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

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.

Keywords: MicF-ompF; Yersinia pestis; intrinsic plasmid; sRNA regulation; translational fusion.

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-null). (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.

Figure 2

Expression of the ompF::gfp fusions upon MicF overexpression in the Yersinia pestis strains with different plasmid combinations. Representative fluorescence images of Y. pestis derivative strains are shown (A–C). The corresponding quantitative results are also shown (D), in which the asterisks indicate statistically significant differences compared to the values detected in strain MG1655 shown in Figure 1.

Figure 3

Detection of MicF expression in various strains of Y. pestis. MicF expression detected by Northern Blot, in which 5S rRNA images from each tested strain were provided as control.

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.