Contact Area and Deformation of Escherichia coli Cells Adhered on a Cationic Surface.

When bacteria adhere to surfaces, the chemical and mechanical character of the cell-substrate interface guides cell function and the development of microcolonies and biofilms. Alternately on bactericidal surfaces, intimate contact is critical to biofilm prevention. The direct study of the buried cell-substrate interfaces at the heart of these behaviors is hindered by the small bacterial cell size and inaccessibility of the contact region. Here, we present a total internal reflectance fluorescence depletion approach to measure the size of the cell-substrate contact region and quantify the gap separation and curvature near the contact zone, providing an assessment of the shapes of the near-surface undersides of adhered bacterial cells. Resolution of the gap height is about 10%, down to a few nanometers at contact. Using 1 and 2 µm silica spheres as calibration standards we report that, for flagella-free Escherichia coli (E. coli) adhering on a cationic poly-l-lysine layer, the cell-surface contact and apparent cell deformation vary with adsorbed cell configuration. Most cells adhere by their ends, achieving small contact areas of 0.15 µm2, corresponding to about 1-2% of the cell's surface. The altered Gaussian curvatures of end-adhered cells suggest the flattening of the envelope within the small contact region. When cells adhere by their sides, the contact area is larger, in the range 0.3-1.1 µm2 and comprising up to ∼12% of the cell's total surface. A region of sharper curvature, greater than that of the cells' original spherocylindrical shape, borders the flat contact region in cases of side-on or end-on cell adhesion, suggesting envelope stress. From the measured curvatures, precise stress distributions over the cell surface could be calculated in future studies that incorporate knowledge of envelope moduli. Overall the small contact areas of end-adhered cells may be a limiting factor for antimicrobial surfaces that kill on contact rather than releasing bactericide.

A Metabolic-Tag-Based Method for Assessing the Permeation of Small Molecules Across the Mycomembrane in Live Mycobacteria.

The general lack of permeability of small molecules observed for Mycobacterium tuberculosis (Mtb) is most ascribed to its unique cell envelope. More specifically, the outer mycomembrane is hypothesized to be the principal determinant for access of antibiotics to their molecular targets. We describe a novel assay that combines metabolic tagging of the peptidoglycan, which sits directly beneath the mycomembrane, click chemistry of test molecules, and a fluorescent labeling chase step, to measure the permeation of small molecules. We showed that the assay workflow was robust and compatible with high-throughput analysis in mycobacteria by testing a small panel of azide-tagged molecules. The general trend is similar across the two types of mycobacteria with some notable exceptions. We anticipate that this assay platform will lay the foundation for medicinal chemistry efforts to understand and improve uptake of both existing drugs and newly-discovered compounds into mycobacteria.

A Far-Red Molecular Rotor Fluorogenic Trehalose Probe for Live Mycobacteria Detection and Drug-Susceptibility Testing.

Increasing the speed, specificity, sensitivity, and accessibility of mycobacteria detection tools are important challenges for tuberculosis (TB) research and diagnosis. In this regard, previously reported fluorogenic trehalose analogues have shown potential, but their green-emitting dyes may limit sensitivity and applications in complex settings. Here, we describe a trehalose-based fluorogenic probe featuring a molecular rotor turn-on fluorophore with bright far-red emission (RMR-Tre). RMR-Tre, which exploits the unique biosynthetic enzymes and environment of the mycobacterial outer membrane to achieve fluorescence activation, enables fast, no-wash, low-background fluorescence detection of live mycobacteria. Aided by the red-shifted molecular rotor fluorophore, RMR-Tre exhibited up to a 100-fold enhancement in M. tuberculosis labeling compared to existing fluorogenic trehalose probes. We show that RMR-Tre reports on M. tuberculosis drug resistance in a facile assay, demonstrating its potential as a TB diagnostic tool.

Cell Wall Damage Reveals Spatial Flexibility in Peptidoglycan Synthesis and a Nonredundant Role for RodA in Mycobacteria.

