The conserved σD envelope stress response monitors multiple aspects of envelope integrity in corynebacteria.

The cell envelope fortifies bacterial cells against antibiotics and other insults. Species in the Mycobacteriales order have a complex envelope that includes an outer layer of mycolic acids called the mycomembrane (MM) and a cell wall composed of peptidoglycan and arabinogalactan. This envelope architecture is unique among bacteria and contributes significantly to the virulence of pathogenic Mycobacteriales like Mycobacterium tuberculosis. Characterization of pathways that govern envelope biogenesis in these organisms is therefore critical in understanding their biology and for identifying new antibiotic targets. To better understand MM biogenesis, we developed a cell sorting-based screen for mutants defective in the surface exposure of a porin normally embedded in the MM of the model organism Corynebacterium glutamicum. The results revealed a requirement for the conserved σD envelope stress response in porin export and identified MarP as the site-1 protease, respectively, that activate the response by cleaving the membrane-embedded anti-sigma factor. A reporter system revealed that the σD pathway responds to defects in mycolic acid and arabinogalactan biosynthesis, suggesting that the stress response has the unusual property of being induced by activating signals that arise from defects in the assembly of two distinct envelope layers. Our results thus provide new insights into how C. glutamicum and related bacteria monitor envelope integrity and suggest a potential role for members of the σD regulon in protein export to the MM.

Physiological characterization of single-gene lysis proteins.

Single-strand RNA (ssRNA) and single-strand DNA phages elicit host lysis using a single gene, in each case designated as sgl. Of the 11 identified Sgls, three have been shown to be specific inhibitors of different steps in the pathway that supplies lipid II to the peptidoglycan (PG) biosynthesis machinery. These Sgls have been called "protein antibiotics" because the lytic event is a septal catastrophe indistinguishable from that caused by cell wall antibiotics. Here, we designate these as type I Sgls. In this formalism, the other eight Sgls are assigned to type II, the best-studied of which is protein L of the paradigm F-specific ssRNA phage MS2. Comparisons have suggested that type II Sgls have four sequence elements distinguished by hydrophobic and polar character. Environmental metatranscriptomics has revealed thousands of new ssRNA phage genomes, each of which presumably has an Sgl. Here, we describe methods to distinguish type I and type II Sgls. Using phase contrast microscopy, we show that both classes of Sgls cause the formation of blebs prior to lysis, but the location of the blebs differs significantly. In addition, we show that L and other type II Sgls do not inhibit the net synthesis of PG, as measured by radio-labeling of PG. Finally, we provide direct evidence that the Sgl from Pseudomonas phage PP7 is a type I Sgl, in support of a recent report based on a genetic selection. This shows that the putative four-element sequence structure suggested for L is not a reliable discriminator for the operational characterization of Sgls. IMPORTANCE: The ssRNA phage world has recently undergone a metagenomic expansion upward of a thousandfold. Each genome likely carries at least one single-gene lysis (sgl) cistron encoding a protein that single-handedly induces host autolysis. Here, we initiate an approach to segregate the Sgls into operational types based on physiological analysis, as a first step toward the alluring goal of finding many new ways to induce bacterial death and the attendant expectations for new antibiotic development.

Chemical Proteomics Strategies for Analyzing Protein Lipidation Reveal the Bacterial O-Mycoloylome.

Protein lipidation dynamically controls protein localization and function within cellular membranes. A unique form of protein O-fatty acylation in Corynebacterium, termed protein O-mycoloylation, involves the attachment of mycolic acids─unusually large and hydrophobic fatty acids─to serine residues of proteins in these organisms' outer mycomembrane. However, as with other forms of protein lipidation, the scope and functional consequences of protein O-mycoloylation are challenging to investigate due to the inherent difficulties of enriching and analyzing lipidated peptides. To facilitate the analysis of protein lipidation and enable the comprehensive profiling and site mapping of protein O-mycoloylation, we developed a chemical proteomics strategy integrating metabolic labeling, click chemistry, cleavable linkers, and a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method employing LC separation and complementary fragmentation methods tailored to the analysis of lipophilic, MS-labile O-acylated peptides. Using these tools in the model organism Corynebacterium glutamicum, we identified approximately 30 candidate O-mycoloylated proteins, including porins, mycoloyltransferases, secreted hydrolases, and other proteins with cell envelope-related functions─consistent with a role for O-mycoloylation in targeting proteins to the mycomembrane. Site mapping revealed that many of the proteins contained multiple spatially proximal modification sites, which occurred predominantly at serine residues surrounded by conformationally flexible peptide motifs. Overall, this study (i) discloses the putative protein O-mycoloylome for the first time, (ii) yields new insights into the undercharacterized proteome of the mycomembrane, which is a hallmark of important pathogens (e.g., Corynebacterium diphtheriae, Mycobacterium tuberculosis), and (iii) provides generally applicable chemical strategies for the proteomic analysis of protein lipidation.

Co-ordinated assembly of the multilayered cell envelope of Gram-negative bacteria.

