Bacterial regulon evolution: distinct responses and roles for the identical OmpR proteins of Salmonella Typhimurium and Escherichia coli in the acid stress response - PubMed (original) (raw)
Bacterial regulon evolution: distinct responses and roles for the identical OmpR proteins of Salmonella Typhimurium and Escherichia coli in the acid stress response
Heather J Quinn et al. PLoS Genet. 2014.
Abstract
The evolution of new gene networks is a primary source of genetic innovation that allows bacteria to explore and exploit new niches, including pathogenic interactions with host organisms. For example, the archetypal DNA binding protein, OmpR, is identical between Salmonella Typhimurium serovar Typhimurium and Escherichia coli, but regulatory specialization has resulted in different environmental triggers of OmpR expression and largely divergent OmpR regulons. Specifically, ompR mRNA and OmpR protein levels are elevated by acid pH in S. Typhimurium but not in E. coli. This differential expression pattern is due to differences in the promoter regions of the ompR genes and the E. coli ompR orthologue can be made acid-inducible by introduction of the appropriate sequences from S. Typhimurium. The OmpR regulon in S. Typhimurium overlaps that of E. coli at only 15 genes and includes many horizontally acquired genes (including virulence genes) that E. coli does not have. We found that OmpR binds to its genomic targets in higher abundance when the DNA is relaxed, something that occurs in S. Typhimurium as a result of acid stress and which is a requirement for optimal expression of its virulence genes. The genomic targets of OmpR do not share a strong nucleotide sequence consensus: we propose that the ability of OmpR to recruit additional genes to its regulon arises from its modest requirements for specificity in its DNA targets with its preference for relaxed DNA allowing it to cooperate with DNA-topology-based allostery to modulate transcription in response to acid stress.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
Figure 1. The promoter region is responsible for the pH sensitivity of ompR in S. Typhimurium.
(A) Quantitative PCR measurements of ompR transcript levels in S. Typhimurium (SL1344) and E. coli (CSH50) and constructs with exchanged ompR regulatory regions (see C) at pH 7 and pH 4.5. Mean (N≥3) values are reported and the error bars represent the standard deviation of the mean. (B) OmpR protein levels in S. Typhimurium (SL1344 ompR::3xFLAG) and E. coli (CSH50 ompR::3xFLAG) at pH 7 and pH 4.5. Anti-FLAG antibody was used to detect the FLAG epitope and DnaK was used as a loading control. (C) Diagrams illustrating the constructs (i–v) used in the study. Bent arrows denote transcription start sites (TSSs) The wild type ompB locus in S. Typhimurium (i). The wild type ompB locus in E. coli (ii). In E. coli ompBS. T. the native E. coli ompR and envZ genes are replaced by the corresponding ompR/envZ genes from S. Typhimurium (dark grey). The native E. coli ompR promoter remains (light grey) (iii). In E. coli P_ompR S_ .T. the ompR promoter in E. coli is replaced by the ompR promoter from S. Typhimurium (dark grey). The native E. coli ompB locus is retained (light grey) (iv). In E. coli P_ompR ompBS_ . T. both the ompR promoter and ompR/envZ genes in E. coli are replaced by the ompR promoter and ompR/envZ genes from S. Typhimurium (iv). (D) Gel electrophoresis of 5′ RACE-amplified ompR cDNA ends analysed on a 3% agarose gel. RNA was extracted after 90 min at pH 7 or pH 4.5. Samples contained either tobacco acid pyrophosphatase (TAP)-treated (+) or untreated (−) RNA, generating 5′ monophosphate for ligation of the RNA-linker (A4; see Table S2) . Ligation of the linker was more efficient in the TAP treated sample. RNA was reverse-transcribed into cDNA and PCR was performed on each cDNA sample using primers RACE_ ompR and JVO-0367; see Table S2. Arrowhead denotes the PCR product found to be TSS-1. M, 100-bp ladder. The white asterisk denotes a non-specific PCR product that was sequenced and identified as a 23S ribosomal RNA product using BLAST (Basic Local Alignment Search Tool). (E) Gel electrophoresis of 5′ RACE-amplified ompR cDNA ends analysed on a 1% agarose gel. RNA was extracted from CSH50 and CSH50 P_ompRS._ T after 90 min at pH 7 or pH 4.5. Arrowheads TSS-1 and TSS-2 denote the PCR products. M, 100-bp ladder. The locations of the transcription start sites are shown in Figure S1.
Figure 2. Genome-wide distribution of OmpR in E. coli and S. Typhimurium.
