Antisense RNA Targeting the First Periplasmic Domain of YidC Appears to Reduce Cell Growth at 30 Degrees Celcius but Does Not Induce a Filamentous Phenotype

YidC is an essential protein in Escherichia coli that is evolutionarily conserved. It is involved in the proper folding and insertion of many membrane proteins. YidC is a transmembrane protein that consists of six transmembrane domains and a major 30 kDa periplasmic domain between transmembrane domains one and two. In this study, we sought to develop a method to study the role of the YidC major periplasmic domain by knocking down expression of full length YidC using antisense RNA designed to bind to the sequence of messenger RNA corresponding to this region of YidC. Mutant forms of YidC (lacking the major periplasmic domain) could then be studied using this system. In our study, we investigated whether a knockdown of YidC would result in a growth defect or defined phenotype. We used an IPTG-inducible antisense RNA targeting the first periplasmic domain of YidC. The antisense RNA product was designed to suppress YidC protein expression by binding to a 248 bp and 50 bp region of mRNA coding for the periplasmic domain of YidC. Uninduced and IPTG induced E. coli DH5α cells harbouring these constructs were compared at 4 hours and 24 hours post-induction. When grown on solid media, cells harbouring the pNEARH2481 antisense RNA construct showed reduced growth when compared to cells harbouring the parental vector. This is consistent with the crucial role of YidC in the insertion of essential proteins in the inner membrane, as decreased expression of YidC lead to reduced cell growth. Microscopic observations of these cells did not show a filamentous morphology relative to their uninduced control suggesting that the expression of the antisense RNA did not impact cell division under the conditions tested.