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Supplementary figures

Elevated levels of σ^S inhibit biofilm formation in Escherichia coli: a role for the Rcs phosphorelay, by L. Ferrières, A. Thompson and D. J. Clarke

Microbiology vol. 155, part 11, pp. 3544-3553

Fig. S1. Mutations in rcsC and rcsD reduce expression of flhDC and motility. (a) The expression of flhDC was measured in different backgrounds using a chromosomal flhDC′-lacZ reporter fusion. β-Galactosidase activity was measured in bacteria grown to mid-exponential phase (OD₆₀₀~0.5) at 30 °C in LB broth with agitation. Strains: BMM920, WT; BMM934, rcsC::Tn10; BMM933, rcsD::Cm; BMM928, rcsC::Tn10 rcsB::Cm; BMM929, (rcsD-rcsB)::Cm; BMM926, rcsB::Cm. Asterisks indicate values that are significantly different from the wild-type (n=3, P<0.05). (b) Derivatives of ZK2686 carrying the same alleles were inoculated at the centre of a 0.3% LB agar plate and incubated at 30 °C. The rate of migration was determined as described in Methods and is expressed as a percentage of the WT migration rate (% WT). Strains: ZK2686, WT; BMM520, rcsC::Tn10; BMM564, rcsD::Cm; BMM552, rcsC::Tn10 rcsB::Cm; BMM921, (rcsD-rcsB)::Cm; BMM532, rcsB::Cm. The values shown are the means of 11 independent experiments and the error bars represent SD.

Fig. S2. Colanic acid production is not responsible for the biofilm defect observed in the rcsC mutant background. Strains ZK2686 (WT), BMM520 (rcsC::Tn10), BMM526 (gmd::λplacMu53) and BMM555 (rcsC::Tn10 gmd::λplacMu53) were grown overnight and inoculated to an OD₆₀₀ of 0.01 in the wells of a PVC microtitre plate and incubated at 30 °C without agitation for 48 h. Biofilm formation was quantified by CV staining, as described in Methods. The values shown are the means of at least three independent experiments and the error bars represent SD.

Fig. S3. The deletion of rprA restores σ^S levels in the rcsC mutant. Strains ZK2686 (WT), BMM533 (rcsC::Cm), BMM940 (rcsC::Cm rprA::Km), BMM938 (rprA::Km) and BMM910 (rpoS::Km) were grown at 30 °C in LB broth with agitation until OD₆₀₀=0.8. The cells were lysed and the proteins were separated using a 12.5% polyacrylamide gel. The proteins were transferred to a nylon membrane and σ^S was detected using σ^S polyclonal antibodies. (a) Typical immunoblot showing the σ^S band in the different backgrounds. (b) The intensity of the σ^S bands was quantified and normalized to the intensity of the band in WT (as described in Methods). The values shown are the means of at least three independent experiments and the error bars represent SD. Statistically significant differences in σ^S levels are indicated.

Fig. S4. The RprA-mediated increase in the level of σ^S is not responsible for the decrease in motility. Strains ZK2686 (WT), BMM533 (rcsC::Cm), BMM938 (rprA::Km) and BMM940 (rcsC::Cm rprA::Km) were inoculated at the centre of a 0.3% (w/v) LB agar plate and incubated at 30 °C. The rate of migration was determined as described in Methods and is expressed as a percentage of the WT migration rate (% WT). The values shown are the means of three independent experiments and the error bars represent SD. Statistically significant differences in the rates of motility are indicated.

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