Inhibition of expression in Escherichia coli of a virulence regulator MglB of Francisella tularensis using external guide sequence technology

Abstract

External guide sequences (EGSs) have successfully been used to inhibit expression of target genes at the post-transcriptional level in both prokaryotes and eukaryotes. We previously reported that EGS accessible and cleavable sites in the target RNAs can rapidly be identified by screening random EGS (rEGS) libraries. Here the method of screening rEGS libraries and a partial RNase T1 digestion assay were used to identify sites accessible to EGSs in the mRNA of a global virulence regulator MglB from Francisella tularensis, a Gram-negative pathogenic bacterium. Specific EGSs were subsequently designed and their activities in terms of the cleavage of mglB mRNA by RNase P were tested in vitro and in vivo. EGS73, EGS148, and EGS155 in both stem and M1 EGS constructs induced mglB mRNA cleavage in vitro. Expression of stem EGS73 and EGS155 in Escherichia coli resulted in significant reduction of the mglB mRNA level coded for the F. tularensis mglB gene inserted in those cells.

Figure 1. Mapping of accessible sites in the target mglB mRNA by RNase T1 and rEGS libraries.

A. Polyacrylamide gel electrophoresis of cleavage products of 5′end labeled mglB mRNA generated by RNase P holoenzyme and rEGSe or rEGSx libraries. The RNase P holoenzyme was reconstituted by 10 nM M1 RNA and 100 nM C5 protein. mglB mRNA cleavage products were separated on 8% polyacrylamide/7 M urea gels together with an alkali ladder and a partial RNase T1 digest of the same RNA. Arrows indicate positions selected for further analyses. The black filled triangles represent decreasing concentrations of rEGSs (1000-, 100-, and 10-fold molar excess to the mglB mRNA) incubated with RNase P holoenzyme. Lane OH represents the alkali ladder. Lanes T1 and M1+C5 represent reaction with RNase T1 and RNase P holoenzyme, respectively. The difference between rEGSe and rEGSx is the length of 3′ end of rEGS mRNA resulted from the BstNI digestion of the DNA fragments for in vitro transcription. The numbers indicate the positions of cleavage relative to the mglB translational initiation site. B. Complexes between the mglB mRNA and EGS52, EGS73, EGS148, and EGS155. The secondary structure of the partial mglB sequence used for the assay was created by the Mfold program . Signal strength is according to the RNase T1 assay.

Figure 2. Cleavage in vitro of the mglB mRNA by the RNase P holoenzyme in the absence or presence of stem EGSs.

A. Activities of stem EGS155 and EGS155R. B. Activities of stem EGS52, EGS73, and EGS148. If needed, 10 nM M1 RNA and 100 nM C5 protein in buffer PA were added to reconstitute the RNase P holoenzyme. The mglB mRNA cleavage products were separated on 8% polyacrylamide/7 M urea gels along with the mglB mRNA alone or the mglB mRNA with the RNase P holoenzyme. Internally labeled pSupS1 ptRNA was used as a positive control to check for activity of the reconstituted RNase P holoenzyme. EGSs were added in 100-, 50-, and 10-fold molar excess to the mglB mRNA, denoted by black triangles.

Figure 3. Cleavage in vitro of the mglB mRNA by the M1 EGSs alone in the presence of high MgCl₂ concentration.

A. Activities of M1 EGS155. B. Activities of M1 EGS52, M1 EGS73, and M1 EGS148. The RNase P holoenzyme was reconstituted by 10 nM M1 RNA and 100 nM C5 protein. If M1 EGS or M1 RNA was added in the absence of the C5 protein, additional 90 mM MgCl₂ was added. The mglB mRNA cleavage products were separated on 8% polyacrylamide/7 M urea gels together with the mglB mRNA alone or the mglB mRNA with M1 and/or C5 components of the RNase P holoenzyme. Internally labeled pSupS1 ptRNA was used as a positive control to check for activity of the reconstituted RNase P holoenzyme or M1 RNA alone in the presence of high MgCl₂ concentration. Stem EGS155 and M1 EGSs (M1 EGS52, M1 EGS73, M1 EGS148, or M1 EGS155) were added in 10-, 50- or 100-fold molar excess to mglB mRNA, denoted by black triangles.

Figure 4. Analysis of stem and M1 EGSs activity in vivo.

A. Northern analysis of the mglB mRNA level in E. coli. The plasmid vector pKB283-Mlac without (−) mglB nor (−) EGS was used as a negative control. The plasmid vector pMlac-mglB with (+) mglB but without (−) EGS was used as a positive control. The plasmids pMlac-mglB-EGS73, pMlac-mglB-EGS148, and pMlac-mglB-EGS155 carried both (+) mglB and EGS73, EGS148, and EGS155, respectively. Total RNAs were prepared from E. coli BL21(DE3) cells in the absence (−) or presence (+) of IPTG to induce expression of the EGSs listed. The RNA samples were separated on 2% agarose gels. The mglB mRNA was probed with 5′ end labeled oligonucleotide RNA155 and the 5S rRNA with 5S1 on the same membrane. The experiments were independently done twice and only a typical figure was shown. B. Average of the relative mglB mRNA level in E. coli. The ratios of the mglB mRNA to the 5S rRNA for cells carrying different plasmids were calculated. The ratio for cells carrying pMlac-mglB was chosen as 100% to calculate the relative mglB mRNA level in other cells listed.