Tuesday, June 4, 2013

Characterizing biofilm formation with the FailSafe™ PCR System

The Hha gene in E. coli encodes the haemolysin expression modulating protein, a gene product that is responsible for pathogenesis in Enterohemorrhagic E. coli (EHEC) strain O157:H7 - a common pathogenic organism that is found in biofilms in hospital medical environments. While molecular mechanisms promoting adherence of this strain on epithelial cells are well characterized, regulatory mechanisms controlling biofilm formation are not fully understood. In order to more fully understand how the Hha gene regulates biofilm formation, researchers at the USDA generated mutant forms of three different E. coli genes, including Hha, using the FailSafe™ PCR System. A hha deletion mutant was developed using deletion recombination to remove the sequence of the Hha open reading frame from the bacterium. The removal of the Hha sequence also had other consequences: the deletion mutant produced large quantities of biofilm compared to the O157:H7 wild-type strain at 30°C and 37°C. Complementation of the hha mutant reduced the level of biofilm formation to that of the wild-type strain, indicating that Hha is a negative regulator of biofilm production. While swimming motility and expression of the flagellar gene fliC were significantly reduced in the hha mutants, the expression of csgA (encoding curlin of curli fimbriae) and the ability to bind dye Congo red were significantly enhanced. The expression of both fliC and csgA and the phenotypes of motility and curli production affected by these two genes, respectively, were restored to wild-type levels in the complemented hha mutant. The csgA deletion abolished biofilm formation in the hha mutant and wild-type strain, and csgA complementation restored biofilm formation to these strains, indicating the importance of csgA and curli in biofilm formation. The regulatory effects of Hha on flagellar and curli gene expression appear to occur via the induction and repression of FlhDC and CsgD, as demonstrated by reduced flhD and increased csgD transcription in the hha mutant, respectively. In gel shift assays Hha interacted with flhDC and csgD promoters. In conclusion, Hha regulates biofilm formation in EHEC O157:H7 by differential regulation of FlhDC and CsgD, the global regulators of motility and curli production, respectively.
ResearchBlogging.orgSharma VK et al. (2013). Hha controls Escherichia coli O157:H7 biofilm formation by differential regulation of global transcriptional regulators FlhDC and CsgD. Applied and environmental microbiology, 79 (7), 2384-96 PMID: 23377937