Pseudomonas chlororaphis L5 and Enterobacter asburiae L95 biocontrol Dickeya soft rot diseases by quenching virulence factor modulating quorum sensing signal

Virulence factor modulating (VFM) is a quorum sensing (QS) signal shared by and specific to Dickeya bacteria, regulating the production of plant cell wall degrading enzymes (PCWDEs) and virulence of Dickeya. High polarity and trace of VFM signal increase the difficulty of signal separation and structure identification, and thus limit the development of quorum quenching strategy to biocontrol bacterial soft rot diseases caused by Dickeya. In order to high-throughput screen VFM quenching bacteria, a vfmE-gfp biosensor VR2 (VFM Reporter) sensitive to VFM signal was first constructed. Subsequently, two bacterial strains with high quenching efficiency were screened out by fluorescence intensity measurement and identified as Pseudomonas chlororaphis L5 and Enterobacter asburiae L95 using multilocus sequence analysis (MLSA). L5 and L95 supernatants reduced the expression of vfm genes, and both strains also decreased the production of PCWDEs of D. zeae MS2 and significantly reduced the virulence of D. oryzae EC1 on rice seedlings, D. zeae MS2 on banana seedlings, D. dadantii 3937 on potato and D. fangzhongdai CL3 on taro. Findings in this study provide a method to high-throughput screen VFM quenching bacteria and characterize novel functions of P. chlororaphis and E. asburiae in biocontrolling plant diseases through quenching VFM QS signal.     

Interplay of classic Exp and specific Vfm quorum sensing systems on the phenotypic features of Dickeya solani strains exhibiting different virulence levels

Bacteria from the genus Dickeya cause severe symptoms on numerous economically important plants. Dickeya solani is the Dickeya species most frequently found on infected potato plants in Europe. D. solani strains from different countries show high genetic homogeneity, but significant differences in their virulence level. Dickeya species possess two quorum sensing (QS) mechanisms: the Exp system based on classic N‐acyl‐homoserine lactone (AHL) signals and a specific system depending on the production and perception of a molecule of unknown structure, Virulence Factor Modulating (VFM). To study the interplay between these two QS systems, five D. solani strains exhibiting different virulence levels were selected. Mutants were constructed by inactivating genes coding for each QS system. Double mutants were obtained by simultaneous inactivation of genes coding for both QS systems. Most of the D. solani mutants showed an attenuation of chicory maceration and a decreased production of plant cell wall‐degrading enzymes (PCWDEs) and motility, but to different degrees depending on the strain. The VFM‐QS system seems to regulate virulence in both D. solani and Dickeya dadantii, but the AHL‐QS system has greater effects in D. solani than in D. dadantii. The inactivation of both QS systems in D. solani did not reveal any additive effect on the tested features. The inactivation of vfm genes generally has a more dominant effect relative to that of exp genes. Thus, VFM‐ and AHL‐QS systems do not work in synergy to modulate the production of diverse virulence factors and the ability to macerate plant tissue.     

Differential regulation of toxoflavin production and its role in the enhanced virulence of Burkholderia gladioli

Burkholderia gladioli is a causal agent of bacterial panicle blight and sheath/grain browning in rice in many countries. Many strains produce the yellow pigment toxoflavin, which is highly toxic to plants, fungi, animals and microorganisms. Although there have been several studies on the toxoflavin biosynthesis system of B. glumae, it is still unclear how B. gladioli activates toxoflavin biosynthesis. In this study, we explored the genomic organization of the toxoflavin system of B. gladioli and its biological functions using comparative genomic analysis between toxoflavin‐producing strains (B. glumae BGR1 and B. gladioli BSR3) and a strain not producing toxoflavin (B. gladioli KACC11889). The latter exhibits normal physiological characteristics similar to other B. gladioli strains. Burkholderia gladioli KACC11889 possesses all the genes involved in toxoflavin biosynthesis, but lacks the quorum‐sensing (QS) system that functions as an on/off switch for toxoflavin biosynthesis. These data suggest that B. gladioli has evolved to use the QS signalling cascade of toxoflavin production (TofI/TofR of QS → ToxJ or ToxR → tox operons) similar to that in B. glumae. However, some strains may have evolved to eliminate toxoflavin production through deletion of the QS genes. In addition, we demonstrate that the toxoflavin biosynthetic system enhances the virulence of B. gladioli. These findings provide another line of evidence supporting the differential regulation of the toxoflavin system in Burkholderia strains.     

