A Genome-Wide Screen for Bacterial Envelope Biogenesis Mutants Identifies a Novel Factor Involved in Cell Wall Precursor Metabolism

Abstract

The cell envelope of Gram-negative bacteria is a formidable barrier that is difficult for antimicrobial drugs to penetrate. Thus, the list of treatments effective against these organisms is small and with the rise of new resistance mechanisms is shrinking rapidly. New therapies to treat Gram-negative bacterial infections are therefore sorely needed. This goal will be greatly aided by a detailed mechanistic understanding of envelope assembly. Although excellent progress in the identification of essential envelope biogenesis systems has been made in recent years, many aspects of the process remain to be elucidated. We therefore developed a simple, quantitative, and high-throughput assay for mutants with envelope biogenesis defects and used it to screen an ordered single-gene deletion library of Escherichia coli. The screen was robust and correctly identified numerous mutants known to be involved in envelope assembly. Importantly, the screen also implicated 102 genes of unknown function as encoding factors that likely impact envelope biogenesis. As a proof of principle, one of these factors, ElyC (YcbC), was characterized further and shown to play a critical role in the metabolism of the essential lipid carrier used for the biogenesis of cell wall and other bacterial surface polysaccharides. Further analysis of the function of ElyC and other hits identified in our screen is likely to uncover a wealth of new information about the biogenesis of the Gram-negative envelope and the vulnerabilities in the system suitable for drug targeting. Moreover, the screening assay described here should be readily adaptable to other organisms to study the biogenesis of different envelope architectures.

Oxidative Stress Enhances Cephalosporin Resistance of Enterococcus faecalis through Activation of a Two-Component Signaling System

“…In wild-type cells, CPRG is excluded from cells by the permeability barrier of the cell envelope so it cannot be cleaved efficiently by intracellular ␤-galactosidase. However, if circumstances arise in which the cell envelope exhibits enhanced permeability (such as a mutation that compromises the envelope barrier), CPRG can penetrate the cells and be cleaved by ␤-galactosidase to yield the red product (39).…”

Novel RpoS-Dependent Mechanisms Strengthen the Envelope Permeability Barrier during Stationary Phase

“…YdcF is predicted to be a cytoplasmic protein, which binds to S-adenosyl-L-methionine, and has been suggested to be directly or indirectly regulated by both the Rcs response and FNR (67,(69)(70)(71). A deletion mutant of elyC (ycbC), but not sanA, was identified through a high-throughput screen as causing cell lysis at room temperature in LB medium with 1% salt (72). Suppressor and complementation assays suggested that ElyC may be involved in balancing undecaprenyl-phosphate (Und-P) use between PG biosynthesis and the biosynthesis of polysaccharides such as ECA (72).…”

“…A deletion mutant of elyC (ycbC), but not sanA, was identified through a high-throughput screen as causing cell lysis at room temperature in LB medium with 1% salt (72). Suppressor and complementation assays suggested that ElyC may be involved in balancing undecaprenyl-phosphate (Und-P) use between PG biosynthesis and the biosynthesis of polysaccharides such as ECA (72). Supporting this role, deletions of genes involved in the biosynthesis of ECA, a glycolipid with trimeric repeats of N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-mannosaminuronic acid (ManNAcA), and 4-acetamido-4,6-dideoxy-D-galactose (Fuc4NAc), have a suppressive or synthetic phenotype on elyC deletion depending on the Und-P utilization of the mutants.…”

“…Supporting this role, deletions of genes involved in the biosynthesis of ECA, a glycolipid with trimeric repeats of N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-mannosaminuronic acid (ManNAcA), and 4-acetamido-4,6-dideoxy-D-galactose (Fuc4NAc), have a suppressive or synthetic phenotype on elyC deletion depending on the Und-P utilization of the mutants. Thus, deletion of wecA acts as a suppressor of lysis with elyC deletion as it prevents Und-P use for ECA synthesis by preventing the formation of Und-P-P-GlcNAc (lipid I ECA ), while deletion of wecE has a synthetic phenotype with elyC deletion as it causes the buildup of Und-P-P-GlcNAc-ManNAcA (lipid II ECA ), preventing the use of Und-P for PG biosynthesis (47,72,73). Although these proteins contain DUF218 domains similar to SanA, deletion of ygjQ, ydcF, or elyC had no effect on the SDS sensitivity of carbon-limited cells (Fig.…”

A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

“…Deletion cassettes of genes involved in envelope biogenesis (bamB, surA, mrcB), peptidoglycan precursor synthesis (dapF), efflux (tolC), and OM stability (tolQRA) were transduced by P1 transduction from previously constructed mutants into the MG1655 ⌬lacI ⌬lacY strain (20,(29)(30)(31)(32)(33)(34). All gene deletion mutants, except the ⌬tolC and ⌬bamB mutants, were previously identified in an HTS assay as mutants with increased envelope permeability or an elevated rate of cell lysis (35). After incubation for 30 min with ONPG, the deletion mutants displayed increased signal production relative to the baseline signal of the MG1655 ⌬lacI ⌬lacY strain (Fig.…”