Defective O-antigen polymerization in tolA and pal mutants of Escherichia coli in response to extracytoplasmic stress

Abstract

We have previously shown that the TolA protein is required for the correct surface expression of the Escherichia coli O7 antigen lipopolysaccharide (LPS). In this work, delta tolA and delta pal mutants of E. coli K-12 W3110 were transformed with pMF19 (encoding a rhamnosyltransferase that reconstitutes the expression of O16-specific LPS), pWQ5 (encoding the Klebsiella pneumoniae O1 LPS gene cluster), or pWQ802 (encoding the genes necessary for the synthesis of Salmonella enterica O:54). Both DeltatolA and delta pal mutants exhibited reduced surface expression of O16 LPS as compared to parental W3110, but no significant differences were observed in the expression of K. pneumoniae O1 LPS and S. enterica O:54 LPS. Therefore, TolA and Pal are required for the correct surface expression of O antigens that are assembled in a wzy (polymerase)-dependent manner (like those of E. coli O7 and O16) but not for O antigens assembled by wzy-independent pathways (like K. pneumoniae O1 and S. enterica O:54). Furthermore, we show that the reduced surface expression of O16 LPS in delta tolA and delta pal mutants was associated with a partial defect in O-antigen polymerization and it was corrected by complementation with intact tolA and pal genes, respectively. Using derivatives of W3110 delta tolA and W3110 delta pal containing lacZ reporter fusions to fkpA and degP, we also demonstrate that the RpoE-mediated extracytoplasmic stress response is upregulated in these mutants. Moreover, an altered O16 polymerization was also detected under conditions that stimulate RpoE-mediated extracytoplasmic stress responses in tol+ and pal+ genetic backgrounds. A Wzy derivative with an epitope tag at the C-terminal end of the protein was stable in all the mutants, ruling out stress-mediated proteolysis of Wzy. We conclude that the absence of TolA and Pal elicits a sustained extracytoplasmic stress response that in turn reduces O-antigen polymerization but does not affect the stability of the Wzy O-antigen polymerase.

FIG. 1.

Analysis of O16 LPS expression in E. coli K-12 strain W3110 and ΔtolA and Δpal isogenic mutants. All strains carry the plasmid pMF19 for O16 antigen expression. LPS preparations in panels A and C were analyzed by tricine-SDS-PAGE followed by silver staining as described in Materials and Methods. (A) Effect of ΔtolA and Δpal on O16 LPS surface expression. (B) Western blot of a similar gel as in panel A using O16-specific antiserum. (C) Complementation analysis of E. coli W3110 and the ΔtolA and Δpal mutants with pEV2 and pEV3 carrying functional tolA and pal genes, respectively.

FIG. 2.

(A) Analysis of O16 LPS expression in E. coli K-12 strain W3110 and the isogenic Δwzz and ΔtolA Δwzz mutants. LPS preparations were analyzed by tricine-SDS-PAGE followed by silver staining. (B) Analysis of O16 LPS expression in E. coli K-12 strain W3110 and the isogenic Δwzz and ΔpalΔwzz mutants. All strains in panels A and B carry the plasmid pMF19 for the expression of the O16 polysaccharide.

FIG. 3.

Complementation of the ΔtolA (A) and Δpal (B) phenotypes with plasmids carrying functional wzz or wzy genes under the control of the P_BAD promoter. All strains carry the plasmid pMF19 for the expression of the O16 polysaccharide and were grown in the presence of 0.1% arabinose.

FIG. 4.

(A) Analysis of LPS in E. coli W3110 and the ΔtolA and Δpal isogenic mutants, each containing pWQ5, which encodes the synthesis of K. pneumoniae O1 antigen. LPS preparations were analyzed by tricine-SDS-PAGE followed by silver staining. (B) Analysis of LPS in E. coli W3110 and the ΔtolA and Δpal isogenic mutants, each containing pWQ802, which encodes the synthesis of S. enterica serovar Borreze O:54 antigen. LPS preparations were separated by tricine-SDS-PAGE, transferred to nitrocellulose, and analyzed by Western blotting with O:54-specific antiserum.

FIG. 5.

β-Galactosidase activities of W3110 and ΔtolA and Δpal derivatives containing dsbA-lacZ, fkpA-lacZ, or degP-lacZ fusions. Bars depict the mean units of activity calculated from five repeated experiments. Asterisks indicate statistically significant induction (P < 0.005).

FIG. 6.

(A) Effect of indole and diphenylamine (DPA) on LPS expression in E. coli K-12 W3110(pMF19). LPS preparations were analyzed by tricine-SDS-PAGE followed by silver staining. (B) Effect of indole and diphenylamine on the β-galactosidase activity of the W3110 derivative GL113 containing a degP-lacZ fusion. Bars depict the mean units of activity calculated from three repeated experiments.

FIG. 7.

(A) Effect of an rseA deletion on the β-galactosidase activity of the W3110 derivative GL113 containing a degP-lacZ fusion. Bars depict the mean units of activity calculated from three repeated experiments. (B) Effect of the rseA deletion on the LPS expression in E. coli W3110, as compared with the LPS expression in the parental W3110, EVV8 (ΔtolA), and EVV9 (Δpal) strains. All strains carry the plasmid pMF19 for the expression of the O16 polysaccharide. LPS preparations were analyzed by tricine-SDS-PAGE followed by silver staining.

FIG. 8.

Effect of overexpression of rseA encoded by pEV30 on LPS expression of ΔtolA and Δpal mutants in E. coli K-12. All strains carry the plasmid pMF19 for O-antigen expression and were also grown in the presence of 0.3% arabinose.

FIG. 9.

Analysis of O16 LPS expression in E. coli K-12 strain W3110 and the ΔtolA, Δpal, ΔdegP, ΔtolA ΔdegP, and Δpal ΔdegP isogenic mutants. LPS preparations were analyzed by tricine-SDS-PAGE followed by silver staining. All strains carry the plasmid pMF19 for the expression of the O16 polysaccharide.

FIG. 10.

Western blot analysis of proteins involved in O-antigen assembly. Inner membrane fractions were obtained as described in Materials and Methods. Proteins were separated by SDS-PAGE and transferred to nitrocellulose. Blots were reacted with the anti-FLAG M2 monoclonal antibody. (A) Detection of Wzy_FLAG3X protein expression. Prestained molecular mass standards in kilodaltons are indicated: myosin (206 kDa), β-galactosidase (119 kDa), bovine serum albumin (96 kDa), ovalbumin (56 kDa), carbonic anhydrase (38 kDa), and soybean trypsin inhibitor (29 kDa). The arrow indicates the position of Wzy_FLAG3X. (B) Detection of WecA_FLAG6xHis and Wzx_FLAG. Prestained molecular mass standards in kilodaltons are indicated: β-galactosidase (113 kDa), bovine serum albumin (96 kDa), ovalbumin (53 kDa), carbonic anhydrase (36 kDa), and soybean trypsin inhibitor (28 kDa). The differences with respect to the markers in panel A are due to different lots of markers used for the two gels. The arrows indicate the positions of Wzx_FLAG and WecA_FLAG6xHis.