Thursday, November 15, 2012

Role of rpoS in E. coli 0157:H7 strain H32 biofilm development and survival

Bacterial strain development can be aided by EZ-Tn5™ Transposomes for generating mutations in certain genes for study of important characteristics. It is well-known that the E. coli strains that harbor the serotype O157:H7 can cause several diseases in humans; the strain studied by Sheldon et al. carries the rpoS gene that controls cell survival in pathogenic E. coli by providing resistance factors to various stressors, and is also responsible for biofilm development. In this study, a custom transposome was constructed using the EZ-Tn5™ Custom Transposome™ Construction Kits in the pMOD-2 vector and contained a Kanamycin resistance gene and a GFP gene. The transposon was complexed with the EZ-Tn5 Transposase to form the Transposome, which was electroporated into the H32 strain of E. coli O157:H7 using standard procedures. Kanamycin-resistant mutants were isolated and the locations of the transposon insertion points in the chromosome determined by sequencing. The GFP functionality allowed easy quantification of microbes in the biofilms by confocal microscopy. A subset of the GFP-containing cells was further modified to be RpoS-deficient using lambda-Red recombinase to inactivate the rpoS gene to provide a suitable control to compare with the GFP-tagged, RpoS wild type E. coli. Both strains of the E. coli cells were further used to study biofilm formation and compared for survivability in samples of water from various sources. Biofilm mass formation and cell survivability were assayed.

The researchers found that the protein RpoS is responsible for mediating cell survival during the stationary phase by conferring cell resistance to various stressors and has been linked to biofilm formation. Confocal scanning laser microscopy revealed a nutrient-dependent role of rpoS in biofilm formation. The enhanced biofilm formation of the rpoS mutant did not translate to increased survival in sterile double-distilled water, filter-sterilized lake water, or unfiltered lake water. While the rpoS recombinant mutant showed an overall cell viability reduction the wild type cells showed significantly less loss of viability, depending on the water source. However, the survival rates of the detached biofilm-derived rpoS+ and rpoS mutant cells were comparable. Under the competitive stress conditions of unfiltered lake water, the advantage conferred by the presence of rpoS was lost, and both the wild-type and knockout forms displayed similar declines in viable counts. These results suggest that rpoS does have an influence on both biofilm formation and survival of E. coli O157:H7 and that the advantage conferred by rpoS is contingent on the environmental conditions.
ResearchBlogging.orgSheldon JR, et al. (2012). Role of rpoS in Escherichia coli O157:H7 Strain H32 Biofilm Development and Survival. Applied and environmental microbiology, 78 (23), 8331-9 PMID: 23001657

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