Isolation And Characterization Of DSF Quorum Quenching Bacteria Pseudomonas Sp.HS-18 And The Study Of Quenching Mechanisms

Quorum sensing(QS)is a cell-cell communication mechanism with which bacteria regulate the expression of virulence genes.Quorum quenching(QQ)is a novel antibacterial strategy,which attenuates bacterial virulence by disrupting or blocking bacterial QS systems.QQ has been regarded as a novel and environmental safe disease control strategy as its function is not based on killing of pathogens and therefore does not excert selective pressure on bacteria to cause antibiotic-resistance and environmental pollution problems..There are only a few reported studies on screening and characterization of quorum quenching mechanisms associated with DSF(diffusible signal factor)signals in gram-negative bacteria.DSF regulates the production of various virulence factors and DSF-dependent pathogens cause substantial economical losses in agriculture.To date,control of these bacterial diseases mainly relies on selection of resistant cultivars and the use of chemical pesticides.However,long term application of chemical pesticides has caused severe pollution and contamination problems,which are harmful to environment,animals and human beings.Previous studies have showed that mutation of the DSF-dependent quorum sensing system could markedly reduce the virulence of the DSF-dependent pathogens,highlighting the potential of using QQ approaches to control bacterial infections.In this study,we isolated a bacterium Pseudomonas sp.HS-18 that can degrade DSF with high efficiency.The putative genes encoding DSF inactivation were located by genome sequencing and amino acid BLAST,and confirmed by deletion and heterogenous expression analysis.Furthermore,the role of the DSF inactivation genes in biocontrol of plant diseases was verified by deletion and complementation analysis.The major results were shown as follows:11 high efficient DSF quenching strains were identified on a minimum medium(MM*)with DSF as sole carbon source.Among them,Pseudomonas sp.HS-18,which can quench 0.5 m M DSF within 12 h as determined by quantitative analysis using HPLC,was selected for further studies.The genetic informations of strain HS-18 were unveiled by genome sequencing.The genome of HS-18 is a circular chromosome with the genome size of 6.49 M and the average G+C content at 64.74%.In addition,the genome of strain HS-18 also contains a circular plasmid of 0.1 M in size and G+C content of 55%.In total,6090 encoding genes are predicted withno t RNA genes,6 genes encoding 5S r RNA,5 genes encoding 16 S r RNA,5 genes encoding 23 S r RNA,and 16 genes encoding s RNA.In addition,strain HS-18 contains all the genes encoding a complete fatty acid metabolic pathway.We hypothesized that the ability of strain HS-18 in DSF inactivation might be related to fatty acid metabolism,according to the structure of DSF and the studies on DSF turnover mechanism.The amino acid sequences of Fad D from E.coli and Rpf B from Xanthomonas campestris pv.campestris(Xcc)were used for sequence alignment to locate the putative DSF inactivation genes.Based on the similarity of amino acid sequence,4 genes in HS-18 were selected and their roles in DSF inactivation were validated by deletion and complementation analyses.These genes were therefore designated as DSF inactivation genes(dig),i.e.,dig1,dig2,dig3,and dig4,respectively.In addition,heterologous expression of dig1,dig2,dig3,and dig4,respectively in E.coli DH5? significantly improved its DSF quenching capacity,which further consolidates the role of the dig genes in QQ against DSF signals.The endogenous DSF production of Xcc mutant XC1?rpf C,which overproduces DSF,was markedly reduced by heterologously expression of dig1,dig2,dig3 and dig4,respectively,as unveiled by HPLC analysis.Similarly,heterologous expression of these dig genes respectively in Xcc wild type strain XC1 reduced the production of key virulence factors,such as extracellular protease,extracellular cellulase,extracellular polygalacturonase,and also impaired the bacterial motility and biofilm formation.The role of dig in biocontrol of DSF-mediated infection was established by comparing the biocontrol efficiency strain HS-18 and its dig deletion mutants.The results showed that strain HS-18 substantially reduced the virulence of strain XC1,whereas in contrast,its dig1,dig2,dig3 and dig4 deletion mutants are much compromised in biocontrol efficiency.Significantly,it was found that the transcriptional expression of dig1,dig2,dig3 and dig4 were significantly increased by exogenous addition of DSF signal moleulces by q-RT PCR(quantitative real-time PCR)analysis.This interesting finding was further confirmed by using a reporter fusion gene driven by the dig promoter.In conclusion,a highly efficient DSF quenching strain Pseudomonas sp.HS-18 was isolated in this study.At least four genes in strain HS-18,i.e.,dig1,dig2,dig3 and dig4,were found encoding DSF inactivation enzymes.The presence of multiple DSF inactivation genes,together with the trait that the dig genes were highly induced by DSF signal molecules,might explain the high efficiency of strain HS-18 in quorum quenching against the DSF signals.This study also demonstrated the promising potentials of strain HS-18 in biocontrol of DSF-mediated bacterial infections,as well as the key roles of dig genes in biocontrol.The findings from this study not only present a promising biocontrol agent and potent DSF inactivation enzymes for biotechnological applications,but may also provide useful clues to dissect molecular mechanisms with which bacteria could detect and respond to the DSF quorum sensing signals produced by other microorganisms in the process of microbe-microbe interactions.