Novel Plasmid-Borne Fimbriae-Associated Gene Cluster Participates in Biofilm Formation in

This study reported the involvement of a gene cluster from a conjugative plasmid in the biofilm formation of . We used a novel EZ-Tn transposon technique to generate a transposon library and used arbitrarily primed PCR to detect the insertion sites in biofilm formation-deficient mutants. To validate the function of candidate biofilm formation genes, the genes were cloned into plasmid pBluescript II SK (+) and transformed into DH5α. Biofilm production from the transformants was then assessed by phenotypic biofilm formation using Crystal Violet staining and microscopy. A total of 3,000 transposon mutants of DH5α-p253 were screened, of which 28 were found to be deficient in biofilm formation. Further characterization revealed that 24/28 mutations were detected with their insertions in chromosome, while the remaining 4 mutations were evidenced that the functional genes for biofilm formation were harbored in the plasmid. Interestingly, the plasmid sequencing showed that these four transposon mutations were all inserted into a fimbriae-associated gene cluster (-cluster). This -cluster is a hybrid segment spanning a 7,949 bp sequence, with a terminal inverted repeat sequence and two coding regions. In summary, we performed a high-efficiency screening to a library constructed with the EZ-Tn5-based transposon approach and identified the gene clusters responsible for the biofilm production of , especially the genes harbored in the plasmid. Further studies are needed to understand the spread of this novel plasmid-mediated biofilm formation gene in clinical isolates and the clinical impacts.