Activation of the Rcs signal transduction system is responsible for the thermosensitive growth defect of an Escherichia coli mutant lacking phosphatidylglycerol and cardiolipin

Abstract

The lethal effect of an Escherichia coli pgsA null mutation, which causes a complete lack of the major acidic phospholipids, phosphatidylglycerol and cardiolipin, is alleviated by a lack of the major outer membrane lipoprotein encoded by the lpp gene, but an lpp pgsA strain shows a thermosensitive growth defect. Using transposon mutagenesis, we found that this thermosensitivity was suppressed by disruption of the rcsC, rcsF, and yojN genes, which code for a sensor kinase, accessory positive factor, and phosphotransmitter, respectively, of the Rcs phosphorelay signal transduction system initially identified as regulating the capsular polysaccharide synthesis (cps) genes. Disruption of the rcsB gene coding for the response regulator of the system also suppressed the thermosensitivity, whereas disruption of cpsE did not. By monitoring the expression of a cpsB'-lac fusion, we showed that the Rcs system is activated in the pgsA mutant and is reverted to a wild-type level by the rcs mutations. These results indicate that envelope stress due to an acidic phospholipid deficiency activates the Rcs phosphorelay system and thereby causes the thermosensitive growth defect independent of the activation of capsule synthesis.

FIG. 1.

Suppression of the thermosensitive growth defect of the pgsA null mutant. The minitransposon insertions in rcsC, rcsF, and yojN identified by a screen for suppressor mutants were transduced by phage P1 into S330. Overnight cultures of S330 (A) and its rcsC::mini-Tn10 cam (B), rcsF::mini-Tn10 cam (C), yojN::mini-Tn10 cam (D), and cpsE::Tn10 (E) transductants were diluted to 3 to 5 Klett units in LB medium and grown at 37°C (open circles) and 42°C (closed circles), and the culture turbidities were monitored.

FIG. 2.

Activation of Rcs phosphorelay signal transduction system in the pgsA null mutant. Cells were grown in buffered LB medium at 30°C to mid-exponential phase (ca. 100 Klett units), and the β-galactosidase activity was measured. The means and standard errors of at least three measurements are shown. The mutations transduced into strain UE29, CL330, or CL332 were rcsC::mini-Tn10 cam (rcsC), rcsF::mini-Tn10 cam (rcsF), rcsB::Tn10Δ16Δ17 (rcsB), yojN::mini-Tn10 cam (yojN), ΔyojN::kan (ΔyojN), mdoH::Tn10 (mdoH), and mdoB::Tn10 (mdoB). Three transductants were isolated and examined. They exhibited essentially the same results. For panel F, the cells were grown in the presence of 1 mM IPTG.

FIG. 3.

The mutations that suppress the thermosensitivity of the pgsA null mutant do not suppress the loss of phosphatidylglycerol and cardiolipin. UE29 (lane 1), CL330 (lane 2), and its rcsC::mini-Tn10 cam (lane 3), rcsF::mini-Tn10 cam (lane 4), yojN::mini-Tn10 cam (lane 5), and rcsB::Tn10Δ16Δ17 (lane 6) transductants were grown to mid-exponential phase (ca. 100 Klett units) in LB medium. The lipids were extracted and separated by thin-layer chromatography, and spots of phospholipids were visualized with Dittmer-Lester reagent. F, solvent front; CL, cardiolipin; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; O, chromatographic origin. The faint spots at the positions between cardiolipin and phosphatidylglycerol in lanes 2 to 6 are phosphatidic acid, which accumulates in small amounts in pgsA null cells (29).

FIG. 4.

RcsF is essential for Rcs signal transduction in response to treatment with chlorpromazine. The experimental procedure was done essentially as described by Conter et al. (11). A cpsB′-lac fusion strain, SG20781 (circles), and its rcsF::mini-Tn10 cam transductant (squares) were grown in NaCl-free LB medium to ca. 25 Klett units at 37°C, and chlorpromazine was added to the indicated concentrations. After 2 h, the turbidity (open symbols) and β-galactosidase activity (closed symbols) were measured. The turbidity was normalized to the value of the culture that was not treated with the drug for each strain (ca. 200 Klett units) and is shown as a percentage.