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Transcriptional regulation of the waaAE-coaD operon by PhoP and RcsAB in Yersinia pestis biovar Microtus.

Liu L, Fang N, Sun Y, Yang H, Zhang Y, Han Y, Zhou D, Yang R - Protein Cell (2014)

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, 100071, China.

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Flea-borne transmission distinguishes Y. pestis from its progenitor Y. pseudotuberculosis, which is a mild food-borne pathogen (Zhou and Yang, )... In Y. pestis, transmission by fleas involves the synthesis of biofilms that physically block the flea’s proventriculus; Y. pseudotuberculosis does not produce biofilms in fleas (Zhou and Yang, )... Phosphorylated RcsB (RcsB-P) acts as a transcriptional regulator either independently or upon binding with an auxiliary protein RcsA, which is present in Y. pseudotuberculosis but not Y. pestis... Based on computational analysis, PhoP and RcsAB are predicted to bind the promoter proximal region of waaAE-coaD, suggesting that they may be transcriptional regulators of the operon... Primer extension experiments (Fig.  1A) indicate that a ΔphoP mutant has significantly lower waaA mRNA levels compared to WT at 26°C... A waaA::lacZ fusion strain (Fig.  1B) showed that waaA promoter activity was significantly reduced in ΔphoP relative to WT... S4C)... This distinction indicated that deletion of waaA resulted in a major decrease in exopolysaccharide production... Thus, RcsAB acts as a master repressor of Yersinia biofilm production through inhibiting the expression of multiple biofilm determinants including at least HmsT and WaaA... Expression of PhoP/PhoQ is induced in flea gut, where it promotes the formation of flea-borne infectious Y. pestis biofilms (Rebeil et al., )... Nevertheless, PhoP/PhoQ has no regulatory effect on the expression of hmsHFRS, an operon responsible for synthesis and translocation of biofilm matrix exopolysaccharides through the cell envelope (Bobrov et al., )... Additionally, there is no regulatory effect on hmsHFRS-depedent pigmentation... Thus, PhoP is an important determinant of biofilm production in Yersinia and may play a role in the difference between the species Y. pestis and Y. pseudotuberculosis.

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Positive regulation ofwaaAE-coaDby PhoP. The positive and minus numbers in the brackets indicated the nucleotide positions upstream and downstream of waaA, respectively. (A) Primer extension. Lanes C, T, A, and G represented Sanger sequencing reactions. The primer extension products and the sequence ladders were analyzed with an 8 mol/L urea-6% acrylamide sequencing gel. The transcriptional start site of waaA was indicated by arrows with nucleotides. (B) LacZ fusion. The waaA:lacZ transcriptional fusion vector was transformed into indicated Y. pestis strains, and then the waaA promoter activities (the miller units of β-galactosidase activity) were determined in the cellular extracts. (C) EMSA. The radioactively labeled DNA fragments were incubated with increasing amounts of purified His-PhoP protein and then subjected to a native 4% polyacrylamide gel electrophoresis. (D) DNase I footprinting. Labeled coding or non-coding DNA probes were incubated with increasing amounts of purified His-PhoP and then subjected to DNase I footprinting assay. The footprint regions were indicated with vertical bars. Lanes G, A, T, and C represented Sanger sequencing reactions
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Fig1: Positive regulation ofwaaAE-coaDby PhoP. The positive and minus numbers in the brackets indicated the nucleotide positions upstream and downstream of waaA, respectively. (A) Primer extension. Lanes C, T, A, and G represented Sanger sequencing reactions. The primer extension products and the sequence ladders were analyzed with an 8 mol/L urea-6% acrylamide sequencing gel. The transcriptional start site of waaA was indicated by arrows with nucleotides. (B) LacZ fusion. The waaA:lacZ transcriptional fusion vector was transformed into indicated Y. pestis strains, and then the waaA promoter activities (the miller units of β-galactosidase activity) were determined in the cellular extracts. (C) EMSA. The radioactively labeled DNA fragments were incubated with increasing amounts of purified His-PhoP protein and then subjected to a native 4% polyacrylamide gel electrophoresis. (D) DNase I footprinting. Labeled coding or non-coding DNA probes were incubated with increasing amounts of purified His-PhoP and then subjected to DNase I footprinting assay. The footprint regions were indicated with vertical bars. Lanes G, A, T, and C represented Sanger sequencing reactions

