Response regulator DegU of Listeria monocytogenes controls temperature-responsive flagellar gene expression in its unphosphorylated state

Abstract

We demonstrate that in Listeria monocytogenes, temperature-responsive transcriptional control of flagellar genes does not rely on the phosphorylation of the conserved phosphorylation site (D55) in the receiver domain of response regulator DegU. Furthermore, proper control of DegU-regulated genes involved in ethanol tolerance and virulence is independent of receiver phosphorylation.

FIG. 1.

(A) Swimming motility of the L. monocytogenes EGD, ΔdegU, ΔdegU*, and degU(D55N) strains. Bacteria from single colonies of the respective strains were stabbed into BHI soft agar, and the plates were incubated for 48 h at 24°C. Swimming halos obtained in a representative experiment are shown. The diameter of halos produced by the L. monocytogenes ΔdegU* and degU(D55N) strains averaged 106% ± 18% and 69% ± 8%, respectively, of the mean diameter of the wild-type strain EGD. (B) Electron micrographs of the L. monocytogenes EGD and degU(D55N) strains. Bacteria were grown without shaking in BHI broth at 24°C and were then examined under a transmission electron microscope (TEM 100; Zeiss) after negative staining with 0.5% uranyl acetate for 1 min. The number of flagella of 50 bacteria were counted per strain. On average, L. monocytogenes EGD exhibited seven flagella per cell, while 4.5 flagella were counted in the case of the degU(D55N) strain. The flagella are indicated by arrowheads. Original magnification, ×20,000.

FIG. 2.

Expression analysis of motility genes in the L. monocytogenes EGD, ΔdegU, and degU(D55N) strains. (A) Slot blot hybridization carried out with equal amounts of RNA extracted from the L. monocytogenes EGD (lane 2), ΔdegU (lane 1), and degU(D55N) (lane 3) strains. Hybridization was performed with flaA- and 16S rRNA-specific probes on RNA from three independent preparations. The 16S rRNA-specific hybridization probe was PCR amplified with primer pair 16S-S5/16S-S3. The results of a representative experiment are shown. (B) The relative changes in transcription of flaA, fliN, fliI, fliF, flgK, cheY, cheR, and mcp in the L. monocytogenes ΔdegU and degU(D55N) mutant strains, compared to the wild-type strain EGD (WT), were assessed by qRT-PCR. The ratios of the transcript amount detected in the respective mutant to that of the wild type (ratio, mutant/WT) are depicted. The indicated ratios represent the means of results of three qRT-PCR experiments performed in duplicate with cDNA which was reverse transcribed from independent RNA preparations extracted from bacteria grown at 24°C. The relative transcription of the genes under study was normalized to that of the housekeeping gene rpoB, as described by Mertins et al. (5). The primer pairs generating the respective amplicons are listed in Table 1. Error bars indicate the standard deviations from the means. (C) Western blot analysis was performed with equal amounts of supernatant proteins prepared from liquid cultures of the L. monocytogenes EGD (lane 2), ΔdegU (lane 1), and degU(D55N) (lane 3) strains grown at 24°C using a polyclonal rabbit antiserum directed against flagella of L. monocytogenes serotype 1/2a. FlaA is indicated by an arrow. The results of one of three independent experiments are shown.

FIG. 3.

Growth of the L. monocytogenes EGD, ΔdegU, and degU(D55N) strains at 37°C in BHI broth in the presence of 5% ethanol. Overnight cultures of the respective strains were diluted 1:50 in BHI broth supplemented with 5% ethanol. The optical densities (in Klett units) were determined at the indicated time points. The growth curve represents the means of results of four independent experiments. Error bars indicate the standard deviations from the means.