Identification of the Lyso-Form N-Acyl Intramolecular Transferase in Low-GC Firmicutes.

Abstract

Bacterial lipoproteins are embedded in the cell membrane of both Gram-positive and Gram-negative bacteria, where they serve numerous functions central to cell envelope physiology. Lipoproteins are tethered to the membrane by an N-acyl-S-(mono/di)-acyl-glyceryl-cysteine anchor that is variously acylated depending on the genus. In several low-GC, Gram-positive firmicutes, a monoacyl-glyceryl-cysteine with an N-terminal fatty acid (known as the lyso form) has been reported, though how it is formed is unknown. Here, through an intergenic complementation rescue assay in Escherichia coli, we report the identification of a common orthologous transmembrane protein in both Enterococcus faecalis and Bacillus cereus that is capable of forming lyso-form lipoproteins. When deleted from the native host, lipoproteins remain diacylated with a free N terminus, as maturation to the N-acylated lyso form is abolished. Evidence is presented suggesting that the previously unknown gene product functions through a novel intramolecular transacylation mechanism, transferring a fatty acid from the diacylglycerol moiety to the α-amino group of the lipidated cysteine. As such, the discovered gene has been named lipoprotein intramolecular transacylase (lit), to differentiate it from the gene for the intermolecular N-acyltransferase (lnt) involved in triacyl lipoprotein biosynthesis in Gram-negative organisms.IMPORTANCE This study identifies a new enzyme, conserved among low-GC, Gram-positive bacteria, that is involved in bacterial lipoprotein biosynthesis and synthesizes lyso-form lipoproteins. Its discovery is an essential first step in determining the physiological role of N-terminal lipoprotein acylation in Gram-positive bacteria and how these modifications impact bacterial cell envelope function.