Isolation of an Escherichia coli K-12 Mutant Strain Able To Form Biofilms on Inert Surfaces: Involvement of a New ompR Allele That Increases Curli Expression

Abstract

Classical laboratory strains of Escherichia coli do not spontaneously colonize inert surfaces. However, when maintained in continuous culture for evolution studies or industrial processes, these strains usually generate adherent mutants which form a thick biofilm, visible with the naked eye, on the wall of the culture apparatus. Such a mutant was isolated to identify the genes and morphological structures involved in biofilm formation in the very well characterizedE. coli K-12 context. This mutant acquired the ability to colonize hydrophilic (glass) and hydrophobic (polystyrene) surfaces and to form aggregation clumps. A single point mutation, resulting in the replacement of a leucine by an arginine residue at position 43 in the regulatory protein OmpR, was responsible for this phenotype. Observations by electron microscopy revealed the presence at the surfaces of the mutant bacteria of fibrillar structures looking like the particular fimbriae described by the Olsén group and designated curli (A. Olsén, A. Jonsson, and S. Normark, Nature 338:652–655, 1989). The production of curli (visualized by Congo red binding) and the expression of the csgA gene encoding curlin synthesis (monitored by coupling a reporter gene to its promoter) were significantly increased in the presence of theompR allele described in this work. Transduction of knockout mutations in either csgA or ompRcaused the loss of the adherence properties of several biofilm-formingE. coli strains, including all those which were isolated in this work from the wall of a continuous culture apparatus and two clinical strains isolated from patients with catheter-related infections. These results indicate that curli are morphological structures of major importance for inert surface colonization and biofilm formation and demonstrate that their synthesis is under the control of the EnvZ-OmpR two-component regulatory system.

Involvement of the Cpx signal transduction pathway of E. coli in biofilm formation

“…The pta-ackA deletion was moved as described in [11]. The 7.25-kb PvuII fragment containing the csgA-uidA fusion of pOV874 [1] was used to transform the recD csgA D355 strain. White colonies were selected on CFA medium and the fusion was then moved by P1 vir transduction in di¡erent genetic backgrounds.…”

“…Adherence of both E. coli K12 and medical isolates depends on curli production [1]. In order to address the possible regulation of the curli operon by cpxA, we used a csgA: :uidA transcriptional fusion which enables us to estimate the synthesis rate of curlin in di¡erent cpxA backgrounds.…”

“…Bio¢lm formation on solid surfaces is a very common phenomenon among bacteria with important economic and medical consequences. A strain of Escherichia coli K-12 able to form a thick bio¢lm on inert surfaces such as glass or polystyrene has previously been isolated from a continuous culture in minimal media [1]. Increased adhesion to solid sur-faces by this mutant is the result of the overproduction of curli, a particular class of pili.…”

“…The presence of the ompR234 allele signi¢cantly increases the expression of the curlin encoding csgA gene. Loss of adherence properties of several strains of adherent E. coli is caused by transduction of knockout mutations in either the csgA or ompR gene [1]. Curli are therefore essential for bio¢lm formation and the EnvZ-OmpR two-component system regulates the synthesis of curlin, their major component.…”

Systematic characterization ofEscherichia coligenes/ORFs affecting biofilm formation

“…Also, none of the genes selected on the basis of the results of their over-expression caused unusual bio¢lm formation in LB or M63-glucose medium or in the presence of varying concentrations of NaCl (0.1^0.4 M) when deleted in W3110 strain. Although it has been reported that curli production is important for the adherence of bio¢lm-forming cells [35], disruptants of csgF and csgG genes for curli transport and assembly formed bio¢lm. We observed that the strain W3110 we used to construct disruptants was ine⁄cient in curli production, judging from the color of its colonies on Congo red plates (data not shown).…”