Results from genome-wide analysis of OmpR binding in E. coli (A) and S. Typhimurium (B) at pH 7 (blue) and pH 4.5 (red). The log2 enrichment ratio (ChIP/input) is plotted on the _y_-axis and the locations of the probes are shown on the _x_-axis. (C) Overlap between OmpR binding sites at pH 7 and pH 4.5 in E. coli and S. Typhimurium. The histogram illustrates the number of significant OmpR binding peaks bound at pH 7 and pH 4.5 in E. coli and S. Typhimurium. The number of targets occupied at pH 7 and pH 4.5 is shown in dark blue (pH sensitive). The number of pH-specific targets occupied at pH 7 or pH 4.5 is shown in light blue (pH insensitive).
Figure 3. The effect of pH and DNA relaxation on OmpR binding across Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2).
(A) Log2 enrichment ratio (ChIP/input) is plotted on the _y_-axis and the locations of the probes are shown on the _x_-axis for SPI-1. Coloured arrows below the _x_-axis show the locations of SPI-1 genes determined using Jbrowse . The inset shows the OmpR binding pattern at the hilC regulatory gene of SPI-1 in more detail. (B) Log2 enrichment ratio (ChIP/input) is plotted on the _y_-axis and location of the probes are shown on the _x_-axis for SPI-2. Genes colour is based on their function regulatory genes are red, effector genes are pink, structural genes are green, translocon genes are orange, chaperone genes are blue and genes of unknown function are grey. The inset shows the OmpR binding pattern at the ssrA ssrB regulatory genes of SPI-2 in more detail. In (A) and (B), the top histogram shows OmpR binding at pH 7 (blue) and pH 4.5 (red) and the bottom histogram shows OmpR binding without (blue) and with (red) novobiocin treatment (25 µg ml−1). Genes are coloured to indicate their function: regulatory genes are red, effector genes are pink, structural genes are green, translocon genes are orange, chaperone genes are blue and genes of unknown function are grey.
Figure 4. The effect of pH on DNA supercoiling in Salmonella Typhimurium strain SL1344 (top) and E. coli strain CSH50 (bottom).
Electrophoretic mobility of plasmid pUC18 topoisomers in agarose gel containing chloroquine at 2.5 µg ml−1. At this concentration of chloroquine more supercoiled topoisomers run further in the gel. Cultures were pelleted after 90 min at pH 7 or pH 4.5 E-minimal medium and plasmids were extracted immediately. Gel image is representative of three independent experiments.
Figure 5. OmpR regulates PhoP-regulated genes.
(A) OmpR binding at mgtC at pH 7 and pH 4.5. Sliding window average of log2 enrichment as calculated by ChIPOTle is shown on the _y-_axis. (B) EMSA analysis showing OmpR binding to the mgtC promoter as well as the ompC promoter (positive control) and the kan R (kanamycin resistance gene; negative control). D, free DNA probe; P+D, protein + DNA complex. OmpR concentrations used were 0, 0.02, 0.04,.078,.16, 0.31, 0.63, 1.25, 2.5 µM. A representative gel image is shown from three independent replicates (C) Quantitative PCR measurements of mgtC transcript levels at pH 7 and pH 4.5 in the wild type strain and an ompR knockout mutant. (D) Quantitative PCR measurements of phoP transcript levels at pH 7 and pH 4.5 in the wild type strain and an ompR knockout mutant and in the ompR hns::kan double mutant. N≥3; standard deviations from the mean are shown as error bars.
Figure 6. The number of high-scoring OmpR sites identified within the ChIP datasets.
Sequences of the binding sites are listed in Table S6. Random DNA datasets were generated as described in Materials and Methods.
Figure 7. The OmpR regulons of S. Typhimurium and E. coli in acid pH.
Members of the OmpR regulon in has evolved since the divergence of these closely-related species. OmpR (pairs of circles) binds to a species-specific regulon in Salmonella and E. coli; examples of these targets are shown. In E. coli, OmpR binds to genes involved in acid resistance e.g. cad operon and general stress resistance e.g. the uspC and emrK genes. In S. Typhimurium, OmpR binds to pathogenicity islands SPI-1, -2, and -4, and to genes regulated by PhoP. OmpR positively regulates phoP expression by an unknown mechanism (denoted by the question mark). The genes identified as the core OmpR regulon (conserved targets; bound by OmpR in both species) encode surface-associated organelles and proteins. Modulation of the cell-surface composition may be an important function of the core OmpR regulon in response to acidic stress.
References
- Karem K, Foster JW (1993) The influence of DNA topology on the environmental regulation of a pH-regulated locus in Salmonella Typhimurium. Mol Microbiol 10: 75–86. -PubMed
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