Mentions: In the present work, the RT-PCR assay indicated that the three consecutive genes waaA, waaE, and coaD were transcribed as a single primary RNA (Fig. S1), and thereby these three genes constituted a three-gene operon in Y. pestis. The relative mRNA levels of waaA were measured using primer extension in the wild-type Y. pestisMicrotus strain 201(WT) grown at 26°C or 37°C (Fig. S2A). This assay detected a single transcriptional start site (nucleotide T) located 26 bp upstream of waaA, and thus a single promoter was identified for waaA under the growth conditions tested. Cells grown at 26°C had dramatically higher levels of waaA mRNA than cells grown at 37°C. These results were recapitulated using a waaA::lacZ fusion vector, containing the promoter for waaA fused to the coding region of lacZ. This upregulation between 37°C and 26°C could correlate with an upregulation in response to movement from the warm-blooded host (37°C) to the flea gut (26°C). Thus WaaA may be important in the transition between the two vectors. For all the following experiments, Y. pestis was cultivated at 26°C. Based on computational analysis, PhoP and RcsAB are predicted to bind the promoter proximal region of waaAE-coaD, suggesting that they may be transcriptional regulators of the operon. Primer extension experiments (Fig. 1A) indicate that a ΔphoP mutant has significantly lower waaA mRNA levels compared to WT at 26°C. A waaA::lacZ fusion strain (Fig. 1B) showed that waaA promoter activity was significantly reduced in ΔphoP relative to WT. The electrophoretic mobility shift assay (EMSA) (Fig. 1C) denoted that the purified His-PhoP protein was able to bind to the waaA promoter-proximal DNA in a dose-dependent manner; in contrast, His-PhoP did not bind the 16S rRNA gene at any concentration. Subsequent DNase I footprinting experiments (Fig. 1D) disclosed that His-PhoP protected a single region, located from 176 bp to 130 bp upstream of waaA in a dose-dependent manner. This region contained a predicted PhoP box-like sequence. Thus, PhoP positively controls waaA transcription through binding to the waaA promoter-proximal region.Figure 1


Transcriptional regulation of the waaAE-coaD operon by PhoP and RcsAB in Yersinia pestis biovar Microtus.

Liu L, Fang N, Sun Y, Yang H, Zhang Y, Han Y, Zhou D, Yang R - Protein Cell (2014)