Heterologous expression of human paraoxonases in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> inhibits biofilm formation and decreases antibiotic resistance

Quorum sensing (QS) regulates virulence and biofilm formation in Pseudomonas aeruginosa and other medically relevant bacteria. Human paraoxonases (hPONs) are a family of closely related enzymes with multiple functions, including inactivation of the QS signal molecule in P. aeruginosa. However, there is no direct evidence to show the functions of hPONs on biofilm formation and antibiotic resistance in P. aeruginosa. In the present study, hPONs (hPON1, hPON2, and hPON3) genes were respectively cloned into the pMEKm12 shuttle vector and transformed into P. aeruginosa strain PAO1. Expression of the three recombinant proteins was confirmed by Western blotting, and growth of the recombinant strains was not affected by the hPONs gene expression. Biofilm formation and antibiotics resistance of the hPONs recombinant strains were analyzed. Our results showed that biofilm formation was significantly inhibited in all of the three hPONs recombinant strains. Interestingly, this inhibition can be reverted by addition of the corresponding hPONs polyclonal antibodies in the culture media, further indicating that the inhibition of biofilm formation was due to hPONs protein expression. In addition, we also demonstrated that hPONs expression decreased resistance of P. aeruginosa to gentamicin and ceftazidima, two antibiotics clinically used for the treatment of P. aeruginosa infection.     

Quorum‐sensing regulation of a capsular polysaccharide receptor for the Rhodobacter capsulatus gene transfer agent (RcGTA)

The gene transfer agent produced by Rhodobacter capsulatus (RcGTA) resembles a small tailed bacteriophage that packages almost random genomic DNA segments that may be transferred to other R. capsulatus cells. Gene transfer agents are produced by a number of prokaryotes; however, no receptors have been identified. We investigated the RcGTA recipient capability of wild‐type R. capsulatus cells at different culture growth phases, and found that the frequency of RcGTA‐dependent acquisition of an allele increases as cultures enter the stationary phase. We also found that RcGTA adsorption to cells follows a similar trend. RcGTA recipient capability and adsorption were found to be dependent on the GtaR/I quorum‐sensing (QS) system. Production of an extracellular polysaccharide was found to be regulated by GtaR/I QS, as was production of the cell capsule. A number of QS‐regulated putative polysaccharide biosynthesis genes were identified, and mutagenesis of two of these genes, rcc01081 and rcc01932, yielded strains that lack a capsule. Furthermore, these mutants were impaired in RcGTA recipient capability and adsorption, as was a non‐encapsulated wild‐type isolate of R. capsulatus. Overall, our results indicate that capsular polysaccharide is a receptor for the gene transfer agent of R. capsulatus, RcGTA.     

