A role for rmoA in IncF plasmid mediated biofilm formation in E. coli K12

Abstract number: 1134_01_250

Nuk M., Reisner A., Neuwirth M., Zechner E.

Objectives:  

Bacterial biofilms cause serious health problems because these sessile cells can withstand immune response and show an increased resistance to antimicrobial agents. Non pathogenic E. coli have a limited ability to form biofilms, but carriage and transmission of conjugative plasmids enhance biofilm formation. We are interested in understanding the role plasmids play in promoting biofilm formation and the molecular mechanisms behind these effects.

Methods:  

To create mutations in biofilm associated genes random transposon mutagenesis of IncF plasmid R1–16 was performed using miniTn5 transposition. To exclude mutations in transfer genes, a selection screen requiring conjugative transfer of mutant plasmids was employed. Interrupted genes were identified using arbitrary PCR and DNA sequencing. Strains harbouring mutant plasmid derivatives were tested for biofilm forming ability in microtiter format. This in vitro model of biofilm formation that relies on removal of planktonic cells and quantification of surface attached bacterial mass by crystal violet staining. Subsequent specific inactivation of the rmoA gene in R1–16 was performed using the lambda Red system of bacteriophage lambda. To obtain information about the expression of the gene of interest Northern Blot analysis and Reverse Transcriptase PCR were performed.

Results:  

As a result of the transposon mutagenesis rmoA was found to be important for biofilm formation. E. coli strains carrying R1–16 with a deletion of rmoA showed a 5-fold decrease in biofilm formation compared to wildtype R1–16. This effect was observed in full and in minimal media. Kinetic experiments of biofilm formation revealed that the rmoA phenotype is already developed in an early stage of biofilm formation. Northern Blot analysis and Reverse Transcriptase PCR indicate that rmoA is transcribed as part of an operon.

Conclusions:  

Genes homologous to rmoA are present in Yersinia enterolitica, Salmonella typhimurium, and on many plasmids including plasmids of pathogenic E. coli, Shigella flexneri and Salmonella typhi. The amino acid sequence of RmoA shows strong homology to the Hha protein of Escherichia coli and the YmoA of Yersinia enterolitica, which are modulators of bacterial gene expression in response to environmental stimuli. Thus, RmoA may act to regulate plasmid or host genes that are important to bacterial biofilm formation.