriching the cooperative and highly infective strains with functional QS system.     

Cell-to-cell signaling in intestinal pathogens

In the conventional view of prokaryotic life, bacteria live a unicellular existence, with responses to external stimuli limited to the detection of chemical and physical signals of environmental origin. This view of bacteriology is now recognized as overly simplistic, because bacteria communicate with each other through small "hormone-like" organic compounds referred to as autoinducers (Als). These bacterial cell-to-cell signaling systems were initially described as mechanisms through which bacteria regulate gene expression via cell density, and, therefore, they have been named quorum sensing. When the Als reach a threshold concentration, they interact with regulatory proteins, thereby driving bacterial gene expression. Bacterial intercellular communication provides a mechanism for the regulation of gene expression resulting in coordinated population behavior. The functions controlled by quorum sensing are varied and reflect the needs of a particular species of bacteria inhabiting a given niche. Quorum sensing-controlled processes include bioluminescence, virulence factor expression, biofilm development, and conjugation among others. Enteric pathogens use quorum sensing to regulate genes involved in virulence, such as motility, and type III secretion. Quorum sensing is utilized to sense the presence of the normal intestinal flora and to warrant successful colonization of the host.     

Type III secretion contributes to the pathogenesis of the soft-rot pathogen Erwinia carotovora: partial characterization of the hrp gene cluster

The virulence of soft-rot Erwinia species is dependent mainly upon secreted enzymes such as pectinases, pectin lyases, and proteases that cause maceration of plant tissue. Some soft-rot Erwinia spp. also harbor genes homologous to the hypersensitive reaction and pathogenesis (hrp) gene cluster, encoding components of the type III secretion system. The hrp genes are essential virulence determinants for numerous nonmacerating gram-negative plant pathogens but their role in the virulence of soft-rot Erwinia spp. is not clear. We isolated and characterized 11 hrp genes of Erwinia carotovora subsp. carotovora. Three putative sigmaL-dependent Hrp box promoter sequences were found. The genes were expressed when the bacteria were grown in Hrp-inducing medium. The operon structure of the hrp genes was determined by mRNA hybridization, and the results were in accordance with the location of the Hrp boxes. An E. carotovora strain with mutated hrcC, an essential hrp gene, was constructed. The hrcC- strain was able to multiply and cause disease in Arabidopsis, but the population kinetics were altered so that growth was delayed during the early stages of infection.     

Production of N-acyl Homoserine Lactones and Virulence Factors of Waterborne Aeromonas hydrophila

Aeromonads are inhabitants of aquatic ecosystems and are described as being involved in intestinal disturbances and other infections. The purpose of this study was to investigate the production of N-acyl-homoserine lactone (AHL) signal molecules and some virulence factors, including hemolysins, proteases, extracellular nucleases production and cytotoxicity by waterborne Aeromonas hydrophila. A total of 24 strains isolated from fresh-water or diseased fish were used in the study. The majority A.hydrophila strains produce two AHL molecules (21/24), one is N-butanoyl homoserine lactone (BHL), and the other is N-hexanoyl homoserine lactone (HHL) according to thin-layer chromatography analysis. Among the virulence factors tested, more than 83 % of the isolates produced β haemolysin when inoculated on sheep blood agar, only 50 % of the isolates displayed DNase activity, 75 % of the isolates shown proteolytic activity on skimmed milk plate, and cytotoxic activity was detected in 20 of 24 of the isolates. The strains producing AHLs possessed one or more virulence factors. In conclusion, the production of quorum sensing signal molecules is common among the strains that we examined, and there seems to some relationships between quorum sensing signal production and virulence factors in A. hydrophila.     

Cooperation of two distinct ExpR regulators controls quorum sensing specificity and virulence in the plant pathogen Erwinia carotovora

Quorum sensing, the population density-dependent regulation mediated by N-acylhomoserine lactones (AHSL), is essential for the control of virulence in the plant pathogen Erwinia carotovora ssp. carotovora (Ecc). In Erwinia carotovora ssp. the AHSL signal with an acyl chain of either 6 or 8 carbons is generated by an AHSL synthase, the expI gene product. This work demonstrates that the AHSL receptor, ExpR1, of Ecc strain SCC3193 has strict specificity for the cognate AHSL 3-oxo-C8-HSL. We have also identified a second AHSL receptor (ExpR2) and demonstrate a novel quorum sensing mechanism, where ExpR2 acts synergistically with the previously described ExpR1 to repress virulence gene expression in Ecc. We show that this repression is released by addition of AHSLs and appears to be largely mediated via the negative regulator RsmA. Additionally we show that ExpR2 has the novel property to sense AHSLs with different acyl chain lengths. The expI expR1 double mutant is able to act in response to a number of different AHSLs, while the expI expR2 double mutant can only respond to the cognate signal of Ecc strain SCC3193. These results suggest that Ecc is able to react both to the cognate AHSL signal and the signals produced by other bacterial species.     