Cell wall peptidoglycan is a heteropolymeric mesh that protects the bacterium from internal turgor and external insults. In many rod-shaped bacteria, peptidoglycan synthesis for normal growth is achieved by two distinct pathways: the Rod complex, comprised of MreB, RodA, and a cognate class B penicillin-binding protein (PBP), and the class A PBPs (aPBPs). In contrast to laterally growing bacteria, pole-growing mycobacteria do not encode an MreB homolog and do not require SEDS protein RodA for in vitro growth. However, RodA contributes to the survival of Mycobacterium tuberculosis in some infection models, suggesting that the protein could have a stress-dependent role in maintaining cell wall integrity. Under basal conditions, we find here that the subcellular distribution of RodA largely overlaps that of the aPBP PonA1 and that both RodA and the aPBPs promote polar peptidoglycan assembly. Upon cell wall damage, RodA fortifies Mycobacterium smegmatis against lysis and, unlike aPBPs, contributes to a shift in peptidoglycan assembly from the poles to the sidewall. Neither RodA nor PonA1 relocalize; instead, the redistribution of nascent cell wall parallels that of peptidoglycan precursor synthase MurG. Our results support a model in which mycobacteria balance polar growth and cell-wide repair via spatial flexibility in precursor synthesis and extracellular insertion. IMPORTANCE Peptidoglycan synthesis is a highly successful target for antibiotics. The pathway has been extensively studied in model organisms under laboratory-optimized conditions. In natural environments, bacteria are frequently under attack. Moreover, the vast majority of bacterial species are unlikely to fit a single paradigm of cell wall assembly because of differences in growth mode and/or envelope structure. Studying cell wall synthesis under nonoptimal conditions and in nonstandard species may improve our understanding of pathway function and suggest new inhibition strategies. Mycobacterium smegmatis, a relative of several notorious human and animal pathogens, has an unusual polar growth mode and multilayered envelope. In this work, we challenged M. smegmatis with cell wall-damaging enzymes to characterize the roles of cell wall-building enzymes when the bacterium is under attack.

Inositol acylation of phosphatidylinositol mannosides: a rapid mass response to membrane fluidization in mycobacteria.

Mycobacteria share an unusually complex, multilayered cell envelope, which contributes to adaptation to changing environments. The plasma membrane is the deepest layer of the cell envelope and acts as the final permeability barrier against outside molecules. There is an obvious need to maintain the plasma membrane integrity, but the adaptive responses of the plasma membrane to stress exposure remain poorly understood. Using chemical treatment and heat stress to fluidize the membrane, we show here that phosphatidylinositol (PI)-anchored plasma membrane glycolipids known as PI mannosides (PIMs) are rapidly remodeled upon membrane fluidization in Mycobacterium smegmatis. Without membrane stress, PIMs are predominantly in a triacylated form: two acyl chains of the PI moiety plus one acyl chain modified at one of the mannose residues. Upon membrane fluidization, we determined the fourth fatty acid is added to the inositol moiety of PIMs, making them tetra-acylated variants. Additionally, we show that PIM inositol acylation is a rapid response independent of de novo protein synthesis, representing one of the fastest mass conversions of lipid molecules found in nature. Strikingly, we found that M. smegmatis is more resistant to the bactericidal effect of a cationic detergent after benzyl alcohol pre-exposure. We further demonstrate that fluidization-induced PIM inositol acylation is conserved in pathogens such as Mycobacterium tuberculosis and Mycobacterium abscessus. Our results demonstrate that mycobacteria possess a mechanism to sense plasma membrane fluidity change. We suggest that inositol acylation of PIMs is a novel membrane stress response that enables mycobacterial cells to resist membrane fluidization.

Metabolic Processing of Selenium-Based Bioisosteres of meso-Diaminopimelic Acid in Live Bacteria.

A primary component of all known bacterial cell walls is the peptidoglycan (PG) layer, which is composed of repeating units of sugars connected to short and unusual peptides. The various steps within PG biosynthesis are targets of potent antibiotics as proper assembly of the PG is essential for cellular growth and survival. Synthetic mimics of PG have proven to be indispensable tools to study the bacterial cell structure, growth, and remodeling. Yet, a common component of PG, meso-diaminopimelic acid (m-DAP) at the third position of the stem peptide, remains challenging to access synthetically and is not commercially available. Here, we describe the synthesis and metabolic processing of a selenium-based bioisostere of m-DAP (selenolanthionine) and show that it is installed within the PG of live bacteria by the native cell wall crosslinking machinery in mycobacterial species. This PG probe has an orthogonal release mechanism that could be important for downstream proteomics studies. Finally, we describe a bead-based assay that is compatible with high-throughput screening of cell wall enzymes. We envision that this probe will supplement the current methods available for investigating PG crosslinking in m-DAP-containing organisms.

Surface Chemistry Guides the Orientations of Adhering E. coli Cells Captured from Flow.

Motivated by observations of cell orientation at biofilm-substrate interfaces and reports that cell orientation and adhesion play important roles in biofilm evolution and function, we investigated the influence of surface chemistry on the orientation of Escherichia coli cells captured from flow onto surfaces that were cationic, hydrophobic, or anionic. We characterized the initial orientations of nonmotile cells captured from gentle shear relative to the surface and flow directions. The broad distribution of captured cell orientations observed on cationic surfaces suggests that rapid electrostatic attractions of cells to oppositely charged surfaces preserve the instantaneous orientations of cells as they rotate in the near-surface shearing flow. By contrast, on hydrophobic and anionic surfaces, cells were oriented slightly more in the plane of the surface and in the flow direction compared with that on the cationic surface. This suggests slower development of adhesion at hydrophobic and anionic surfaces, allowing cells to tip toward the surface as they adhere. Once cells were captured, the flow was increased by 20-fold. Cells did not reorient substantially on the cationic surface, suggesting a strong cell-surface bonding. By contrast, on hydrophobic and anionic surfaces, increased shear forced cells to tip toward the surface and align in the flow direction, a process that was reversible upon reducing the shear. These findings suggest mechanisms by which surface chemistry may play a role in the evolving structure and function of microbial communities.

