Genome-wide Screening And Regulation Mechanism Of Biofilm Related Genes Of Stenotrophomonas Maltophilia

Stenotrophomonas maltophilia is a gram-negative bacteria and it often causes theimmunodeficiency or immunosuppressed patients infection including respiratory tract,meningitis, endocarditis and bacteremia, etc. The reported mortality rates for S. maltophiliainfected patients in intensive care units (ICU) is more than70%. It has become one of themost important pathogenic bacteria in nosocomial infection. Most of the clinical isolates ofS. maltophilia easily form biofilms on biotic or abiotic surfaces, which makes it gain higheradaptability, such as multiple drug resistance, by changing the physiological metabolism ofitself. However, very few reports are on the molecular and regulatory mechanismsresponsible for S. maltophilia biofilm formation.Here S. maltophilia transposon insertional mutant library was constructed by using theTn5transposon and a total of14208kanamycin resistant transformants were obtained. Bycrystal violet dying method,74mutants exhibiting remarkable changes in biofilm wereidentified. The franking fragments of the mutants were obtained by TAIL-PCR. The resultsshowed that mutant insertion sites belonged to forty-six genes. And the other four mutantswere inserted in intergenic regions and two multi-copies mutants were included. Flagellabiosynthesis, extracellular polysaccharide production, purine synthesis, oxidoreduction,transportation, gene regulation, peptide and lipid synthesis were involved in biofilmformation. Of these genes,20genes putatively contributed to flagella biosynthesis, whichindicated flagella plays a critical role in S.maltophilia biofilm formation.In particular，Smlt2299which encodes an orphan response regulator, FsnR, played animportant role in flagella assembly, cell motility and biofilm formation. Semi-RT-PCRshowed that FsnR as a typical regulation factors directly or indirectly regulated ten flagellagene transcription except Smlt0710.The EMSA test demonstrated that FsnR activatedtranscription of at least two flagella-associated operons by directly binding to theirpromoters and affected the expression of these genes.In conclusion, the transposon insertion mutant library of S. maltophilia ATCC13637was constructed for the first time in this paper. The genes involved in biofilm formationwere identified successfully and the Smlt2299affected biofilm formation through regulating flagellum assembly was also confirmed. These results provide a genetic basis tosystematically study biofilm formation of S. maltophilia.