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Intrinsic plasmids influence MicF-mediated translational repression of ompF in Yersinia pestis.

Liu Z, Wang H, Wang H, Wang J, Bi Y, Wang X, Yang R, Han Y - Front Microbiol (2015)

Bottom Line: Unexpectedly, upon MicF overexpression, the slightly upregulated expression of OmpF were found in the wild-type strain, which contradicted the previously established model.Further examination showed that plasmid pPCP1 is likely the main contributor to the abolishment of MicF-mediated translational repression of endogenous or plasmid-borne ompF.It represents that the possible roles of intrinsic plasmids should be considered upon investigating sRNA-mediated gene regulation, at least in Y. pestis, even if the exact mechanism is not fully understood.

View Article: PubMed Central - PubMed

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

ABSTRACT
Yersinia pestis, which is the causative agent of plague, has acquired exceptional pathogenicity potential during its evolution from Y. pseudotuberculosis. Two laterally acquired plasmids, namely, pMT1 and pPCP1, are specific to Y. pestis and are critical for pathogenesis and flea transmission. Small regulatory RNAs (sRNAs) commonly function as regulators of gene expression in bacteria. MicF, is a paradigmatic sRNA that acts as a post-transcriptional repressor through imperfect base pairing with the 5'-UTR of its target mRNA, ompF, in Escherichia coli. The high sequence conservation and minor variation in the RNA duplex of MicF-ompF has been reported in Yersinia. In this study, we utilized super-folder GFP reporter gene fusion to validate the post-transcriptional MicF-mediated regulation of target mRNA ompF in Y. pestis. Unexpectedly, upon MicF overexpression, the slightly upregulated expression of OmpF were found in the wild-type strain, which contradicted the previously established model. Interestingly, the translational repression of ompF target fusions was restored in the intrinsic plasmids-cured Y. pestis strain, suggesting intrinsic plasmids influence the MicF-mediated translational repression of ompF in Y. pestis. Further examination showed that plasmid pPCP1 is likely the main contributor to the abolishment of MicF-mediated translational repression of endogenous or plasmid-borne ompF. It represents that the possible roles of intrinsic plasmids should be considered upon investigating sRNA-mediated gene regulation, at least in Y. pestis, even if the exact mechanism is not fully understood.

No MeSH data available.


Related in: MedlinePlus

Expression of the ompF::gfp fusions upon MicF overexpression in Escherichia coli and Yersinia pestis strains. (A) Layout map of bacterial strains carrying the control plasmid without sRNA overexpression (denoted as “vector”) or MicF-overexpression plasmid (denoted as “MicF”). Duplicate images of representative strains are shown in parallel on plates. (B) Representative fluorescence images of E. coli strain MG1655. The image obtained under visible light mode is shown at the left panel and that of the same plate under the fluorescence mode at the right panel. (C) Representative fluorescence images of Y. pestis WT strain (201) and its plasmid-cured derivative strain (201-). (D) Quantitative measurements of fluorescence produced by the tested strains. Fold changes are provided as the ratio of fluorescence values detected in MicF-overexpressed bacterial strains divided by those detected in strains carrying pBAD blank vectors. Values presented are means ± standard deviations of two independent experiments. The asterisks indicate statistically significant differences compared to the values detected in strain MG1655.
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Figure 1: Expression of the ompF::gfp fusions upon MicF overexpression in Escherichia coli and Yersinia pestis strains. (A) Layout map of bacterial strains carrying the control plasmid without sRNA overexpression (denoted as “vector”) or MicF-overexpression plasmid (denoted as “MicF”). Duplicate images of representative strains are shown in parallel on plates. (B) Representative fluorescence images of E. coli strain MG1655. The image obtained under visible light mode is shown at the left panel and that of the same plate under the fluorescence mode at the right panel. (C) Representative fluorescence images of Y. pestis WT strain (201) and its plasmid-cured derivative strain (201-). (D) Quantitative measurements of fluorescence produced by the tested strains. Fold changes are provided as the ratio of fluorescence values detected in MicF-overexpressed bacterial strains divided by those detected in strains carrying pBAD blank vectors. Values presented are means ± standard deviations of two independent experiments. The asterisks indicate statistically significant differences compared to the values detected in strain MG1655.

Mentions: The inhibitory translation of ompF-gfp fusion upon MicF overexpression (approximately five-fold repression) was observed in E. coli strain MG1655. Only the 1.8-fold repression was observed in the Y. pestis strain 201 that is cured of all the endogenous plasmids (Figure 1). This finding is consistent with the results previously reported on E. coli (Mizuno et al., 1984) and also confirms that MicF-mediated ompF repression occurs in the 5′-UTR. Interestingly, the inhibitory effect was not found in the Y. pestis WT strain 201. Instead, more than three-fold upregulation of OmpF-GFP expression was observed under the same conditions (Figure 1). The relative quantification of fluorescence value was also measured in Y. pestis strains with different combinatorial plasmids grown to exponential phase in LB medium followed by arabinose induction for 1 h. Similar tendency was found in Y. pestis strains grown in liquid medium as that found in solid medium (Figures 1, 2 and Figure S2). To exclude the possibility that the effect was caused by the translational reporter system, we construct plasmid pXG-1 which sfGFP expression is constitutively PLtetO-controlled. No changes in fluorescence intensity were found between pBAD-MicF and pBAD-vector groups in Y. pestis strain 201 and 201- carrying pXG-1 (Figure S3). The different regulatory consequences in Y. pestis strains carrying or cured of plasmids indicated that the virulence-associated plasmids might have been recruited to sRNA-mediated regulatory networks in the chromosome during evolution.