Positive regulation ofwaaAE-coaDby PhoP. The positive and minus numbers in the brackets indicated the nucleotide positions upstream and downstream of waaA, respectively. (A) Primer extension. Lanes C, T, A, and G represented Sanger sequencing reactions. The primer extension products and the sequence ladders were analyzed with an 8 mol/L urea-6% acrylamide sequencing gel. The transcriptional start site of waaA was indicated by arrows with nucleotides. (B) LacZ fusion. The waaA:lacZ transcriptional fusion vector was transformed into indicated Y. pestis strains, and then the waaA promoter activities (the miller units of β-galactosidase activity) were determined in the cellular extracts. (C) EMSA. The radioactively labeled DNA fragments were incubated with increasing amounts of purified His-PhoP protein and then subjected to a native 4% polyacrylamide gel electrophoresis. (D) DNase I footprinting. Labeled coding or non-coding DNA probes were incubated with increasing amounts of purified His-PhoP and then subjected to DNase I footprinting assay. The footprint regions were indicated with vertical bars. Lanes G, A, T, and C represented Sanger sequencing reactions
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Fig1: Positive regulation ofwaaAE-coaDby PhoP. The positive and minus numbers in the brackets indicated the nucleotide positions upstream and downstream of waaA, respectively. (A) Primer extension. Lanes C, T, A, and G represented Sanger sequencing reactions. The primer extension products and the sequence ladders were analyzed with an 8 mol/L urea-6% acrylamide sequencing gel. The transcriptional start site of waaA was indicated by arrows with nucleotides. (B) LacZ fusion. The waaA:lacZ transcriptional fusion vector was transformed into indicated Y. pestis strains, and then the waaA promoter activities (the miller units of β-galactosidase activity) were determined in the cellular extracts. (C) EMSA. The radioactively labeled DNA fragments were incubated with increasing amounts of purified His-PhoP protein and then subjected to a native 4% polyacrylamide gel electrophoresis. (D) DNase I footprinting. Labeled coding or non-coding DNA probes were incubated with increasing amounts of purified His-PhoP and then subjected to DNase I footprinting assay. The footprint regions were indicated with vertical bars. Lanes G, A, T, and C represented Sanger sequencing reactions
Mentions: In the present work, the RT-PCR assay indicated that the three consecutive genes waaA, waaE, and coaD were transcribed as a single primary RNA (Fig. S1), and thereby these three genes constituted a three-gene operon in Y. pestis. The relative mRNA levels of waaA were measured using primer extension in the wild-type Y. pestisMicrotus strain 201(WT) grown at 26°C or 37°C (Fig. S2A). This assay detected a single transcriptional start site (nucleotide T) located 26 bp upstream of waaA, and thus a single promoter was identified for waaA under the growth conditions tested. Cells grown at 26°C had dramatically higher levels of waaA mRNA than cells grown at 37°C. These results were recapitulated using a waaA::lacZ fusion vector, containing the promoter for waaA fused to the coding region of lacZ. This upregulation between 37°C and 26°C could correlate with an upregulation in response to movement from the warm-blooded host (37°C) to the flea gut (26°C). Thus WaaA may be important in the transition between the two vectors. For all the following experiments, Y. pestis was cultivated at 26°C. Based on computational analysis, PhoP and RcsAB are predicted to bind the promoter proximal region of waaAE-coaD, suggesting that they may be transcriptional regulators of the operon. Primer extension experiments (Fig. 1A) indicate that a ΔphoP mutant has significantly lower waaA mRNA levels compared to WT at 26°C. A waaA::lacZ fusion strain (Fig. 1B) showed that waaA promoter activity was significantly reduced in ΔphoP relative to WT. The electrophoretic mobility shift assay (EMSA) (Fig. 1C) denoted that the purified His-PhoP protein was able to bind to the waaA promoter-proximal DNA in a dose-dependent manner; in contrast, His-PhoP did not bind the 16S rRNA gene at any concentration. Subsequent DNase I footprinting experiments (Fig. 1D) disclosed that His-PhoP protected a single region, located from 176 bp to 130 bp upstream of waaA in a dose-dependent manner. This region contained a predicted PhoP box-like sequence. Thus, PhoP positively controls waaA transcription through binding to the waaA promoter-proximal region.Figure 1

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, 100071, China.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Flea-borne transmission distinguishes Y. pestis from its progenitor Y. pseudotuberculosis, which is a mild food-borne pathogen (Zhou and Yang, )... In Y. pestis, transmission by fleas involves the synthesis of biofilms that physically block the flea’s proventriculus; Y. pseudotuberculosis does not produce biofilms in fleas (Zhou and Yang, )... Phosphorylated RcsB (RcsB-P) acts as a transcriptional regulator either independently or upon binding with an auxiliary protein RcsA, which is present in Y. pseudotuberculosis but not Y. pestis... Based on computational analysis, PhoP and RcsAB are predicted to bind the promoter proximal region of waaAE-coaD, suggesting that they may be transcriptional regulators of the operon... Primer extension experiments (Fig.  1A) indicate that a ΔphoP mutant has significantly lower waaA mRNA levels compared to WT at 26°C... A waaA::lacZ fusion strain (Fig.  1B) showed that waaA promoter activity was significantly reduced in ΔphoP relative to WT... S4C)... This distinction indicated that deletion of waaA resulted in a major decrease in exopolysaccharide production... Thus, RcsAB acts as a master repressor of Yersinia biofilm production through inhibiting the expression of multiple biofilm determinants including at least HmsT and WaaA... Expression of PhoP/PhoQ is induced in flea gut, where it promotes the formation of flea-borne infectious Y. pestis biofilms (Rebeil et al., )... Nevertheless, PhoP/PhoQ has no regulatory effect on the expression of hmsHFRS, an operon responsible for synthesis and translocation of biofilm matrix exopolysaccharides through the cell envelope (Bobrov et al., )... Additionally, there is no regulatory effect on hmsHFRS-depedent pigmentation... Thus, PhoP is an important determinant of biofilm production in Yersinia and may play a role in the difference between the species Y. pestis and Y. pseudotuberculosis.

Show MeSH
Related in: MedlinePlus