Thursday, November 1, 2012

Transposon mutagenesis identifies uropathogenic Escherichia coli biofilm factors

Transposon mutagenesis using EZ-Tn5™ Transposomes has been a common method for determining gene activity and location of pathogenesis features in medically important bacteria. Hadjifrangiskou et al. demonstrate the use of the <EZ-Tn5 <R6Kγori/KAN-2> Tnp Transposome Kit to identify and rescue a specific pathogenesis marker in uropathogenic E. coli that appears to be responsible for biofilm formation in catheters and biotic surfaces. The biofilms in these environments are responsible for rapid formation of intracellular bacterial communities within bladder epithelial cells and protects the communities from the host immune system; what remains unknown is the nature of the interaction and relationship between the intracellular bacterial communities and what factors are responsible for biofilm buildup inside the human host. It is also well known that the presence of flagella and pili are essential for biofilm formation, and using transposon mutagenesis with the concept of knocking out flagellar “motor” genes may have an impact on biofilm formation.

To search for markers responsible for this phenomenon, three different strains were transformed with the EZ-Tn5 <R6Kλori/KAN-2> Transposome. Mutant E. coli that showed biofilm defects after mutagenesis were selected and the insertion points of the transposon identified through multiple rounds of sequencing using a modified Random Amplification of Transposon Ends (RATE procedure). Other assays performed to determine loss of biofilm formation included motility assays, Hemagglutination assays, immunoblot analysis, and mouse infections followed by microscopy to characterize any molecular changes that arose from the mutagenesis procedure.  The results showed numerous changes in biofilm formation and bacterial consortia assembly characteristics due to the transposon insertional mutagenesis, resulting in reduced to total loss of the ability to form bacterial communities, and a subgroup of these mutants generated with the EZ-Tn5 Transposome were unable to form  biofilms when tested in a mouse model system.

The experiments reveal that a complex variety of regulatory and effector genes that express under multiple conditions, again demonstrating the utility of Transposon mutagenesis using EZ-Tn5 Transposomes for elucidating bacterial pathogenesis factors.

ResearchBlogging.orgHadjifrangiskou, M. et al. (2012). Transposon Mutagenesis Identifies Uropathogenic Escherichia coli Biofilm Factors. Journal of bacteriology, 194 (22), 6195-205 PMID: 22984258

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