Depletion forces drive reversible capture of live bacteria on non-adhesive surfaces.

Because bacterial adhesion to surfaces is associated with infections and biofilm growth, it has been a longstanding goal to develop coatings that minimize biomolecular adsorption and eliminate bacteria adhesion. We demonstrate that, even on carefully-engineered non-bioadhesive coatings such as polyethylene glycol (PEG) layers that prevent biomolecule adsorption and cell adhesion, depletion interactions from non-adsorbing polymer in solution (such as 10 K PEG or 100 K PEO) can cause adhesion and retention of Escherichia coli cells, defeating the antifouling functionality of the coating. The cells are immobilized and remain viable on the timescale of the study, at least up to 45 minutes. When the polymer solution is replaced by buffer, cells rapidly escape from the surface, consistent with expectations for the reversibility of depletion attractions. The dissolved polymer additionally causes cells to aggregate in solution and aggregates rapidly dissociate to singlets upon tenfold dilution in buffer, also consistent with depletion. Hydrodynamic forces can substantially reduce the adhesion of aggregates on surfaces in conditions where single cells adhere via depletion. The findings reported here suggest that because bacteria thrive in polymer-rich environments both in vivo and in situ, depletion interactions may make it impossible to avoid bacterial retention on surfaces.

Photoactivatable Glycolipid Probes for Identifying Mycolate-Protein Interactions in Live Mycobacteria.

Mycobacteria have a distinctive glycolipid-rich outer membrane, the mycomembrane, which is a critical target for tuberculosis drug development. However, proteins that associate with the mycomembrane, or that are involved in its metabolism and host interactions, are not well-characterized. To facilitate the study of mycomembrane-related proteins, we developed photoactivatable trehalose monomycolate analogues that metabolically incorporate into the mycomembrane in live mycobacteria, enabling in vivo photo-cross-linking and click-chemistry-mediated analysis of mycolate-interacting proteins. When deployed in Mycobacterium smegmatis with quantitative proteomics, this strategy enriched over 100 proteins, including the mycomembrane porin (MspA), several proteins with known mycomembrane synthesis or remodeling functions (CmrA, MmpL3, Ag85, Tdmh), and numerous candidate mycolate-interacting proteins. Our approach is highly versatile, as it (i) enlists click chemistry for flexible protein functionalization; (ii) in principle can be applied to any mycobacterial species to identify endogenous bacterial proteins or host proteins that interact with mycolates; and (iii) can potentially be expanded to investigate protein interactions with other mycobacterial lipids. This tool is expected to help elucidate fundamental physiological and pathological processes related to the mycomembrane and may reveal novel diagnostic and therapeutic targets.

Engineering the Mycomembrane of Live Mycobacteria with an Expanded Set of Trehalose Monomycolate Analogues.

Mycobacteria and related organisms in the Corynebacterineae suborder are characterized by a distinctive outer membrane referred to as the mycomembrane. Biosynthesis of the mycomembrane occurs through an essential process called mycoloylation, which involves antigen 85 (Ag85)-catalyzed transfer of mycolic acids from the mycoloyl donor trehalose monomycolate (TMM) to acceptor carbohydrates and, in some organisms, proteins. We recently described an alkyne-modified TMM analogue (O-AlkTMM-C7) which, in conjunction with click chemistry, acted as a chemical reporter for mycoloylation in intact cells and allowed metabolic labeling of mycoloylated components of the mycomembrane. Here, we describe the synthesis and evaluation of a toolbox of TMM-based reporters bearing alkyne, azide, trans-cyclooctene, and fluorescent tags. These compounds gave further insight into the substrate tolerance of mycoloyltransferases (e.g., Ag85s) in a cellular context and they provide significantly expanded experimental versatility by allowing one- or two-step cell labeling, live cell labeling, and rapid cell labeling via tetrazine ligation. Such capabilities will facilitate research on mycomembrane composition, biosynthesis, and dynamics. Moreover, because TMM is exclusively metabolized by Corynebacterineae, the described probes may be valuable for the specific detection and cell-surface engineering of Mycobacterium tuberculosis and related pathogens. We also performed experiments to establish the dependence of probe incorporation on mycoloyltransferase activity, results from which suggested that cellular labeling is a function not only of metabolic incorporation (and likely removal) pathway(s), but also accessibility across the envelope. Thus, whole-cell labeling experiments with TMM reporters should be carefully designed and interpreted when envelope permeability may be compromised. On the other hand, this property of TMM reporters can potentially be exploited as a convenient way to probe changes in envelope integrity and permeability, facilitating drug development studies.