Detection of quorum sensing signal molecules in the family Vibrionaceae

Aims: The aim of this study was to detect the production of three kinds of quorum sensing (QS) signal molecules, i.e. the N‐acyl‐homoserine lactone (AHL), the autoinducer‐2 (AI‐2) and the cholerae autoinducer‐1‐like (CAI‐1‐like) molecules in 25 Vibrionaceae strains. Methods and Results: The QS signal molecules in 25 Vibrionaceae strains were detected with different biosensors. Except Salinivibrio costicola VIB288 and Vibrio natriegens VIB299, all the other 23 Vibrionaceae strains could produce one or more kinds of detectable QS signal molecules. Twenty‐one of the 25 strains were found to produce AHL signal molecules by using Vibrio harveyi JMH612 and Agrobacterium tumefaciens KYC55 (pJZ372; pJZ384; pJZ410) as biosensors. The AHL fingerprints of eight strains were detected by thin‐layer chromatography with Ag. tumefaciens KYC55, and two of them, i.e. V. mediterranei VIB296 and Aliivibrio logei VIB414 had a high diversity of AHLs. Twenty of the 25 strains were found to have the AI‐2 activity, and the luxS gene sequences in 18 strains were proved to be conserved by PCR amplification and sequencing. Only six (five Vibrio strains and A. logei VIB414) of the 25 strains possessed the CAI‐1‐like activity. A. logei VIB414, V. campbellii VIB285, V. furnissii VIB293, V. pomeroyi LMG20537 and two V. harveyi strains VIB571 and VIB645 were found to produce all the three kinds of QS signal molecules. Conclusions: The results indicated that the QS signal molecules, especially AHL and AI‐2 molecules, were widespread in the family Vibrionaceae. Significance and Impact of the Study: In response to a variety of environmental conditions and selection forces, the family Vibrionaceae produced QS signal molecules with great diversity and complexity. The knowledge we obtained from this study will be useful for further research on the roles of different QS signal molecules in this family.     

Distinguishing bacterial pathogens of potato using a genome-wide microarray approach

A set of 9676 probes was designed for the most harmful bacterial pathogens of potato and tested in a microarray format. Gene‐specific probes could be designed for all genes of Pectobacterium atrosepticum, c. 50% of the genes of Streptomyces scabies and c. 30% of the genes of Clavibacter michiganensis ssp. sepedonicus utilizing the whole‐genome sequence information available. For Streptomyces turgidiscabies, 226 probes were designed according to the sequences of a pathogenicity island containing important virulence genes. In addition, probes were designed for the virulence‐associated nip (necrosis‐inducing protein) genes of P. atrosepticum, P. carotovorum and Dickeya dadantii and for the intergenic spacer (IGS) sequences of the 16S–23S rRNA gene region. Ralstonia solanacearum was not included in the study, because it is a quarantine organism and is not presently found in Finland, but a few probes were also designed for this species. The probes contained on average 40 target‐specific nucleotides and were synthesized on the array in situ, organized as eight sub‐arrays with an identical set of probes which could be used for hybridization with different samples. All bacteria were readily distinguished using a single channel system for signal detection. Nearly all of the c. 1000 probes designed for C. michiganensis ssp. sepedonicus, c. 50% and 40% of the c. 4000 probes designed for the genes of S. scabies and P. atrosepticum, respectively, and over 100 probes for S. turgidiscabies showed significant signals only with the respective species. P. atrosepticum, P. carotovorum and Dickeya strains were all detected with 110 common probes. By contrast, the strains of these species were found to differ in their signal profiles. Probes targeting the IGS region and nip genes could be used to place strains of Dickeya to two groups, which correlated with differences in virulence. Taken together, the approach of using a custom‐designed, genome‐wide microarray provided a robust means for distinguishing the bacterial pathogens of potato.     

Bacterial cell‐to‐cell signaling promotes the evolution of resistance to parasitic bacteriophages

The evolution of host–parasite interactions could be affected by intraspecies variation between different host and parasite genotypes. Here we studied how bacterial host cell‐to‐cell signaling affects the interaction with parasites using two bacteria‐specific viruses (bacteriophages) and the host bacterium Pseudomonas aeruginosa that communicates by secreting and responding to quorum sensing (QS) signal molecules. We found that a QS‐signaling proficient strain was able to evolve higher levels of resistance to phages during a short‐term selection experiment. This was unlikely driven by demographic effects (mutation supply and encounter rates), as nonsignaling strains reached higher population densities in the absence of phages in our selective environment. Instead, the evolved nonsignaling strains suffered relatively higher growth reduction in the absence of the phage, which could have constrained the phage resistance evolution. Complementation experiments with synthetic signal molecules showed that the Pseudomonas quinolone signal (PQS) improved the growth of nonsignaling bacteria in the presence of a phage, while the activation of las and rhl quorum sensing systems had no effect. Together, these results suggest that QS‐signaling can promote the evolution of phage resistance and that the loss of QS‐signaling could be costly in the presence of phages. Phage–bacteria interactions could therefore indirectly shape the evolution of intraspecies social interactions and PQS‐mediated virulence in P. aeruginosa.     