Intrinsic plasmids influence MicF-mediated translational repression of ompF in Yersinia pestis.

Liu Z, Wang H, Wang H, Wang J, Bi Y, Wang X, Yang R, Han Y - Front Microbiol (2015)

Expression of the ompF::gfp fusions upon MicF overexpression in Escherichia coli and Yersinia pestis strains. (A) Layout map of bacterial strains carrying the control plasmid without sRNA overexpression (denoted as “vector”) or MicF-overexpression plasmid (denoted as “MicF”). Duplicate images of representative strains are shown in parallel on plates. (B) Representative fluorescence images of E. coli strain MG1655. The image obtained under visible light mode is shown at the left panel and that of the same plate under the fluorescence mode at the right panel. (C) Representative fluorescence images of Y. pestis WT strain (201) and its plasmid-cured derivative strain (201-). (D) Quantitative measurements of fluorescence produced by the tested strains. Fold changes are provided as the ratio of fluorescence values detected in MicF-overexpressed bacterial strains divided by those detected in strains carrying pBAD blank vectors. Values presented are means ± standard deviations of two independent experiments. The asterisks indicate statistically significant differences compared to the values detected in strain MG1655.
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Related In: Results  -  Collection

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Figure 1: Expression of the ompF::gfp fusions upon MicF overexpression in Escherichia coli and Yersinia pestis strains. (A) Layout map of bacterial strains carrying the control plasmid without sRNA overexpression (denoted as “vector”) or MicF-overexpression plasmid (denoted as “MicF”). Duplicate images of representative strains are shown in parallel on plates. (B) Representative fluorescence images of E. coli strain MG1655. The image obtained under visible light mode is shown at the left panel and that of the same plate under the fluorescence mode at the right panel. (C) Representative fluorescence images of Y. pestis WT strain (201) and its plasmid-cured derivative strain (201-). (D) Quantitative measurements of fluorescence produced by the tested strains. Fold changes are provided as the ratio of fluorescence values detected in MicF-overexpressed bacterial strains divided by those detected in strains carrying pBAD blank vectors. Values presented are means ± standard deviations of two independent experiments. The asterisks indicate statistically significant differences compared to the values detected in strain MG1655.
Mentions: The inhibitory translation of ompF-gfp fusion upon MicF overexpression (approximately five-fold repression) was observed in E. coli strain MG1655. Only the 1.8-fold repression was observed in the Y. pestis strain 201 that is cured of all the endogenous plasmids (Figure 1). This finding is consistent with the results previously reported on E. coli (Mizuno et al., 1984) and also confirms that MicF-mediated ompF repression occurs in the 5′-UTR. Interestingly, the inhibitory effect was not found in the Y. pestis WT strain 201. Instead, more than three-fold upregulation of OmpF-GFP expression was observed under the same conditions (Figure 1). The relative quantification of fluorescence value was also measured in Y. pestis strains with different combinatorial plasmids grown to exponential phase in LB medium followed by arabinose induction for 1 h. Similar tendency was found in Y. pestis strains grown in liquid medium as that found in solid medium (Figures 1, 2 and Figure S2). To exclude the possibility that the effect was caused by the translational reporter system, we construct plasmid pXG-1 which sfGFP expression is constitutively PLtetO-controlled. No changes in fluorescence intensity were found between pBAD-MicF and pBAD-vector groups in Y. pestis strain 201 and 201- carrying pXG-1 (Figure S3). The different regulatory consequences in Y. pestis strains carrying or cured of plasmids indicated that the virulence-associated plasmids might have been recruited to sRNA-mediated regulatory networks in the chromosome during evolution.

Bottom Line: Unexpectedly, upon MicF overexpression, the slightly upregulated expression of OmpF were found in the wild-type strain, which contradicted the previously established model.Further examination showed that plasmid pPCP1 is likely the main contributor to the abolishment of MicF-mediated translational repression of endogenous or plasmid-borne ompF.It represents that the possible roles of intrinsic plasmids should be considered upon investigating sRNA-mediated gene regulation, at least in Y. pestis, even if the exact mechanism is not fully understood.

View Article: PubMed Central - PubMed

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

ABSTRACT
Yersinia pestis, which is the causative agent of plague, has acquired exceptional pathogenicity potential during its evolution from Y. pseudotuberculosis. Two laterally acquired plasmids, namely, pMT1 and pPCP1, are specific to Y. pestis and are critical for pathogenesis and flea transmission. Small regulatory RNAs (sRNAs) commonly function as regulators of gene expression in bacteria. MicF, is a paradigmatic sRNA that acts as a post-transcriptional repressor through imperfect base pairing with the 5'-UTR of its target mRNA, ompF, in Escherichia coli. The high sequence conservation and minor variation in the RNA duplex of MicF-ompF has been reported in Yersinia. In this study, we utilized super-folder GFP reporter gene fusion to validate the post-transcriptional MicF-mediated regulation of target mRNA ompF in Y. pestis. Unexpectedly, upon MicF overexpression, the slightly upregulated expression of OmpF were found in the wild-type strain, which contradicted the previously established model. Interestingly, the translational repression of ompF target fusions was restored in the intrinsic plasmids-cured Y. pestis strain, suggesting intrinsic plasmids influence the MicF-mediated translational repression of ompF in Y. pestis. Further examination showed that plasmid pPCP1 is likely the main contributor to the abolishment of MicF-mediated translational repression of endogenous or plasmid-borne ompF. It represents that the possible roles of intrinsic plasmids should be considered upon investigating sRNA-mediated gene regulation, at least in Y. pestis, even if the exact mechanism is not fully understood.

No MeSH data available.


Related in: MedlinePlus