1887

Abstract

The Rcs phosphorelay is composed of RcsC, RcsD and the response regulator RcsB, and this signalling pathway has been implicated in virulence and biofilm formation in many enteric bacteria. It was previously shown that a mutation in resulted in defective biofilm formation in [ Ferrières, L. & Clarke, D. J. (2003) , 1665–1682 ]. To identify the molecular mechanisms underlying the observed biofilm defect we carried out a screen looking for suppressor mutants that restored biofilm formation in the mutant background. One of the mutants was identified to be in , a gene encoding a small RNA molecule that is involved in the post-transcriptional control of the alternative sigma factor, . The expression of is regulated by the Rcs phosphorelay, and there are elevated levels present in the mutant due to the overexpression of in this background. Using different approaches, we have established that the increase in levels is responsible for the biofilm defect. Therefore, the Rcs phosphorelay is involved in maintaining appropriate levels of during biofilm formation in .

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2009-11-01
2019-10-23
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vol. , part 11, pp. 3544-3553

IMAGE

Mutations in and reduce expression of and motility. (a) The expression of was measured in different backgrounds using a chromosomal ′- reporter fusion. β-Galactosidase activity was measured in bacteria grown to mid-exponential phase (OD ~0.5) at 30 °C in LB broth with agitation. Strains: BMM920, WT; BMM934, ::Tn ; BMM933, ::Cm; BMM928, ::Tn ::Cm; BMM929, ( )::Cm; BMM926, ::Cm. Asterisks indicate values that are significantly different from the wild-type ( =3, <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, ::Tn ; BMM564, ::Cm; BMM552, ::Tn ::Cm; BMM921, ( )::Cm; BMM532, ::Cm. The values shown are the means of 11 independent experiments and the error bars represent SD.

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Colanic acid production is not responsible for the biofilm defect observed in the mutant background. Strains ZK2686 (WT), BMM520 ( ::Tn ), BMM526 ( ::λp Mu53) and BMM555 ( ::Tn ::λp Mu53) were grown overnight and inoculated to an OD 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.

IMAGE

The deletion of restores σ levels in the mutant. Strains ZK2686 (WT), BMM533 ( ::Cm), BMM940 ( ::Cm ::Km), BMM938 ( ::Km) and BMM910 ( ::Km) were grown at 30 °C in LB broth with agitation until OD =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 was detected using σ polyclonal antibodies. (a) Typical immunoblot showing the σ band in the different backgrounds. (b) The intensity of the σ 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 levels are indicated.

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