Intercellular and intracellular signalling systems that globally control the expression of virulence genes in plant pathogenic bacteria

Plant pathogenic bacteria utilize complex signalling systems to control the expression of virulence genes at the cellular level and within populations. Quorum sensing (QS), an important intercellular communication mechanism, is mediated by different types of small molecules, including N‐acyl homoserine lactones (AHLs), fatty acids and small proteins. AHL‐mediated signalling systems dependent on the LuxI and LuxR family proteins play critical roles in the virulence of a wide range of Gram‐negative plant pathogenic bacteria belonging to the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria. Xanthomonas spp. and Xylella fastidiosa, members of the Gammaproteobacteria, however, possess QS systems that are mediated by fatty acid‐type diffusible signal factors (DSFs). Recent studies have demonstrated that Ax21, a 194‐amino‐acid protein in Xanthomonas oryzae pv. oryzae, plays dual functions in activating a rice innate immune pathway through binding to the rice XA21 pattern recognition receptor and in regulating bacterial virulence and biofilm formation as a QS signal molecule. In xanthomonads, DSF‐mediated QS systems are connected with the signalling pathways mediated by cyclic diguanosine monophosphate (c‐di‐GMP), which functions as a second messenger for the control of virulence gene expression in these bacterial pathogens.     

Revisiting the virulence hallmarks of Pseudomonas aeruginosa: a chronicle through the perspective of quorum sensing

Pseudomonas aeruginosa is an opportunistic pathogen and the leading cause of mortality among immunocompromised patients in clinical setups. The hallmarks of virulence in P. aeruginosa encompass six biologically competent attributes that cumulatively drive disease progression in a multistep manner. These multifaceted hallmarks lay the principal foundation for rationalizing the complexities of pseudomonal infections. They include factors for host colonization and bacterial motility, biofilm formation, production of destructive enzymes, toxic secondary metabolites, iron-chelating siderophores and toxins. This arsenal of virulence hallmarks is fostered and stringently regulated by the bacterial signalling system called quorum sensing (QS). The central regulatory functions of QS in controlling the timely expression of these virulence hallmarks for adaptation and survival drive the disease outcome. This review describes the intricate mechanisms of QS in P. aeruginosa and its role in shaping bacterial responses, boosting bacterial fitness. We summarize the virulence hallmarks of P. aeruginosa, relating them with the QS circuitry in clinical infections. We also examine the role of QS in the development of drug resistance and propose a novel antivirulence therapy to combat P. aeruginosa infections. This can prove to be a next-generation therapy that may eventually become refractory to the use of conventional antimicrobial treatments.     

CqsA–CqsS quorum‐sensing signal‐receptor specificity in Photobacterium angustum

Quorum sensing (QS) is a process of bacterial cell–cell communication that relies on the production, detection and population‐wide response to extracellular signal molecules called autoinducers. The QS system commonly found in vibrios and photobacteria consists of the CqsA synthase/CqsS receptor pair. Vibrio cholerae CqsA/S synthesizes and detects (S)‐3‐hydroxytridecan‐4‐one (C10‐CAI‐1), whereas Vibrio harveyi produces and detects a distinct but similar molecule, (Z)‐3‐aminoundec‐2‐en‐4‐one (Ea‐C8‐CAI‐1). To understand the signalling properties of the larger family of CqsA–CqsS pairs, here, we characterize the Photobacterium angustum CqsA/S system. Many photobacterial cqsA genes harbour a conserved frameshift mutation that abolishes CAI‐1 production. By contrast, their cqsS genes are intact. Correcting the P. angustum cqsA reading frame restores production of a mixture of CAI‐1 moieties, including C8‐CAI‐1, C10‐CAI‐1, Ea‐C8‐CAI‐1 and Ea‐C10‐CAI‐1. This signal production profile matches the P. angustum CqsS receptor ligand‐detection capability. The receptor exhibits a preference for molecules with 10‐carbon tails, and the CqsS Ser¹⁶⁸ residue governs this preference. P. angustum can overcome the cqsA frameshift to produce CAI‐1 under particular limiting growth conditions presumably through a ribosome slippage mechanism. Thus, we propose that P. angustum uses CAI‐1 signalling for adaptation to stressful environments.     

