Screening And Identification Of Autoninducer Synthesis Related Gene Of Quorum-Sensing Systems In Sinorhzibium Sp.1128

The ability to produce autoninducer of Sinorhizobium sp. 1128 was investigated by a ultrasensitive AHL bioassay strain KYC55 (pJZ372) (pJZ384) (pJZ410). The quantitative LacZ activity and qualitative TLC detection for autoinucer showed that Sinorhizobium sp. 1128 could produce higher active and various AHLs.To investigate quorum-sensing systems in Sinorizobium sp. 1128, we screened and characterized two transposon insertion mutants (YW1 and YW2) that were partially deficient in autoinducer production using transposon mutagenesis. Compared with wild type strain, the two mutants were deficient in LacZ activity and AHL contents. In an effort to characterize the transposon insertions of two autoinducer-deficient strains, we performed an arbitrary PCR and subclone techniques to identify insertion sites. The sequence analysis result showed that the transposon inserted between 277th and 278th bp of one ORF in autoinducer-deficient mutant YW1, coding for amino acid is homologous to (99% identity) the autoinducer synthase TraI in Sinorhizobium medicae WSM419. The transposon in the mutant YW2 inserted between 1257th and 1258th bp of one ORF, coding for amino acid is homologous to (100% identity) the hypothetical conserved protein KaiC in Sinorhizobium medicae WSM419. Interestingly, regulator KaiC is necessary to remain physiological cycle in cyanobacteria, but it might be involved in autoinducer synsthesis of quorum-sensing in Sinorhizobium sp. 1128.In order to confirm that traI gene function as autoinducer production, we cloned the traI into broad host range expression vectors pYC12 and transformed into Escherichia coli DH5a. The pupative AHL synthase gene was overexpressed in E.coli, and autoinducer could be detected in the supernatant of the positive recombinant strain by LacZ activity assay and TLC. Cloned into pVIK112 and transformed into Escherichia coli BW20676, the internal traI intergrated into the chromosome of a wild type strain by homologous recombination. Thus the autoinducer-deficient YW3 was successfully constructed. Recombinant plasmid carring intact traI was conjugated into YW3, then the complement clone YW5 was obtained. AHL contents detected by TLC showed that the ability to produce autoinducer of YW5 have restored. All of dataes indicated that traI, autoinducer sythase gene, could produce two kinds of AHLs, other autoinducers that YW1 mutant was not deficient in might be synthesized by other autoinducer synthase gene.