Taxonomic Analysis Of Two Novel Bacteria With Quorum Quenching Activity And Their Related Properties

Quorum sensing (QS) is a way of cell-cell comunication in bacteria to coordinatecellular activities by the production, accumulation and response to signal molecules.N-Acyl-homoserine lactones (AHLs) are the signal molecules of most Gram-negativebacteria and closely related to the pathogenicity of many pathogens. Quorumquenching (QQ) refers to the interruption of QS by enzymes or small-moleculeinhibitors, which can turn off the expression of virulence factors without killing thepathogenic bacteria and provide a promising alternative to antibiotics. Theidentification of novel QQ bacteria and QQ substance has great meaning indeveloping eco-friendly treatment of pathogens and a sustainable way of aquaculture.In this study, two novel AHL-degrading bacteria have been investigated in ataxonomic analysis by a polyphasic approach, and the whole genome of QQ strainTh78Thas been sequenced and analysed. Furthermore, the QQ enzyme in strainTh78Thas been expressed in Escherichia coli. All these results have provided furtherinsight into the application of these two quorum quenching bacteria in aquaculture.Strain Th78Tis a Gram-staining-negative, aerobic, yellow-pigmented, rod-shapedand AHL-degrading bacterium isolated from the intestine of cultured flounder(Paralichthys olivaceus). Phylogenetic analysis based on16S rRNA gene sequencesindicates that strain Th78Tbelongs to the family Flavobacteriaceae (Bacteroidetes)and shows the highest16S rRNA gene similarity to Flaviramulus basaltis H35T(96.70%). The isolate is motile by gliding and unable to produce flexirubin pigment.The major fatty acids are iso-C15:03-OH, iso-C15:1G and iso-C17:03-OH. The majorpolar lipids are phosphatidylethanolamine, one unidentified aminolipid and threeunidentified polar lipids. Menaquinone6(MK-6) is the only respiratory quinone. TheDNA G+C content of strain Th78Tis31.5mol%. On the basis of polyphasic analysis,strain Th78Tis considered as a novel species in the genus Flaviramulus, for which thename Flaviramulus ichthyoenteri sp. nov. is proposed. Strain T202Tis a Gram-staining-negative, aerobic and rod-shaped bacteriumisolated from the gill of flounder which shows weak degradative ability of C6-HSL.Strain T202Tshows highest16S rRNA gene similarity with Thalassomonasagariperforans M-M1T(96.58%). Optimal growth occurs in the presence of2–4%(w/v) NaCl, at pH7.0-8.0and at28oC. The major fatty acids are C16:0, C16:1ω7cand/or C16:1ω6c and C16:1ω9c. Ubiquinone-8(Q-8) is the predominent respiratoryquinone. The major polar lipids are phosphatidylethanolamine and phosphatidyl-glycerol. The DNA G+C content of strain T202Tis37.0mol%. On the basis ofpolyphasic analysis, strain T202Tis considered as a novel species in a novel genusThalassotalea, for which the name Thalassotalea piscium gen. nov., sp. nov. isproposed.The whole genome of strain Th78Thas been sequenced by Illumina Hiseq2000platform. The genome is3,953,230bp with3,451predicted protein coding genes,40tRNAs and6rRNAs. Two potential AHL-lactonases in metallo-β-lactamase superfamily have been found by searching the whole genome of strain Th78Tagainstknown AHL-lactonase sequences with BLAST program. Besides, coding genes forvarious digestive enzymes which might have a beneficial effect in the digestiveprocess of fish have been found in the genome of strain Th78T. Strain Th78Talsoshows its ablity to utilize various sugars such as sialic acids in the intestine. Thecomparative analysis of strain Th78Tand another two strains (Gaetbulibacter saeman-kumensis DSM17032Tand Lacinutrix sp.5H-3-7-4) in Flavobacteriaceae indicatesthat strain Th78Tis quite different from them in carbohydrate transport andmetabolism.The potential AHL-lactonase coding gene GL001211in strain Th78Thas beencloned and expressed in Escherichia coli, and the resulting protein with QQ activityhas been named FiaL. The predicted molecular mass of FiaL is32.6kDa and thetheoretical pI value is7.58. Coresponding with the QQ properties of strain Th78T,FiaL can degrade AHLs with different acyl-chain length and modification. Thehomologous protein of FiaL has been found in many marine bacteria inFlavobacteriaceae, which indicates that FiaL may play an important role in the communication of marine bacteria and thus can be applied in aquaculture to combatthe pathogens.