植物病原黄单胞菌退出群体感应生理状态的分子机制和生物学意义

黄单胞菌是一类引起多种作物病害的病原细菌总称。它们利用自身产生的DSF(Diffusible signaling factor)-家族群体感应(quorum sensing,QS)信号分子感应群体密度,调控致病相关基因的表达。当黄单胞菌培养达到对数生长后期时,培养体系中DSF信号分子浓度迅速降低,呈现一种典型的群体感应信号反转(turnover)现象。编码脂酰辅酶A连接酶的基因rpfB在黄单胞菌生长后期表达量显著提高,敲除rpfB导致DSF生物合成显著提高,因此,RpfB参与DSF-家族QS信号分子的降解,在群体感应后期引导黄单胞菌退出群体感应状态。DSF信号通过RpfC/RpfG双组分系统、环二鸟苷酸和全局性转录因子Clp负调控rpfB的表达。DSF群体感应信号关键降解基因rpfB存在于多种细菌中,表明该信号反转机制相对保守,但其调控的生物学功能因菌而异。     

PpoR is a conserved unpaired LuxR solo of Pseudomonas putida which binds N-acyl homoserine lactones

Background  

Only a small number of Pseudomonas putida strains possess the typical N-acyl homoserine lactone quorum sensing system (AHL QS) that consists of a modular LuxR family protein and its cognate LuxI homolog that produces the AHL signal. Moreover, AHL QS systems in P. putida strains are diverse in the type of AHLs they produce and the phenotypes that they regulate.     

Reversible non‐genetic phenotypic heterogeneity in bacterial quorum sensing

Bacteria co‐ordinate their social behaviour in a density‐dependent manner by production of diffusible signal molecules by a process known as quorum sensing (QS). It is generally assumed that in homogenous environments and at high cell density, QS synchronizes cells in the population to perform collective social tasks in unison which maximize the benefit at the inclusive fitness of individuals. However, evolutionary theory predicts that maintaining phenotypic heterogeneity in performing social tasks is advantageous as it can serve as a bet‐hedging survival strategy. Using Pseudomonas syringae and Xanthomonas campestris as model organisms, which use two diverse classes of QS signals, we show that two distinct subpopulations of QS‐responsive and non‐responsive cells exist in the QS‐activated population. Addition of excess exogenous QS signal does not significantly alter the distribution of QS‐responsive and non‐responsive cells in the population. We further show that progeny of cells derived from these subpopulations also exhibited heterogeneous distribution patterns similar to their respective parental strains. Overall, these results support the model that bacteria maintain QS‐responsive and non‐responsive subpopulations at high cell densities in a bet‐hedging strategy to simultaneously perform functions that are both positively and negatively regulated by QS to improve their fitness in fluctuating environments.     

Quorum sensing regulates ‘swim-or-stick’ lifestyle in the phycosphere

Interactions between phytoplankton and bacteria play major roles in global biogeochemical cycles and oceanic nutrient fluxes. These interactions occur in the microenvironment surrounding phytoplankton cells, known as the phycosphere. Bacteria in the phycosphere use either chemotaxis or attachment to benefit from algal excretions. Both processes are regulated by quorum sensing (QS), a cell–cell signalling mechanism that uses small infochemicals to coordinate bacterial gene expression. However, the role of QS in regulating bacterial attachment in the phycosphere is not clear. Here, we isolated a Sulfitobacter pseudonitzschiae F5 and a Phaeobacter sp. F10 belonging to the marine Roseobacter group and an Alteromonas macleodii F12 belonging to Alteromonadaceae, from the microbial community of the ubiquitous diatom Asterionellopsis glacialis. We show that only the Roseobacter group isolates (diatom symbionts) can attach to diatom transparent exopolymeric particles. Despite all three bacteria possessing genes involved in motility, chemotaxis, and attachment, only S. pseudonitzschiae F5 and Phaeobacter sp. F10 possessed complete QS systems and could synthesize QS signals. Using UHPLC–MS/MS, we identified three QS molecules produced by both bacteria of which only 3-oxo-C_16:1-HSL strongly inhibited bacterial motility and stimulated attachment in the phycosphere. These findings suggest that QS signals enable colonization of the phycosphere by algal symbionts.     

