Regulation of Salmonella enterica pathogenicity island 1 by DNA adenine methylation

Abstract

DNA adenine methylase (Dam(-)) mutants of Salmonella enterica are attenuated in the mouse model and present multiple virulence-related defects. Impaired interaction of Salmonella Dam(-) mutants with the intestinal epithelium has been tentatively correlated with reduced secretion of pathogenicity island 1 (SPI-1) effectors. In this study, we show that S. enterica Dam(-) mutants contain lowered levels of the SPI-1 transcriptional regulators HilA, HilC, HilD, and InvF. Epistasis analysis indicates that Dam-dependent regulation of SPI-1 requires HilD, while HilA, HilC, and InvF are dispensable. A transcriptional hilDlac fusion is expressed at similar levels in Dam(+) and Dam(-) hosts. However, lower levels of hilD mRNA are found in a Dam(-) background, thus providing unsuspected evidence that Dam methylation might exert post-transcriptional regulation of hilD expression. This hypothesis is supported by the following lines of evidence: (i) lowered levels of hilD mRNA are found in Salmonella Dam(-) mutants when hilD is transcribed from a heterologous promoter; (ii) increased hilD mRNA turnover is observed in Dam(-) mutants; (iii) lack of the Hfq RNA chaperone enhances hilD mRNA instability in Dam(-) mutants; and (iv) lack of the RNA degradosome components polynucleotide phosphorylase and ribonuclease E suppresses hilD mRNA instability in a Dam(-) background. Our report of Dam-dependent control of hilD mRNA stability suggests that DNA adenine methylation plays hitherto unknown roles in post-transcriptional control of gene expression.

Figure 1.—

Diagram showing the transcriptional units of Salmonella enterica SPI-1 and the regulatory circuits under the control of transcription factors HilA, HilD, HilC, RtsA, and InvF (adapted from Lostroh and Lee 2001; Ellermeier and Slauch 2003; Altier 2005; Jones 2005).

Figure 2.—

Levels of HilA, HilC, HilD, and InvF in protein extracts from Dam⁺ and Dam⁻ isogenic strains. Epitope-tagged proteins were detected by Western blotting with either anti-FLAG or anti-HA commercial antibodies, as appropriate. The charge control was GroEL in all cases. Strains were SV5456 (hilA∷3× FLAG), SV5874 (hilA∷3× FLAG Dam⁻), SV5455 (hilC∷3× FLAG), SV5873 (hilC∷3× FLAG Dam⁻), SV5624 (hilD∷HA), SV5625 (hilD∷HA Dam⁻), SV5457 (invF∷3× FLAG), and SV5875 (invF∷3× FLAG Dam⁻).

Figure 3.—

β-Galactosidase activities of hilA∷lac, invF∷lac, sipB∷lac, hilC∷lac, and invH∷lac fusions in the presence and in the absence of individual transcription factors involved in SPI-1 control. Solid histograms represent β-galactosidase activities measured in a Dam⁺ background. Open histograms represent β-galactosidase activities measured in a Dam⁻ background. Strains were SV5284 (hilA∷lac), SV5285 (hilA∷lac Dam⁻), SV5401 (hilA∷lac HilC⁻), SV5402 (hilA∷lac HilC⁻ Dam⁻), SV5399 (hilA∷lac HilD⁻), SV5400 (hilA∷lac HilD⁻ Dam⁻), SV5297 (invF∷lac), SV5298 (invF∷lac Dam⁻), SV5403 (invF∷lac HilA⁻), SV5404 (invF∷lac HilA⁻ Dam⁻), SV5405 (invF∷lac HilC⁻), SV5406 (invF∷lac HilC⁻ Dam⁻), SV5407 (invF∷lac HilD⁻), SV5408 (invF∷lac HilD⁻ Dam⁻), SV5542 (invF∷lac RtsA⁻), SV5543 (invF∷lac RtsA⁻ Dam⁻), SV5382 (sipB∷lac), SV5383 (sipB∷lac Dam⁻), SV5316 (sipB∷lac HilA⁻), SV5308 (sipB∷lac HilA⁻ Dam⁻), SV5318 (sipB∷lac HilC⁻), SV5310 (sipB∷lac HilC⁻ Dam⁻), SV5320 (sipB∷lac HilD⁻), SV5312 (sipB∷lac HilD⁻ Dam⁻), SV5540 (sipB∷lac RtsA⁻), SV5541 (sipB∷lac RtsA⁻ Dam⁻), SV5322 (sipB∷lac InvF⁻), SV5314 (sipB∷lac InvF⁻ Dam⁻), SV5384 (hilC∷lac), SV5385 (hilC∷lac Dam⁻), SV5386 (hilC∷lac HilD⁻), SV5387 (hilC∷lac HilD⁻ Dam⁻), SV5301 (invH∷lac), SV5302 (invH∷lac Dam⁻), SV5419 (invH∷lac HilA⁻), SV5420 (invH∷lac HilA⁻ Dam⁻), SV5417 (invH∷lac HilC⁻), SV5418 (invH∷lac HilC⁻ Dam⁻), SV5415 (invH∷lac HilD⁻), and SV5416 (invH∷lac HilD⁻ Dam⁻). Data are averages and standard deviations from three experiments.

