Construction Of Transposon Insertion Mutants Library Of Escherichia Coli And Screening Of Genes Involved In Biofilm Formation

Escherichia coli(E.coli)is a Gram-negative short bacilli,which is one of the most important zoonotic pathogens in global public health.E.coli can be affected by various factors including infecting hosts and persisting in the environment for a long time.As a bacterium producing biofilm,this property is essential to help E.coli adapt to the host and non-host environments.During the formation of biofilm,the E.coli will change its physiological metabolism,which will enhance its adaptability,such as tolerance to adverse external environments,antimicrobial resistance and so on.However,little is known about the functional genes on plasmid involved in biofilm formation,and their regulatory mechanisms remain elusive.In this study,we found a widely prevalent E.coli strain harbouring a multi-drug resistant IncF plasmid isolated from food animals in Guangdong province.Conjugation assay showed that the IncF plasmid was successfully transferred into recipient E.coli C600 strains.Interestingly,the transconjugants not only obtained a multi-drug resistant phenotype,but also got the ability to produce biofilm.Thus,we hypothesized that there were unknown functional genes located on this IncF plasmid and could be related to biofilm formation.A transposon mutagenesis method using EZ-Tn5 was performed to identify the functional genes involved in biofilm formation.The EZ-Tn5 transposition complexes were electrotransformed and randomly inserted into the bacterial chromosome and plasmid genes of E.coli that containing plasmid 253.A saturation mutagenesis library containing 30000 mutants were obtained.The biofilm-forming ability of the mutants was further evaluated using crystal violet staining.A total of 28 mutants were found descend biofilm-forming ability.The flanking sequence of insertion site was further identified by two rounds of semi-arbitrary PCR technique.The results showed that the insertion sites were located on the chromosome of E.coli in 24 out of 28 mutants,and only four of them were located on the plasmid.The insertion sites were further confirmed by PCR and/or southern blotting.Sequence analysis showed that the inactivated chromosomal genes involved in the synthesis of flagella and fimbriae,polysaccharide synthesis,energy generation and transformation,and regulation of gene processes,most of which had been speculated to be associated with the formation of biofilm.The other four insertions were aligned to the same segment on the plasmid.Furthermore,high-throughput sequencing was performed and the sequence of plasmid was assembled.A 7949 bp contig containing the four insertion sites was obtained,which encoded a pilus gene cluster fimA-fimC bracketed with a truncated IS1.Interestingly,the pilus gene cluster fimA-fimC probably derived from the chromosome Enterobacter cloacae.In order to decipher the real role of the pilus gene cluster in biofilm formation,the whole contig and the partial gene fimA,fimC,fimA+fimC were subcloned into pBS and further transformed into DH5?.The results showed that the single gene clone didn't regain the ability of biofilm formation except for the clone that containing the whole pilus gene cluster,implying that the plasmid-mediated biofilm formation was collectively controlled by multiple gene clusters that encode pilus.In this study,a transposon insertion mutant library was constructed using EZ-Tn5 transposition complex,and a pilus gene cluster on plasmid involved in biofilm formation were successfully identified.The intrinsic mechanism between pilus and biofilm formation and its evolution and transfer need further exploration.