粘质沙雷氏菌AS-1中SpnR功能及卤化呋喃对其群体感应的抑制

通过分泌和感知一系列信号分子,细菌能够根据自身菌体密度的变化调控基因的表达,从而控制一系列重要的表现型,包括毒力因子的产生,生物膜的形成以及菌体发光等.这种广泛存在的信号机制被称为群体感应.在沙雷氏菌种中已经发现了多套群体感应机制.粘质沙雷氏菌AS-1从土壤中分离,其中含有LuxI/LuxR的同类蛋白,被称为SpnI/SpnR.粘质沙雷氏菌AS-1合成AHLs分子N-hexanoy1-L-homoserinelactone(C6-HSL)和N-(3.oxohexanoyl)-L-homoserine lactone(3-oxo-C6-HSL)作为其信号分子,通过群体感应感知菌体密度来控制基因的表达.通过基因替代的方法制得了spnR基因破坏的变异株,命名为粘质沙雷氏菌AS-1R.对粘质沙雷氏菌AS-1R的研究表明SpnR蛋白消极的调控沙雷氏菌红色色素的产生,运动性以及生物膜的形成等一系列由群体感应控制的性状:另一方面,作为一种天然的群体感应抑制剂,卤化呋喃能够有效的抑制粘质沙雷氏菌AS-1的群体感应,但并不干扰AHL-SpnR的相互作用.为运用粘质沙雷氏菌群体感应调节抑制其致病性提供了方法和依据,同时也为卤化呋喃对群体感应抑制机理的研究提供了新的思路.     

Quorum-sensing blockade as a strategy for enhancing host defences against bacterial pathogens

Conventional antibiotics target the growth and the basal life processes of bacteria leading to growth arrest and cell death. The selective force that is inherently linked to this mode of action eventually selects out antibiotic-resistant variants. The most obvious alternative to antibiotic-mediated killing or growth inhibition would be to attenuate the bacteria with respect to pathogenicity. The realization that Pseudomonas aeruginosa, and a number of other pathogens, controls much of their virulence arsenal by means of extracellular signal molecules in a process denoted quorum sensing (QS) gave rise to a new 'drug target rush'. Recently, QS has been shown to be involved in the development of tolerance to various antimicrobial treatments and immune modulation. The regulation of virulence via QS confers a strategic advantage over host defences. Consequently, a drug capable of blocking QS is likely to increase the susceptibility of the infecting organism to host defences and its clearance from the host. The use of QS signal blockers to attenuate bacterial pathogenicity, rather than bacterial growth, is therefore highly attractive, particularly with respect to the emergence of multi-antibiotic resistant bacteria.     

Identification, characterization and regulation of two secreted polygalacturonases of the emerging rice pathogen Burkholderia glumae

Burkholderia glumae is an emerging seed-borne rice pathogen in many areas around the world. Previous studies have demonstrated that B. glumae produces two major virulence factors: the phytotoxin toxoflavin and a secreted lipase. This synthesis of both of these factors is regulated by an N-acyl homoserine lactone (AHL)-dependent, cell-density-dependent quorum-sensing regulation system. This study reports the production and secretion of two highly similar endo-polygalacturonases (designated PehA and PehB) by B. glumae. The two enzymes were purified to homogeneity and the corresponding genetic determinants were identified and characterized. When either polygalacturonase gene was inactivated, B. glumae retained rice virulence comparable to that of the wild-type parent strain. Furthermore, the role of AHL-dependent quorum sensing and of plant cell wall degradation compounds in their regulation was investigated.     

Identification of a Pseudomonas sp. that Inhibits RHL System of Quorum Sensing

The production of many Pseudomonas aeruginosa virulence factors and secondary metabolites is regulated in concert with cell density by quorum sensing (QS). Therefore, strategies designed to inhibit QS are promising for the control of diseases. Here, we succeeded in isolating soil bacteria (56 out of 7,000 isolates) capable of inhibiting violacein production by Chromobacterium violaceum CV026. We focused on an isolate identified as a Pseudomonas sp. based on its 16S rRNA nucleotide sequence. A partially purified inhibitor factor(s) derived from culture supernatants consisted of at least three major components by HPLC analysis. A more highly purified preparation (16 μg/ml) specifically inhibited rhl-controlled pyocyanin and rhamnolipid production by wild type P. aeruginosa PAO1 (PAO1) and a QS double mutant PAO-MW1, without affecting growth. A significant inhibitory effect on elastase, protease and biofilm was also observed. These results provide compelling evidence that the inhibitor(s) interferes with the QS system. The identities of the inhibitors remain to be established.     

Halogenated furanones from the red alga, Delisea pulchra, inhibit carbapenem antibiotic synthesis and exoenzyme virulence factor production in the phytopathogen Erwinia carotovora

The plant pathogen Erwinia carotovora regulates expression of virulence factors and antibiotic production via an N-3-oxohexanoyl-L-homoserine lactone (3-oxo-C6-HSL) dependent quorum sensing mechanism. The marine alga Delisea pulchra produces halogenated furanones known to antagonise 3-oxo-C6-HSL activity. We have tested the effects of a halogenated furanone on the production of carbapenem, cellulase and protease in E. carotovora. Despite differences in the regulatory mechanisms controlling carbapenem and exoenzyme production each was inhibited by the algal metabolite. We present evidence to suggest that the furanone dependent inhibition of carbapenem production is a result of the disruption of the 3-oxo-C6-HSL dependent expression of the carABCDEFGH operon.