Figure 4.—

(A) β-Galactosidase activity of a hilD∷lac transcriptional fusion in Dam⁺ (SV5286) and Dam⁻ (SV5288) isogenic hosts. Data are averages and standard deviations from three experiments. (B) β-Galactosidase activity of the same hilD∷lac transcriptional fusion in Dam⁺ HilD⁺ (SV5592), Dam⁺ HilD⁻ (SV5594), Dam⁻ HilD⁺ (SV5596), and Dam⁻ HilD⁻ (SV5598) isogenic merodiploids (averages of three experiments). (C) Relative amounts of hilD mRNA in Dam⁺ (ATCC 14028) and Dam⁻ (SV5264) strains, normalized to ompA mRNA. Two primer pairs, complementary to 5′ and 3′ hilD regions, were used. Histograms represent the averages from three independent experiments.

Figure 5.—

(A) Relative amounts of hilD mRNA in Dam⁺ (solid histograms) and Dam⁻ (open histograms) isogenic strains expressing hilD from a heterologous, tetracycline-dependent promoter. Levels of hilD mRNA were normalized to ompA mRNA, as above. Strains were SV5828 (P_tetA-hilD), and SV5829 (dam P_tetA-hilD). Data are averages and standard deviations from three independent experiments. (B) Transcription levels of two SPI-1 genes under HilD control (invF and sipB) in Dam⁺ (solid histograms) and Dam⁻ (open histograms) strains that express hilD from a heterologous, tetracycline-dependent promoter. Strains were SV5297 (invF∷lac), SV5298 (invF∷lac Dam⁻), SV5335 (P_tetA-hilD invF∷lac), SV5336 (P_tetA-hilD invF∷lac Dam⁻), SV5382 (sipB∷lac), SV5383 (sipB∷lac Dam⁻), SV5826 (P_tetA-hilD sipB∷lac), and SV5827 (P_tetA-hilD sipB∷lac Dam⁻). Data are averages and standard deviations from three independent experiments.

Figure 6.—

Stability of hilD mRNA in Dam⁺ (ATCC 14028) and Dam⁻ (SV5264) isogenic hosts. Values are averages from four independent qRT–PCR reactions. Error bars are not shown because the standard deviations were extremely small.

