Screening And Identification Of Autoinducer Synthesis Related Gene Quorum-Sensing In Mesorhizobium Huakuii And Its Effect On Symbiotic Nodulation

The ability to produce autoninducer of Mesorhizobium huakuii As9 was investigated by a ultrasensitive Acyl-homoserine lactone bioassay strain JZA1.The quantitative LacZ activity and qualitative TLC detection for autoinucer showed that Mesorhizobium huakuii As9 can produce high concentrated AHLs molecular.To investigate quorum-sensing systems in Mesorhizobium huakuii As9,we screened and characterized autoinducer production deficient mutants using transposon mutagenesis.Two quorum-sensing deficient mutants named YQ1 and YQ2 were isolated.We performed an arbitrary PCR to identify insertion sites.Sequence analysis of the flanking region of transporson insertions showed that the transposon inserted in a ORF(639bp) in autoinducer-deficient mutant YQ1,coding for a autoinducer synthase named mrhI which is homologous(99%identity) to mlr5638 in Mesorhizobium loti MAFF303099.The transposon in the mutant YQ2 inserted the ORF,coding for the regulate protein named MrhR which is homologous(97%identity) to Mlr5637 in Mesorhizobium loti MAFF303099.In order to study the expression of the AHL synthase gene mrhI,mrhI-lacZ transcriptional fusion(YQ3) was constructed by cloning mrhI internal fragment into pVIK 112 and intergrated into the chromosome of a wild-type strain by homologous recombianation.We found that expression of the quorum-sensing regulators is autoinduced, as mrhI was fully induced only when the supernatant prepared from the cell-free spent medium of the wild-type culture which contains the maximal amount of AHLs was added.Compared with the wild-type strains,mrhI quorum-sensing-deficient mutant was defective in nodule formation on their host plant,Astragalus sinicus L.This means that quorum sensing plays a role in the M.huakuii symbiotic process,but the exactly mechanism is not clear now.