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Abstract

The investigation of biofilm formation is critical to reducing infection rates as more than 60% of the clinically infectious diseases are associated with biofilm formation. Here, we used a plasmid pRL27, having an R6Kγ origin of replication, to generate transposon mutants altered in biofilm development in Escherichia coli 2443 (K-12 strain). The R6Kγ origin of replication requires the pir gene encoding the π protein, which binds at a 22-bp recognition sequence to initiate DNA replication. As the Escherichia coli 2443 is a pir negative strain, the plasmid pRL27 cannot replicate in it and this makes the plasmid an effective suicide vector for genome wide transposon mutagenesis. The plasmid was maintained in MFDpir strain, which is a diaminopimelate (DAP) auxotrophic mutant (dap?) of Escherichia coli and produces the π protein. The plasmid pRL27 was conjugatively transferred from MFDpir to Escherichia coli 2443 on minimal medium plates. Transconjugants were selected by plating the mating mixtures onto LB plates supplemented with dap (300 μg/ml) and kanamycin (50 μg/ml). The transposon insertion mutants obtained were screened for altered biofilm formation using crystal violet staining. The biofilm formation was normalized to planktonic growth, thus obtaining the biofilm formation index (BFI). Based on the results two mutants RM 56 and RM 69, showing significantly lower and higher BFI respectively, were selected. These mutants will be characterized further to identify the site of the transposon insertions and the underlying mechanism altering biofilm formation. This may be of benefit to develop a better understanding of biofilm formation.

Biography

Akash Kumar is pursuing PhD in Department of Biotechnology, Indian Institute of Technology Kharagpur. He has published one research paper in Research in Microbiology.