A Novel Signal Transduction Pathway that Modulates rhl Quorum Sensing and Bacterial Virulence in Pseudomonas aeruginosa

The rhl quorum-sensing (QS) system plays critical roles in the pathogenesis of P. aeruginosa. However, the regulatory effects that occur directly upstream of the rhl QS system are poorly understood. Here, we show that deletion of gene encoding for the two-component sensor BfmS leads to the activation of its cognate response regulator BfmR, which in turn directly binds to the promoter and decreases the expression of the rhlR gene that encodes the QS regulator RhlR, causing the inhibition of the rhl QS system. In the absence of bfmS, the Acka-Pta pathway can modulate the regulatory activity of BfmR. In addition, BfmS tunes the expression of 202 genes that comprise 3.6% of the P. aeruginosa genome. We further demonstrate that deletion of bfmS causes substantially reduced virulence in lettuce leaf, reduced cytotoxicity, enhanced invasion, and reduced bacterial survival during acute mouse lung infection. Intriguingly, specific missense mutations, which occur naturally in the bfmS gene in P. aeruginosa cystic fibrosis (CF) isolates such as DK2 strains and RP73 strain, can produce BfmS variants (BfmS_L181P, BfmS_L181P/E376Q, and BfmS_R393H) that no longer repress, but instead activate BfmR. As a result, BfmS variants, but not the wild-type BfmS, inhibit the rhl QS system. This study thus uncovers a previously unexplored signal transduction pathway, BfmS/BfmR/RhlR, for the regulation of rhl QS in P. aeruginosa. We propose that BfmRS TCS may have an important role in the regulation and evolution of P. aeruginosa virulence during chronic infection in CF lungs.     

In vitro and in vivo efficacy of rosmarinic acid on quorum sensing mediated biofilm formation and virulence factor production in Aeromonas hydrophila

Rosmarinic acid (RA) was assessed for its quorum sensing inhibitory (QSI) potential against Aeromonas hydrophila strains AH 1, AH 12 and MTCC 1739. The pathogenic strains of A. hydrophila were isolated from infected zebrafish and identified through biochemical analysis and amplification of a species-specific gene (rpsL). The biofilm inhibitory concentration (BIC) of RA against A. hydrophila strains was found to be 750 μg ml^?1. At this concentration, RA reduced the QS mediated hemolysin, lipase and elastase production in A. hydrophila. In FT-IR analysis, RA treated A. hydrophila cells showed a reduction in cellular components. Gene expression analysis confirmed the down-regulation of virulence genes such as ahh1, aerA, lip and ahyB. A. hydrophila infected zebrafish upon treatment with RA showed increased survival rates. Thus, the present study demonstrates the use of RA as a plausible phytotherapeutic compound to control QS mediated biofilm formation and virulence factor production in A. hydrophila.     

Quorum sensing regulation in <Emphasis Type="Italic">Pseudomonas</Emphasis>

Expression of many bacterial genes is regulated in a cell density-dependent manner via small signal molecules known as autoinducers; this type of regulation is termed quorum sensing (QS). The QS systems that employ N-acyl-homoserine lactones (HSLs) are best un derstood in Gram-negative bacteria. QS regulates expression of various genes, including the genes responsible for the production of virulence factors, synthesis of exoenzymes and antibiotics, antagonistic properties of bacteria, etc. The QS systems of the genus Pseudomonas are linked to other global regulatory networks of the cell, and their functions are controlled by numerous additional regulatory factors. Such regulators and the QS systems together form an intricate multifactorial cascade regulatory network. The review considers the QS systems of several Pseudomonas species, their interaction with other regulatory systems, and their roles in the regulation of cell processes.