Figure 7.—

(A) Enhancement of hilD mRNA instability in the absence of Hfq. Solid histograms are for Dam⁺ strains, and open histograms are for their Dam⁻ derivatives. RNA levels were normalized to either ompA mRNA or gmk mRNA. Strains were ATCC 14208 (wild type), SV5264 (Dam⁻), SV5646 (Hfq⁻), and SV5847 (Hfq⁻ Dam⁻). Values are averages and standard deviations from three independent experiments. (B) Enhancement of the SPI-1 expression defect of S. enterica Dam⁻ mutants by hfq null mutations. Solid histograms are for Dam⁺ strains, and open histograms are for their Dam⁻ derivatives. To facilitate visual perception of differences, the β-galactosidase activities of individual lac fusions in Dam⁺ hosts have been normalized to 100. Strains were as follows: SV5284 (hilA∷lac), SV5285 (hilA∷lac Dam⁻), SV5278 (sicA∷lac), SV5279 (sicA∷lac Dam⁻), SV5297 (invF∷lac), SV5298 (invF∷lac Dam⁻), SV5382 (sipB∷lac), SV5383 (sipB∷lac Dam⁻), SV5293 (sipC∷lac), SV5294 (sipC∷lac Dam⁻), SV5848 (hilA∷lac Hfq⁻), SV5849 (hilA∷lac Hfq⁻ Dam⁻), SV5856 (sicA∷lac Hfq⁻), SV5857 (sicA∷lac Hfq⁻ Dam⁻), SV5850 (invF∷lac Hfq⁻), SV5851 (invF∷lac Hfq⁻ Dam⁻), SV5852 (sipB∷lac Hfq⁻), SV5853 (sipB∷lac Hfq⁻ Dam⁻), SV5854 (sipC∷lac Hfq⁻), and SV5855 (sipC∷lac Hfq⁻ Dam⁻). Data are averages and standard deviations from three experiments.

Figure 8.—

(A) Suppression of hilD mRNA instability in the absence of degradosome components ribonuclease E (Rne) and polynucleotide phosphorylase (Pnp). Solid histograms are for Dam⁺ strains, and open histograms are for their Dam⁻ derivatives. RNA levels were normalized to either ompA mRNA or gmk mRNA. Strains were ATCC 14028 (wild type), SV5264 (Dam⁻), SV5961 (Rne⁻), SV5962 (Rne⁻ Dam⁻), SV5963 (Pnp⁻), and SV5964 (Pnp⁻ Dam⁻). Values are averages and standard deviations from three independent experiments. (B) Suppression of the SPI-1 expression defect of S. enterica Dam⁻ mutants by rne and pnp mutations. Solid histograms are for Dam⁺ strains, and open histograms are for their Dam⁻ derivatives. To facilitate visual perception of differences, the β-galactosidase activities of lac fusions in individual SPI-1 genes in Dam⁺ hosts have been normalized to 100. Strains were as follows: SV5284 (hilA∷lac), SV5285 (hilA∷lac Dam⁻), SV5278 (sicA∷lac), SV5279 (sicA∷lac Dam⁻), SV5297 (invF∷lac), SV5298 (invF∷lac Dam⁻), SV5382 (sipB∷lac), SV5383 (sipB∷lac Dam⁻), SV5293 (sipC∷lac), SV5294 (sipC∷lac Dam⁻), SV5965 (hilA∷lac Rne⁻), SV5966 (hilA∷lac Rne⁻ Dam⁻), SV5967 (sicA∷lac Rne⁻), SV5968 (sicA∷lac Rne⁻ Dam⁻), SV5969 (invF∷lac Rne⁻), SV5970 (invF∷lac Rne⁻ Dam⁻), SV5971 (sipB∷lac Rne⁻), SV5972 (sipB∷lac Rne⁻ Dam⁻), SV5973 (sipC∷lac Rne⁻), SV5974 (sipC∷lac Rne⁻ Dam⁻), SV5975 (hilA∷lac Pnp⁻), SV5976 (hilA∷lac Pnp⁻ Dam⁻), SV5977 (sicA∷lac Pnp⁻), SV5978 (sicA∷lac Pnp⁻ Dam⁻), SV5979 (invF∷lac Pnp⁻), SV5980 (invF∷lac Pnp⁻ Dam⁻), SV5981 (sipB∷lac Pnp⁻), SV5982 (sipB∷lac Pnp⁻ Dam⁻), SV5983 (sipC∷lac Pnp⁻), and SV5984 (sipC∷lac Pnp⁻ Dam⁻). Data are averages and standard deviations from three experiments.