The PrpC serine-threonine phosphatase and PrkC kinase have opposing physiological roles in stationary-phase Bacillus subtilis cells

Abstract

Loss of the PrpC serine-threonine phosphatase and the associated PrkC kinase of Bacillus subtilis were shown to have opposite effects on stationary-phase physiology by differentially affecting cell density, cell viability, and accumulation of beta-galactosidase from a general stress reporter fusion. These pleiotropic effects suggest that PrpC and PrkC have important regulatory roles in stationary-phase cells. Elongation factor G (EF-G) was identified as one possible target of the PrpC and PrkC pair in vivo, and purified PrpC and PrkC manifested the predicted phosphatase and kinase activities against EF-G in vitro.

FIG. 1.

Effect of prpC and prkC alleles on stationary-phase cell density. Cultures were grown at 37°C in shake flasks containing BLB medium; densities were measured at the times indicated using a Klett-Summerson photoelectric colorimeter with a number 66 (red) filter. Cultures entered stationary phase at 2.5 h. Symbols: ○, PB198 wild type; •, PB703 lacking the phosphatase activity (prpCΔ1); ▴, PB706 lacking the kinase activity (prkCΔ1); ▪, PB723 lacking both activities (prpC-prkCΔ1).

FIG. 2.

Detection of an additional phosphorylated protein in the strain lacking PrpC phosphatase activity. (A) Extracts of cells growing logarithmically in BLB medium were subjected to SDS-PAGE. A Western blot made from this 10% gel was probed with antiphosphothreonine antibody. Lane 1, PB198 wild type; lane 2, PB706 lacking the kinase activity (prkCΔ1); lane 3, PB703 lacking the phosphatase activity (prpCΔ1); lane 4, PB723 lacking both activities (prpCΔ1 prkCΔ1). The arrow indicates the new signal present in lane 3. Mobilities of the molecular weight standards are shown on the left (in thousands). (B) SDS-PAGE analysis of the proteins precipitated from B. subtilis cell extracts by anti-E. coli EF-G antibody. Lanes 1 and 2 were stained with Coomassie blue and each manifested a band with the mobility of B. subtilis EF-G. Lane 1, PB703 lacking the phosphatase activity (prpCΔ1); lane 2, PB198 wild type. Lanes 3 and 4 were analyzed by Western blotting, and lane 3 manifested a band recognized by antiphosphothreonine antibody (arrow). Lane 3, PB703 lacking the phosphatase activity (prpCΔ1); lane 4, PB198 wild type. Mobilities of the molecular weight standards are on the left.

FIG. 3.

Purified PrpC phosphatase and PrkC kinase are active against EF-G in vitro. (A) Lanes 1 to 3 show the kinase assay. Purified proteins were incubated at 37°C in kinase buffer together with [γ-³²P]ATP and then separated by SDS-PAGE. Lane 1, PrkC alone; lane 2, EF-G alone; lane 3, PrkC and EF-G. Lanes 4 to 7 show the phosphatase assay. After completion of the kinase reaction, labeled PrkC and EF-G were separated from unincorporated [γ-³²P]ATP, resuspended in phosphatase buffer, incubated at 37°C, and then separated on an SDS-PAGE gel. Lane 4 shows a 60-min incubation in the absence of the PrpC phosphatase; lanes 5 to 7 show 15-, 30-, and 60-min incubations in the presence of purified PrpC. Arrows here and in panel B denote the positions of unlabeled PrkC and EF-G included as standards. (B) Immune precipitation of EF-G from a kinase labeling reaction. Purified proteins were incubated with [γ-³²P]ATP as described for panel A and then mixed with anti-E. coli EF-G antibody and protein A beads. The resulting precipitate was subjected to SDS-PAGE. Lane 1, the kinase reaction yielding labeled PrkC and EF-G; lane 2, the labeled EF-G recovered by immune precipitation; lane 3, a longer exposure of lane 2. (C) Phosphatase release assay. The immune precipitate shown in lanes 2 and 3 of panel B was washed and resuspended in phosphatase buffer and then incubated at 37°C together with PrpC. Samples were removed at the times indicated, treated with cold trichloroacetic acid, centrifuged, and counted to determine the amount of label released into the supernatant fraction. (D) Determination of antibody specificity. Purified PrkC and EF-G were incubated in a standard kinase reaction with 1 mM cold ATP and no labeled ATP. The reaction was terminated, divided into three equal parts, and analyzed by SDS-PAGE and Western blotting with antiphosphothreonine antibody. Lane 1, antibody alone; lane 2, antibody after preincubation with 20 mM phosphothreonine; lane 3, antibody after preincubation with 20 mM phosphoserine. The PrkC and EF-G signals are indicated by arrows.