[Screening for autoinducer synthase gene in Sinorhizobium meliloti and analysis of the autoinducer produced by recombinant expression in Escherichia coli].
The plasmid pJZ290 containing mariner transposon was used to mutagenize Sinorhizobium meliloti and 3000 transposon insertion mutants were subsequently screened for autoinducer-deficient (AI-deficient) mutants. One AI-deficient mutant YW1 was obtained by quantitative activity assay and qualitative TLC detection. In an effort to characterize the transposon insertions of autoinducer-deficient mutant YW1, we performed an arbitrary PCR and sequencing techniques to identify insertion sites. The sequence analysis result showed that the transposon inserted between the 277th bp and the 278th bp of one 621bp ORF in autoinducer-deficient mutant YW1. The 612-bp ORF encodes a putative protein of 206 amino acids that is highly homologous (99% identity) to AHL-synthase traI of Sinorhizobium medicae WSM419. Cloned into the broad host range expression vectors pYC12 and transformed into Escherichia coli DH5alpha, the putative AI synthase gene was overexpressed in E. coli, and four different autoinducers could be detected in the supernatant of the positive recombinant strain by TLC, among which the two AHL molecules that were deficient in AI-deficient mutant YW1 could be found. All of these showed that the 621bp ORF was an AI synthase gene. This study paved the way of further studying quorum sensing systems in S. meliloti.
Wang
Yang
Y
Key Lab of Microbiological Engineering Agricultural Environment, Ministry of Agriculture, College of Life Science, Nanjing Agricultural University, Nanjing 210095, China.
Zheng
Hui-Ming
HM
Yang
Meng-Hua
MH
Zhong
Zeng-Tao
ZT
Zhu
Jun
J
D054742:Acyl-Butyrolactones / Q000032:analysis; D001426:Bacterial Proteins / Q000235:genetics*; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics*; D010957:Plasmids; D053038:Quorum Sensing; D011994:Recombinant Proteins / Q000096:biosynthesis*; D016962:Sinorhizobium meliloti / Q000201:enzymology / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*
D054742:Acyl-Butyrolactones; D001426:Bacterial Proteins; D004251:DNA Transposable Elements; C085427:LuxI protein, Bacteria; D011994:Recombinant Proteins; D014157:Transcription Factors