The pgaABCD locus of Escherichia coli promotes the synthesis of a polysaccharide adhesin required for biofilm formation

Abstract

Production of a polysaccharide matrix is a hallmark of bacterial biofilms, but the composition of matrix polysaccharides and their functions are not widely understood. Previous studies of the regulation of Escherichia coli biofilm formation suggested the involvement of an unknown adhesin. We now establish that the pgaABCD (formerly ycdSRQP) locus affects biofilm development by promoting abiotic surface binding and intercellular adhesion. All of the pga genes are required for optimal biofilm formation under a variety of growth conditions. A pga-dependent cell-bound polysaccharide was isolated and determined by nuclear magnetic resonance analyses to consist of unbranched beta-1,6-N-acetyl-D-glucosamine, a polymer previously unknown from the gram-negative bacteria but involved in adhesion by staphylococci. The pga genes are predicted to encode envelope proteins involved in synthesis, translocation, and possibly surface docking of this polysaccharide. As predicted, if poly-beta-1,6-GlcNAc (PGA) mediates cohesion, metaperiodate caused biofilm dispersal and the release of intact cells, whereas treatment with protease or other lytic enzymes had no effect. The pgaABCD operon exhibits features of a horizontally transferred locus and is present in a variety of eubacteria. Therefore, we propose that PGA serves as an adhesin that stabilizes biofilms of E. coli and other bacteria.

FIG. 1.

Transposon insertions in the pga locus of E. coli K-12 and their effects on biofilm formation. (A) Insertions are numbered relative to the first nucleotide (+1) of the coding region of the corresponding gene. The coordinates of this locus on the E. coli K-12 genome (6) are shown. (B) Biofilm formation in polystyrene microtiter plates. Isogenic strains are represented by bars numbered as follows: 1, MG1655; 2, XWC880 (pgaC880::cam); 3, XWA672 (pgaA672::cam); 4, XWC880(pUC19); 5, XWC880(pPGA372)(pgaABCD), 6, XWA672(pUC19); and 7, XWA672 (pPGA372). (C) Strains were as in panel B, except that they were csrA mutants. All biofilms were grown in LB medium at 26°C for 24 h. The asterisks denote significant differences relative to the corresponding parent strain (P < 0.001 [Tukey multigroup analysis]).

FIG. 2.

Effects of nonpolar pga gene disruptions on crystal violet binding of biofilms grown in polystyrene microtiter wells (A and B) and adherence of cells to borosilicate coverslips (C). Panels A and C depict results in the MG1655 strain background. Strains are represented by bars in panels A and B as follows: 1, MG1655; 2, XWMGΔABCD (ΔpgaABCD); 3, XWMGΔA (ΔpgaA); 4, XWMGΔB (ΔpgaB); 5, XWMGΔC (ΔpgaC); and 6, XWD146 (pgaD146::cam). (B) Strain identities were the same as in panel A, except that the strains were csrA mutants. Cultures were grown in LB medium at 26°C for 24 h. Asterisks denote significant differences relative to the corresponding parental strain (P < 0.001 [Tukey multigroup analysis]).

FIG. 3.

Fractionation of polysaccharide extracts by gel filtration FPLC. Extracts from strain TRXWEC (MG1655 csrA cpsE pgaC880) containing either pPGA372 (pgaABCD) (A) or pUC19 (B) were fractionated by using a Sephacryl S-200 (HiPrep 16/60) column. Fractions (1.6 ml) were analyzed for neutral-sugar (▴) and, after hydrolysis, for hexosamine (▪). The column void volume, as determined with 2-MDa blue dextran, is indicated by a horizontal line.

FIG. 4.

500/125 MHz ¹H/¹³C-HMQC spectrum of E. coli pga-dependent polysaccharide with projected ¹H (1.55 to 4.85 ppm) and ¹³C (0 to 124) spectra.

FIG. 5.

Time course of adherence to coverslips by strains defective for PGA or other surface factors. The parent strain TRMG1655 and isogenic mutants defective in PGA production (pgaC880), type I pili (ΔfimB-H), motility (ΔmotB), or curli (csgA2::Tn105) were inoculated in parallel into petri dishes containing CFA medium and sterile borosilicate glass coverslips. Cultures were incubated at 26°C, and attached cells were analyzed at the indicated times. Representative fields are shown. The quantitative results of crystal violet staining of 24-h biofilms, grown in a polystyrene microtiter plate under similar conditions, are shown for comparison.