Role of rpoS in Escherichia coli O157:H7 strain H32 biofilm development and survival

Abstract

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. In this study, the role of the rpoS gene in Escherichia coli O157:H7 biofilm formation and survival in water was investigated. Confocal scanning laser microscopy of biofilms established on coverslips revealed a nutrient-dependent role of rpoS in biofilm formation, where the biofilm biomass volume of the rpoS mutant was 2.4- to 7.5-fold the size of its rpoS(+) wild-type counterpart in minimal growth medium. The enhanced biofilm formation of the rpoS mutant did not, however, translate to increased survival in sterile double-distilled water (ddH(2)O), filter-sterilized lake water, or unfiltered lake water. The rpoS mutant had an overall reduction of 3.10 and 5.30 log(10) in sterile ddH(2)O and filter-sterilized lake water, respectively, while only minor reductions of 0.53 and 0.61 log(10) in viable counts were observed for the wild-type form in the two media over a 13-day period, respectively. 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.

Fig 1

Planktonic growth of E. coli O157:H7 strains H32 (●), H32-gfp (○), and H32-gfpΔrpoS::gm (▼) in a rich medium (LB) (top graph) and a minimal medium (MSMG) (bottom graph) at 37°C over 24 h. Bars indicate standard deviations at a 95% confidence level.

Fig 2

Representative CSLM images of biofilm cells of E. coli O157:H7 strains H32, H32-gfp, and H32-gfpΔrpoS::gm (KO) after 24 h of growth in TSB or MSMG. The biofilms were cultivated in TSB at 37°C (left column), MSMG at 37°C (middle column), and MSMG at 22°C (right column). All three E. coli strains were stained with SYTO 9 and visualized with a 60× objective lens.

Fig 3

Biofilm development of E. coli O157:H7 strain H32 (●), H32-gfp (○), and H32-gfpΔrpoS::gm (▼) in MSMG at 22°C. All three E. coli strains were stained with SYTO 9. Bars indicate standard deviations at a 95% confidence level.

Fig 4

Survival of 48-h-old E. coli O157:H7 strain H32-gfp (○) and H32-gfpΔrpoS::gm (▼) biofilm cells exposed to sterile distilled water, filter-sterilized lake water, and unfiltered lake water at 22°C. Day 0 represents the time when the biofilms were first exposed to various water samples. The column on the left represents the total numbers of biofilm cells, including both attached and detached biofilm cells. The middle and right columns represent the cell densities of attached and detached biofilm cells, respectively. Bars indicate standard deviations at a 95% confidence level.

Fig 5

CSLM images of H32-gfp (A) and H32-gfpΔrpoS::gm (B) biofilms in sterile distilled water (ddH₂O), filter-sterilized lake water (FW), and unfiltered lake water (LW). Day 0 represents the time when E. coli O157:H7 biofilms that were established over 48 h were first exposed to various water samples, whereas day 13 represents the exposure of the biofilm cells to the water samples for 13 days. No stain (NS) represents E. coli cells expressing green fluorescence from green fluorescent protein (GFP) only. SYTO 9 (S9) shows all adhered cells, including background organisms. Live/Dead staining (LD) illustrates the viability of all adhered cells, with green and red representing viable and nonviable cells, respectively.