Antisense RNA Targeting the First Periplasmic Domain of YidC in Escherichia coli Appears to Induce Filamentation but Does Not Affect Cell Viability

09/05/2015

Riaaz Lalani, Nathaniel Susilo, Elisa Xiao, Andrea Xu

Department of Microbiology and Immunology, University of British Columbia

Volume 19
Fall 2014 / Winter 2015

YidC is an essential membrane protein in Escherichia coli. It plays a crucial role in the proper insertion and localization of many membrane proteins. We aimed to design a vector which expresses an antisense RNA (asRNA) against yidC mRNA, which could prevent wild-type yidC expression. In further research, this vector can be co-transformed with a construct carrying yidC bearing mutations within the periplasmic region in order to study the role of this region of the protein. In this paper, we constructed plasmids which contain an antisense sequence targeting the segment of yidC mRNA corresponding to the region encoding the first YidC periplasmic region to prevent YidC translation. The region targeted was downstream of the ribosomal binding site. Two different lengths of antisense inserts, 50 base pairs (bp) and 248 base pairs (bp), were ligated into pHN678 vector, which contains an isopropyl-beta-thiogalactopyranoside (IPTG)-inducible promoter. We successfully generated pNEARH50, pNEARH2481, and pNEARH2482 which were transformed into Escherichia coli DH5α. Sequencing results showed that the fragments were inserted in the correct orientation. Plating and growth curve analysis did not show significant differences between the IPTG-induced and uninduced samples. However, the microscopic observations showed filamentous morphology in IPTG-induced DH5α containing the pNEARH50 or pNEARH2482 constructs. This suggests that asRNA expression may have resulted in a partial YidC knockdown phenotype. The partial YidC knockout might be due to the asRNA targeting a sequence downstream to the ribosomal binding site, which is less efficient in preventing translation. This partial knockdown may result in reduced cell division and a filamentous phenotype. We propose that the biogenesis of membrane proteins potentially important for cell division, such as FtsQ, may have been impacted by YidC knockdown.