Characterization of yejM: Essential gene of unknown function in Escherichia coli

Abstract

Gram-negative bacteria have cell envelopes made up of an inner membrane, a peptidoglycan cell wall, and an outer membrane. The outer membrane is made of lipopolysaccharides, β-barrel proteins, lipoproteins, and phospholipids. The mechanisms of trafficking lipopolysaccharides, β-barrel proteins, and lipoproteins are well studied and characterized. However, there is a gap in knowledge regarding the trafficking of phospholipids. This study was undertaken to investigate the essential gene yejM and how it may relate to phospholipid trafficking to the outer membrane in E. coli. A truncated form of the gene, yejM569::kan has previously been shown to cause temperature sensitivity and an outer membrane permeability defect. In this work, spontaneous suppressors of this yejM569::kan phenotype have been isolated and characterized for growth and antibiotic sensitivity. Whole genome sequencing was used to discover the associated mutations in these suppressors. Mutations in lpxC, pgpB, yajR, ygbM, and ygbN were observed in multiple suppressors each and are of unique interest for their relation to yejM. In relation to the mutations in lpxC, LPS levels were analyzed as a possible mechanism of suppression of the TS/outer membrane defect phenotype. Further research is needed to better understand the role of yejM, but evidence from this study indicate that it may be involved in phospholipid trafficking. Deletions of yejM in the suppressor strains would show if the suppressor mutations are sufficient to compensate for yejM deletion. Transposon insertion sequencing should also be done, using the newly created pBAD18::yejM plasmid and strain carrying the yejM569::cam truncation, to identify genes that are synthetic lethal with the yejM truncation.