Functional Analysis Of Luxi Gene And Determination Of QS Signal Molecules Of Erwinia Amylovora

Erwinia amylovora is a necrogenic phytopathogenic bacterium, the causal agent of fire blight in pear, apple and other members of the Rosaceae family, causing serious economic losses. It is one of the oldest known bacterial diseases that was first discovered in Northeast America in1780. Since1900, the disease has been widely distributed in many countries and regions in the world. So far, there has not yet found Erwinia amylovora in China.Bacterial quorum sensing system is one of the most important advances in the field of microbial population studies of the past20years. QS is one of the sophisticated mechanisms of cell-cell communications, by which bacteria count their own number by producing, detecting, and responding to the accumulation of signaling molecules that they export into their environment. LuxI, the enzyme responsible for synthesis of autoinducers, and LuxR, the transcriptional activator that recognizes this specific autoinducers are the main component of LuxI/LuxR circus in quorum sensing (QS) of Gram-negative bacteria. In this study, a luxl deletion mutant EaAluxl was successfully generated by homologous recombination. In comparison with the wild-type strain NCPPB1665(Ea1665),the mutant EaAluxI impaired virulence on pear leaves, tolerance of hydrogen peroxide, extracellular polysaccharide(EPS) production and motility. However, the mutant EaΔluxl showed no difference with the wild type in the tolerance to antibiotics, growth and HR.Though we found that the autoinducers synthase gene luxl was contained in genome of Erwinia amylovora by BLAST, We couldn’t detect the activity of AHL autoinducers from supernatant of wild type Erwinia amylovora NCPPB1665with a small amount of culture in the laboratory. To investigate whether luxl was a functional AHL-synthase gene, the luxl gene was cloned into the expression vector pET30and the E.coli BL21(DE3) was used as a heterogeneous host to express the luxl gene. Then we detected the AHL autoinducers from the supernatant of wild type Ea1665, the mutant EaAluxI and the E.coli BL21(DE3)/pET30a-I. The experiments showed that Ea1665can produce at least a3-oxo-C6-HSL signal molecule. But the abundance is relatively low.