Polar localization of a soluble methyl-accepting protein of Pseudomonas aeruginosa

Abstract

A soluble methyl-accepting chemotaxis protein (MCP) of Pseudomonas aeruginosa, McpS, showed polar localization by immunofluorescence microscopy. Overexpression of McpS resulted in a dominant-negative effect on chemotaxis and caused a loss of polar clustering of the general MCP population. The polar localization of a soluble MCP defines a third, and unexpected, paradigm for cellular MCP localization.

FIG. 1.

Overexpression of His-McpS in P. aeruginosa. (A) Immunoblot showing induction of His-McpS with arabinose. Whole-cell lysates of PAO1(pJN105) or PAO1(pSB46) (His-McpS) grown in the presence of increasing amounts of arabinose. Following 1 h of induction with the indicated arabinose concentration, cells were grown in Luria-Bertani medium at 30°C and harvested at an approximate optical density at 600 nm of 0.4. His-McpS was detected using a 1/2,000 dilution of anti-His antibody (Sigma) followed by rabbit anti-mouse secondary antibody (Pierce). Relevant molecular masses are shown (in kilodaltons). (B) Overexpression of His-McpS inhibits motility on swarm agar plates. The migration of PAO1 harboring an empty vector (pJN105; black) and two clones of PAO1(pSB46) (white and gray) on soft-agar tryptone broth plates supplemented with 50 μg · ml⁻¹ gentamicin and indicated levels of arabinose were measured after 18 h at 30°C. The average diameters (in centimeters) from five independent measurements (± standard errors of the means) are shown. (C) Overexpression of His-McpS does not inhibit twitching motility. The migration of PAO1 harboring an empty vector (pJN105; black) and two clones of PAO1 harboring pSB46 (His-McpS) (white and gray) were measured. Cells were inoculated with toothpicks into LB plates containing 1% (wt/vol) agar supplemented with 50 μg · ml⁻¹ gentamicin and indicated levels of arabinose and incubated for 24 h at 37°C. The average diameters of migration (in centimeters) from five independent measurements (± standard errors of the means) are shown.

FIG. 2.

Cellular localization of His-McpS through fractionation. PAO1 cells expressing His-McpS were lysed by three passages through a French press. Centrifugation at 8,000 × g removed debris, and membranes were pelleted by centrifugation at 180,000 × g for 1 h. Lane markings are as follows: D, debris; S, soluble fraction; M, membrane fraction. Similar results were seen for cells induced under low- and high-induction conditions (0.01% and 0.1% arabinose, respectively); only higher induction conditions are shown here. Relevant molecular masses are shown (in kilodaltons).

FIG. 3.

Localization of general MCPs and His-McpS by immunofluorescence in PAO1 and PAO1 harboring His-McpS. The general population of MCPs (as detected with anti-Tsr antibodies) localize to the poles of wild-type and vector control cells. Increasing expression of His-McpS in PAO1 results in a decrease in the clustering the general MCP population while having a minimal effect on the polar localization or clustering of His-McpS.