The pgaABCD Locus of Escherichia coli Promotes the Synthesis of a Polysaccharide Adhesin Required for Biofilm Formation

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   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]).
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   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]).
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   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.
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   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.
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   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.

• American Society for Microbiology