Molecular Regulation Mechanism Underlying The Quorum Sensing Of Pseudoalteromonas Sp. Rf-1

Quorum sensing is an important mechanism by which bacteria regulate its physiological metabolism activities.Bacterial release,detect and response to signal molecules that called autoindusers to regulate gene expression,such as genes encoding bioluminescence and virulence factors.In this study,we try to explore the molecular regulation mechanism underlying the quorum sensing of Pseudoalteromonas sp.Rf-1 by Random insertion mutagenesis.First,an AHL was detected in Pseudoalteromonas sp.Rf-1 and a high sensitivity detection method of AHL with plate streaking was established.Then,a mutant library was constructed through the conjugation of E.coli S17-1(??)and Pseudoalteromonas sp.Rf-1.The E.coli S17-1(??)contains a suicide plasmid pLOF/Km which bearing a transposon mini-Tn10,the transposon mini-Tn10 was transferred into Pseudoalteromonas sp.Rf-1 by conjugation.More than two thousand individual transposon inserted mutants were gained.Among them,seven mutants of strain Rf-1 with AHL deficience were screened out with plate streak technique.The DNA sequence flanking transposon in the mutant was determined using thermal asymmetric PCR.The inserted sequence in mutant B7 could be matched with the gene from P.rubra SCSIO 6842,with high identity(85%)to a stringent starvation protein gene(sspB).Finally,genetic complementation was conducted by cloning sspB into plasmid pBBR1MCS-5 to construct the recombinant plasmid pBBR1MCS-5sspB which was subsequently transferred into the mutant B7.After complementation,the mutant B7 was almost totally restored to produce AHL.These results suggest that quorum sensing in Rf-1 is controlled by sspB gene.