Plasmid Mediated Complementation of YidC Using a YidC Variant Fused to Green Fluorescence Protein Supports Growth of Escherichia coli BL21 at 30 Degrees Celcius but not at 37 Degrees Celcius (Note: Erratum associated with this manuscript.)

ERRATUM: Follow up studies of the strain described in this study have shown that they are Rhizobium and not E. coli (Hooshmand et al, JEMI, Volume 21, pages 101 – 106).  The described biological characteristics of these strains are therefore not valid.  Nevertheless, the approach designed to build these strains may be of interest to future researchers.   YidC is an essential membrane protein in Gram-negative bacteria involved in translocation and folding of proteins into the inner membrane. Previous studies have shown that the depletion of YidC disrupts folding of penicillin binding proteins leading to defects in peptidoglycan biosynthesis and cell division. YidC is a 60 kDa inner membrane protein with 6 transmembrane domains and a 30 kDa soluble domain (termed P1) located between transmembrane domains 1 and 2 that extends into the periplasm. P1 is not essential for cell viability as mutant strains of Escherichia coli expressing in frame deletions of P1 are viable. The function of P1is unknown. Given the relatively large size of P1 and its evolutionary conservation in Gram-negative bacteria, it is likely to have an important biological function. In this experiment, we designed a chromosomal yidC knock out strain of Escherichia coli by complementing expression witha YidC variant fused at its C-terminus to green fluorescence protein encoded on a plasmid. We compared the phenotype of an Escherichia coli strain expressing a P1 deleted form of YidC with a strain expressing a wildtype copy of YidC. Our preliminary observations suggest that the strain expression of the P1 deleted YidC protein forms rod-shaped cells similar to the wild type strain. These data suggest that the P1 domain is not involved in maintaining cell shape. Interestingly, the knockout strain of Escherichia coli expressing YidC fused to GFP grow at 30oC but not at 37oC suggesting a temperature sensitive phenotype, which may be useful when constructing YidC mutants in future structure-function studies.