The transcriptional antiterminator RfaH represses biofilm formation in Escherichia coli

Abstract

We investigated the influence of regulatory and pathogenicity island-associated factors (Hha, RpoS, LuxS, EvgA, RfaH, and tRNA5Leu) on biofilm formation by uropathogenic Escherichia coli (UPEC) strain 536. Only inactivation of rfaH, which encodes a transcriptional antiterminator, resulted in increased initial adhesion and biofilm formation by E. coli 536. rfaH inactivation in nonpathogenic E. coli K-12 isolate MG1655 resulted in the same phenotype. Transcriptome analysis of wild-type strain 536 and an rfaH mutant of this strain revealed that deletion of rfaH correlated with increased expression of flu orthologs. flu encodes antigen 43 (Ag43), which mediates autoaggregation and biofilm formation. We confirmed that deletion of rfaH leads to increased levels of flu and flu-like transcripts in E. coli K-12 and UPEC. Supporting the hypothesis that RfaH represses biofilm formation through reduction of the Ag43 level, the increased-biofilm phenotype of E. coli MG1655rfaH was reversed upon inactivation of flu. Deletion of the two flu orthologs, however, did not modify the behavior of mutant 536rfaH. Our results demonstrate that the strong initial adhesion and biofilm formation capacities of strain MG1655rfaH are mediated by both increased steady-state production of Ag43 and likely increased Ag43 presentation due to null rfaH-dependent lipopolysaccharide depletion. Although the roles of rfaH in the biofilm phenotype are different in UPEC strain 536 and K-12 strain MG1655, this study shows that RfaH, in addition to affecting the expression of bacterial virulence factors, also negatively controls expression and surface presentation of Ag43 and possibly another Ag43-independent factor(s) that mediates cell-cell interactions and biofilm formation.

FIG. 1.

Analysis of the adhesive and biofilm-forming capacities of planktonic cells of uropathogenic E. coli strain 536 and K-12 strain MG1655 and derivatives of these strains. (A) Initial adhesive capacity as measured using a sea sand column system. (B) Mature biofilm development as measured with a flow culture microfermentor system. The results of a representative experiment are shown.

FIG. 2.

Analysis of the importance of different flu orthologs for the RfaH-dependent increased biofilm formation capacity of E. coli strain 536. (A) Biofilm development of strain 536 and its derivatives in microfermentors after 24 h of growth at 37°C in M63B1 glucose medium. The results of a representative experiment are shown. (B) Average results of at least four experiments plotted as a histogram. The error bars indicate standard errors of the means. The level of biofilm formed by wild-type strain 536 (wt) was defined as 100%.

FIG. 3.

Analysis of the importance of flu for the RfaH-dependent increased biofilm formation capacity of E. coli strain MG1655. (A) Biofilm development of K-12 strain MG1655 and its mutants in microfermentors after 48 h of growth at 37°C in M63B1 glucose medium. The images are representative images of the bottoms of the microfermentors and of the Pyrex spatulas on which biofilms formed. (B) Average results of at least four experiments plotted as a histogram. The error bars indicate standard errors of the means. The level of biofilm formed by wild-type strain MG1655 (wt) was defined as 100%.

FIG. 4.

Comparison of flu transcript levels of E. coli wild-type strains and their rfaH derivatives. (A) Comparison of flu transcript levels of uropathogenic E. coli strain 536 and K-12 strain MG1655. The uropathogenic and K-12 wild-type strains, as well as their rfaH mutants, were cultivated at 37°C. The flu transcript levels of exponentially growing, planktonic cells were compared by RT-PCR. 16S RNA levels were used as internal controls of the quantity of RNA used for RT-PCR. (B) Comparison of the flu ortholog transcript levels of uropathogenic E. coli strain 536 and its rfaH mutant. The strains were cultivated at 37°C. The ORF52_III-536 and ORF47_V-536 transcripts of exponentially growing, planktonic cells were reverse transcribed and amplified by RT-PCR. The cDNA was consecutively digested with SacII and BstEII in order to distinguish between the ORF52_III-536- and ORF47_V-536-specific cDNA fractions. The resulting restriction fragments were separated on an agarose gel. The localization of the SacII and BstEII restriction sites and the sizes of the resulting cDNA fragments are indicated. wt, wild type.

FIG. 5.

Comparison of Ag43 expression in E. coli wild-type strains and their rfaH derivatives. Heat-extracted surface appendages were separated by SDS-polyacrylamide gel electrophoresis. Passenger domains of Ag43 of strain MG1655 (A) or of the Ag43 variants of strain 536 (ORF52_III and ORF47_V) (B) were detected using a polyclonal antiserum raised against MG1655-specific Ag43. The results of a representative experiment are shown.

FIG. 6.

Comparison of biofilm structures formed by strains 536 (A) and MG1655 (B). The phenotypic analysis of the structure of strains 536 and MG1655 and their derivatives was performed in microfermentors with bacteria grown for 48 h at 37°C in M63B1 glucose medium. Thermanox slides were stuck on Pyrex spatulas and removed after biofilm growth for scanning electron microscopy analysis. The images are representative images.

FIG. 7.

Unshielding of Ag43 by LPS depletion causes increasing biofilm formation. (A) Biofilm development for strain MG1655 and its derivatives in microfermentors after 48 h of growth at 37°C in M63B1 glucose medium. The results of a representative experiment are shown. (B) Average results of at least four experiments plotted as a histogram. The error bars indicate standard errors of the means. The level of biofilm formed by wild-type strain MG1655 (wt) was defined as 100%. (C) LPS patterns of the strains.