Differential expression of the Escherichia coli autoaggregation factor antigen 43 - PubMed (original) (raw)

Differential expression of the Escherichia coli autoaggregation factor antigen 43

Mark A Schembri et al. J Bacteriol. 2003 Apr.

Abstract

Antigen 43 (Ag43) is a self-recognizing surface adhesin found in most Escherichia coli strains. Due to its excellent cell-to-cell aggregation characteristics, Ag43 expression confers clumping and fluffing of cells and promotes biofilm formation. Ag43 expression is repressed by the cellular redox sensor OxyR. Here we used mutant versions of OxyR that are locked in either the reduced or the oxidized form as well as the addition of a simple redox-changing chemical to show that the redox state of OxyR influences Ag43 expression. Furthermore, the redox state of OxyR influences the biofilm-forming potential of E. coli. Finally, we demonstrated that Ag43-mediated cell aggregation confers significant protection against hydrogen peroxide killing.

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Figures

FIG. 1.

H2O2 response profiles monitored by adding identical aliquots of H2O2 to the center of plates incubated with oxyR strain MGJ1 harboring various plasmids. (A to D) The plasmids were pBAD/myc-HisA (A), pMGJ1 (wild-type OxyR) (B), pMAS141 (OxyRRed,C199S) (C), and pMAS142 (OxyROx,H198R) (D). Note the presence of bubbles in MGJ1(pMAS142) (D), likely due to enhanced catalase activity resulting from priming of the oxidative stress response regulon. (E) Block diagram showing the mean sizes of the zones of inhibition and standard deviations from eight independent experiments. WT, wild type.

FIG. 2.

Coomassie brilliant blue-stained SDS-polyacrylamide gel (left panel) and Western immunoblot (right panel) of cell lysates of oxyR strain MGJ1 harboring various plasmids. Lane 1, pBAD/myc-HisA; lane 2, pMGJ1 (wild-type OxyR); lane 3, pMAS141 (OxyRRed,C199S); lane 4, pMAS142 (OxyROx,H198R). Western blotting was performed with a polyclonal serum raised against the α subunit of Ag43. The position of the Ag43 α-subunit protein is indicated. Molecular weight size markers (in thousands) are shown at the left.

FIG. 3.

Settling profiles of strain MGJ1 harboring plasmids pBAD/myc-HisA (triangles), pMAS141 (OxyRRed,C199S) (crosses), pMGJ1 (wild-type OxyR) (squares), and pMAS142 (OxyROx,H198R) (circles).

FIG. 4.

Ag43 expression and associated protection against H2O2. (A) Colony morphology of strains examined by phase-contrast microscopy after overnight growth on solid media with 0.2% arabinose. Colonies were photographed at the same magnification. Panel 1, E. coli strain MGJ1 containing plasmid pMAS141 (OxyRRed,C199S); panel 2, E. coli strain MGJ1 containing plasmid pMAS142 (OxyROx,H198R). (B) Phase-contrast microscopy of cells. Panel 1, E. coli strain MS528 (fim flu) containing the vector control (pBR322); panel 2, E. coli strain MS528 containing the _flu_-carrying plasmid pHHA147. (C) Ag43-mediated cell aggregation enhances the ability to survive lethal doses of hydrogen peroxide. Approximately 109 cells of each strain were exposed to a series of H2O2 concentrations and plated on solid medium to obtain viable counts. Shown are the numbers of surviving cells after exposure to 0.275 M H2O2. Two strain sets were used, each differing only in their Ag43 expression. The first set included E. coli strain MS528 containing the vector control and plasmid pHHA147 (flu+); the second set included a non-Ag43-expressing variant (BD1511) and an Ag43-expressing variant (BD1512) of E. coli strain BD1428. Error bars indicate standard deviations.

FIG. 5.

Influence of DTT on cell growth and Ag43 expression. (A) E. coli MG1655 fim grown in LB medium (squares) and the same strain grown in LB medium but pulsed at regular 15-min intervals with 100 μl of either 1.0 M (triangles) or 2.0 M (circles) DTT. (B) Western blot of concentrated heat-extracted cell surface proteins obtained from cultures grown to an OD600 of 1.0. Lane 1, E. coli MG1655 fim; lane 2, E. coli MG1655 fim grown with 100-μl pulses of 1.0 M DTT every 15 min; lane 3, E. coli MG1655 fim grown with 100-μl pulses of 2.0 M DTT every 15 min. Western blotting was performed with a polyclonal serum raised against the α subunit of Ag43. The position of the Ag43 α-subunit protein is indicated.

FIG. 6.

Effect of OxyR redox status on biofilm formation by MGJ1 hosts containing various plasmids. Plasmids were as follows, from left to right: pBAD/myc-HisA (vector control), pMGJ1 (wild-type OxyR), pMAS141 (OxyRRed,C199S), and pMAS142 (OxyROx,H198R). Strains were grown on polystyrene microtiter plates. Adherent cells were stained with 0.1% crystal violet. Shown is the quantification of results after the determination of _A_600 readings. Error bars indicate standard deviations.

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Differential expression of the Escherichia coli autoaggregation factor antigen 43 - PubMed (original) (raw)