Bacteria surround themselves with complex cell envelopes to maintain their integrity and protect against external insults. The envelope of Gram-negative organisms is multilayered, with two membranes sandwiching the periplasmic space that contains the peptidoglycan cell wall. Understanding how this complicated surface architecture is assembled during cell growth and division is a major fundamental problem in microbiology. Additionally, because the envelope is an important antibiotic target and determinant of intrinsic antibiotic resistance, understanding the mechanisms governing its assembly is relevant to therapeutic development. In the last several decades, most of the factors required to build the Gram-negative envelope have been identified. However, surprisingly, little is known about how the biogenesis of the different cell surface layers is co-ordinated. Here, we provide an overview of recent work that is beginning to uncover the links connecting the different envelope biosynthetic pathways and assembly machines to ensure uniform envelope growth.

A potential space-making role in cell wall biogenesis for SltB1and DacB revealed by a beta-lactamase induction phenotype in Pseudomonas aeruginosa.

Pseudomonas aeruginosa encodes the beta-lactamase AmpC, which promotes resistance to beta-lactam antibiotics. Expression of ampC is induced by anhydro-muropeptides (AMPs) released from the peptidoglycan (PG) cell wall upon beta-lactam treatment. AmpC can also be induced via genetic inactivation of PG biogenesis factors such as the endopeptidase DacB that cleaves PG crosslinks. Mutants in dacB occur in beta-lactam-resistant clinical isolates of P. aeruginosa, but it has remained unclear why DacB inactivation promotes ampC induction. Similarly, the inactivation of lytic transglycosylase (LT) enzymes such as SltB1 that cut PG glycans has also been associated with ampC induction and beta-lactam resistance. Given that LT enzymes are capable of producing AMP products that serve as ampC inducers, this latter observation has been especially difficult to explain. Here, we show that ampC induction in sltB1 or dacB mutants requires another LT enzyme called MltG. In Escherichia coli, MltG has been implicated in the degradation of nascent PG strands produced upon beta-lactam treatment. Accordingly, in P. aeruginosa sltB1 and dacB mutants, we detected the MltG-dependent production of pentapeptide-containing AMP products that are signatures of nascent PG degradation. Our results therefore support a model in which SltB1 and DacB use their PG-cleaving activity to open space in the PG matrix for the insertion of new material. Thus, their inactivation mimics low-level beta-lactam treatment by reducing the efficiency of new PG insertion into the wall, causing the degradation of some nascent PG material by MltG to produce the ampC-inducing signal. IMPORTANCE: Inducible beta-lactamases like the ampC system of Pseudomonas aeruginosa are a common determinant of beta-lactam resistance among gram-negative bacteria. The regulation of ampC is elegantly tuned to detect defects in cell wall synthesis caused by beta-lactam drugs. Studies of mutations causing ampC induction in the absence of drug therefore promise to reveal new insights into the process of cell wall biogenesis in addition to aiding our understanding of how resistance to beta-lactam antibiotics arises in the clinic. In this study, the ampC induction phenotype for mutants lacking a glycan-cleaving enzyme or an enzyme that cuts cell wall crosslinks was used to uncover a potential role for these enzymes in making space in the wall matrix for the insertion of new material during cell growth.

FacZ is a GpsB-interacting protein that prevents aberrant division-site placement in Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive pathogen responsible for antibiotic-resistant infections. To identify vulnerabilities in cell envelope biogenesis that may overcome resistance, we enriched for S. aureus transposon mutants with defects in cell surface integrity or cell division by sorting for cells that stain with propidium iodide or have increased light-scattering properties, respectively. Transposon sequencing of the sorted populations identified more than 20 previously uncharacterized factors impacting these processes. Cells inactivated for one of these proteins, factor preventing extra Z-rings (FacZ, SAOUHSC_01855), showed aberrant membrane invaginations and multiple FtsZ cytokinetic rings. These phenotypes were suppressed in mutants lacking the conserved cell-division protein GpsB, which forms an interaction hub bridging envelope biogenesis factors with the cytokinetic ring in S. aureus. FacZ was found to interact directly with GpsB in vitro and in vivo. We therefore propose that FacZ is an envelope biogenesis factor that antagonizes GpsB function to prevent aberrant division events in S. aureus.

Synthesis of lipid-linked precursors of the bacterial cell wall is governed by a feedback control mechanism in Pseudomonas aeruginosa.

Many bacterial surface glycans such as the peptidoglycan (PG) cell wall are built from monomeric units linked to a polyprenyl lipid carrier. How this limiting carrier is distributed among competing pathways has remained unclear. Here we describe the isolation of hyperactive variants of Pseudomonas aeruginosa MraY, the enzyme that forms the first lipid-linked PG precursor. These variants result in the elevated production of the final PG precursor lipid II in cells and are hyperactive in vitro. The activated MraY variants have substitutions that map to a cavity on the extracellular side of the dimer interface, far from the active site. Our structural and molecular dynamics results suggest that this cavity is a binding site for externalized lipid II. Overall, our results support a model in which excess externalized lipid II allosterically inhibits MraY, providing a feedback mechanism that prevents the sequestration of lipid carrier in the PG biogenesis pathway.