|Strobaugh Jr, Terence|
|DUDLEY, EDWARD - Pennsylvania State University|
|Nguyen, Ly Huong|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 2/20/2013
Publication Date: 5/18/2013
Citation: Chen, C., Uhlich, G.A., Strobaugh Jr, T.P., Cottrell, B.J., Dudley, E.G., Hofmann, C.S., Nguyen, L.T. 2013. Mutations in the rpoS gene are the major limiting factor for biofilm formation in Escherichia coli serotype O157:H7 clinical isolates. Meeting Abstract. MA.
Technical Abstract: Background: Biofilm formation is a complex process that is highly regulated through a battery of transcriptional regulators, small regulatory RNAs, and environmental conditions. RpoS sigma factor along with MlrA protein directly regulate the expression of the curli key regulator CsgD. In most serotype O157:H7 strains, mlrA is the insertion site for a prophage carrying the stx1 gene and mlrA disruption was thought to be the major factor resulting in diminished biofilm formation in that serotype. However, our study found that nearly 50% of the O157:H7 clinical isolates failed to be complemented by a plasmid overexpressing mlrA, suggesting other lesions in the genome are responsible for this failure. Materials and Methods: A total of 55 E. coli O157:H7 strains were used in this study, including 53 clinical isolates and two food isolates. A 1.8-kb DNA fragment containing the rpoS gene and the flanking sequences was amplified and sequenced using Big Dye terminator. Biofilm forming capability was assessed by Congo red (CR) indicator plates and crystal violet (CV) 96-well plate assays. Catalase activities were performed using hydrogen peroxide test. Results: Only 12 strains (21.8%) sequenced carried no mutations in the rpoS coding region. At the protein level (deduced from nucleotide sequence), 15 strains carried WT RpoS, 7 strains carried single amino acid substitutions, 1 strain carried an in-frame 2-aa insertion, and 32 strains had truncations resulting from single-base changes, insertions or deletions. Several mutation hot-spots were found. In the group of 15 strains that were trans-complemented by pUC19::mlrA for both CR and CV binding, 10 were predicted to produce WT RpoS, four strains carry truncated RpoS and 1 had a substitution in RpoS; only two in this group had weaker catalase activity. Twenty-three of the 24 strains (~96%) that failed to be complemented by pUC19::mlrA plasmid were found to produce a mutated RpoS protein and have low catalase activity. Conclusion: Mutations in the rpoS gene have been reported, however, the huge diversity found in the rpoS coding region and in the catalase activity in O157:H7 clinical isolates was unexpected. The highly mutatable nature of the rpoS gene may provide distinct advantages for virulence, survival, and biofilm formation for serotype O157:H7.