Limits...
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

Abundance detection of endogenous ompF transcript and OmpF protein in various strains of Y. pestis. Northern Blot was used to detect the chromosome-encoded ompF transcript in various strains of Y. pestis grown under the same conditions as those shown in Figure 3. Meanwhile, the anti-OmpF rabbit multiclonal antibody was used in Western Blot to detect the endogenous OmpF protein in the indicated strains, in which GroEL protein images from each strain were provided as control. The numbers indicated below each panel represent the fold changes of mRNA or protein abundance detected in strains carrying pBAD-MicF divided by that of the corresponding strains carrying pBAD control vector.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4543863&req=5

Figure 4: Abundance detection of endogenous ompF transcript and OmpF protein in various strains of Y. pestis. Northern Blot was used to detect the chromosome-encoded ompF transcript in various strains of Y. pestis grown under the same conditions as those shown in Figure 3. Meanwhile, the anti-OmpF rabbit multiclonal antibody was used in Western Blot to detect the endogenous OmpF protein in the indicated strains, in which GroEL protein images from each strain were provided as control. The numbers indicated below each panel represent the fold changes of mRNA or protein abundance detected in strains carrying pBAD-MicF divided by that of the corresponding strains carrying pBAD control vector.

Mentions: To test whether the intrinsic plasmids interfere with the MicF-mediated regulation of the chromosome-encoded target gene ompF, the abundances of endogenous ompF transcript and OmpF protein were validated in various strains of Y. pestis by Northern Blot and Western Blot, respectively (Figure 4). In agreement with the results of gene fusion reporter systems, the abundance of ompF transcript or OmpF protein were found decreased 2.0–5.0 fold in four pPCP1-cured strains (122-, 122-pCD1+pMT1+, 201-pCD1+, and 201-pMT1+) upon MicF overexpression relative to that in the control strains. Strikingly, the ompF transcript was stable, but approximate three-fold decrease was found in the expression level of OmpF in the strain 122-pMT1+pPCP1+, which is also consistent with the findings presented in Figure 2. However, only the comparable levels of ompF and OmpF were found among three groups of pPCP-containing strains (122, 122-pPCP1, and 122-pCD1+pPCP1+). The downregulation phenomenon is roughly consistent with that of plasmid-borne gfp fusion. The discrepancy is likely due to the different sensitivity between translational fusion assay and Western blot. However, no obvious upregulation was found in strain 201. Maybe the actual activation was magnified by translational fusion assay or veiled by detection threshold of Western blot or Northern Blot. Taken together, this observation further confirmed the conclusion that intrinsic plasmids have the potential impacts on abolishment of MicF-mediated ompF regulation in Y. pestis.


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)

Abundance detection of endogenous ompF transcript and OmpF protein in various strains of Y. pestis. Northern Blot was used to detect the chromosome-encoded ompF transcript in various strains of Y. pestis grown under the same conditions as those shown in Figure 3. Meanwhile, the anti-OmpF rabbit multiclonal antibody was used in Western Blot to detect the endogenous OmpF protein in the indicated strains, in which GroEL protein images from each strain were provided as control. The numbers indicated below each panel represent the fold changes of mRNA or protein abundance detected in strains carrying pBAD-MicF divided by that of the corresponding strains carrying pBAD control vector.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4543863&req=5

Figure 4: Abundance detection of endogenous ompF transcript and OmpF protein in various strains of Y. pestis. Northern Blot was used to detect the chromosome-encoded ompF transcript in various strains of Y. pestis grown under the same conditions as those shown in Figure 3. Meanwhile, the anti-OmpF rabbit multiclonal antibody was used in Western Blot to detect the endogenous OmpF protein in the indicated strains, in which GroEL protein images from each strain were provided as control. The numbers indicated below each panel represent the fold changes of mRNA or protein abundance detected in strains carrying pBAD-MicF divided by that of the corresponding strains carrying pBAD control vector.
Mentions: To test whether the intrinsic plasmids interfere with the MicF-mediated regulation of the chromosome-encoded target gene ompF, the abundances of endogenous ompF transcript and OmpF protein were validated in various strains of Y. pestis by Northern Blot and Western Blot, respectively (Figure 4). In agreement with the results of gene fusion reporter systems, the abundance of ompF transcript or OmpF protein were found decreased 2.0–5.0 fold in four pPCP1-cured strains (122-, 122-pCD1+pMT1+, 201-pCD1+, and 201-pMT1+) upon MicF overexpression relative to that in the control strains. Strikingly, the ompF transcript was stable, but approximate three-fold decrease was found in the expression level of OmpF in the strain 122-pMT1+pPCP1+, which is also consistent with the findings presented in Figure 2. However, only the comparable levels of ompF and OmpF were found among three groups of pPCP-containing strains (122, 122-pPCP1, and 122-pCD1+pPCP1+). The downregulation phenomenon is roughly consistent with that of plasmid-borne gfp fusion. The discrepancy is likely due to the different sensitivity between translational fusion assay and Western blot. However, no obvious upregulation was found in strain 201. Maybe the actual activation was magnified by translational fusion assay or veiled by detection threshold of Western blot or Northern Blot. Taken together, this observation further confirmed the conclusion that intrinsic plasmids have the potential impacts on abolishment of MicF-mediated ompF regulation in Y. pestis.

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