Regulation of Clostridium difficile Spore Formation by the SpoIIQ and SpoIIIA Proteins.

Sporulation is an ancient developmental process that involves the formation of a highly resistant endospore within a larger mother cell. In the model organism Bacillus subtilis, sporulation-specific sigma factors activate compartment-specific transcriptional programs that drive spore morphogenesis. σG activity in the forespore depends on the formation of a secretion complex, known as the "feeding tube," that bridges the mother cell and forespore and maintains forespore integrity. Even though these channel components are conserved in all spore formers, recent studies in the major nosocomial pathogen Clostridium difficile suggested that these components are dispensable for σG activity. In this study, we investigated the requirements of the SpoIIQ and SpoIIIA proteins during C. difficile sporulation. C. difficile spoIIQ, spoIIIA, and spoIIIAH mutants exhibited defects in engulfment, tethering of coat to the forespore, and heat-resistant spore formation, even though they activate σG at wildtype levels. Although the spoIIQ, spoIIIA, and spoIIIAH mutants were defective in engulfment, metabolic labeling studies revealed that they nevertheless actively transformed the peptidoglycan at the leading edge of engulfment. In vitro pull-down assays further demonstrated that C. difficile SpoIIQ directly interacts with SpoIIIAH. Interestingly, mutation of the conserved Walker A ATP binding motif, but not the Walker B ATP hydrolysis motif, disrupted SpoIIIAA function during C. difficile spore formation. This finding contrasts with B. subtilis, which requires both Walker A and B motifs for SpoIIIAA function. Taken together, our findings suggest that inhibiting SpoIIQ, SpoIIIAA, or SpoIIIAH function could prevent the formation of infectious C. difficile spores and thus disease transmission.

Imaging bacterial peptidoglycan with near-infrared fluorogenic azide probes.

Fluorescent probes designed for activation by bioorthogonal chemistry have enabled the visualization of biomolecules in living systems. Such activatable probes with near-infrared (NIR) emission would be ideal for in vivo imaging but have proven difficult to engineer. We present the development of NIR fluorogenic azide probes based on the Si-rhodamine scaffold that undergo a fluorescence enhancement of up to 48-fold upon reaction with terminal or strained alkynes. We used the probes for mammalian cell surface imaging and, in conjunction with a new class of cyclooctyne D-amino acids, for visualization of bacterial peptidoglycan without the need to wash away unreacted probe.

Subpolar addition of new cell wall is directed by DivIVA in mycobacteria.

Mycobacteria are surrounded by a complex multilayered envelope and elongate at the poles. The principles that organize the coordinated addition of chemically diverse cell wall layers during polar extension remain unclear. We show that enzymes mediating the terminal cytosolic steps of peptidoglycan, arabinogalactan, and mycolic acid synthesis colocalize at sites of cell growth or division. The tropomyosin-like protein, DivIVA, is targeted to the negative curvature of the pole, is enriched at the growing end, and determines cell shape from this site. In contrast, cell wall synthetic complexes are concentrated at a distinct subpolar location. When viewed at subdiffraction resolution, new peptidoglycan is deposited at this subpolar site, and inert cell wall covers the DivIVA-marked tip. The differentiation between polar tip and cell wall synthetic complexes is also apparent at the biochemical level. Enzymes that generate mycolate precursors interact with DivIVA, but the final condensation of mycolic acids occurs in a distinct protein complex at the site of nascent cell wall addition. We propose an ultrastructural model of mycobacterial polar growth where new cell wall is added in an annular zone below the cell tip. This model may be broadly applicable to other bacterial and fungal organisms that grow via polar extension.

Osmosensory signaling in Mycobacterium tuberculosis mediated by a eukaryotic-like Ser/Thr protein kinase.

Bacteria are able to adapt to dramatically different microenvironments, but in many organisms, the signaling pathways, transcriptional programs, and downstream physiological changes involved in adaptation are not well-understood. Here, we discovered that osmotic stress stimulates a signaling network in Mycobacterium tuberculosis regulated by the eukaryotic-like receptor Ser/Thr protein kinase PknD. Expression of the PknD substrate Rv0516c was highly induced by osmotic stress. Furthermore, Rv0516c disruption modified peptidoglycan thickness, enhanced antibiotic resistance, and activated genes in the regulon of the alternative σ-factor SigF. Phosphorylation of Rv0516c regulated the abundance of EspA, a virulence-associated substrate of the type VII ESX-1 secretion system. These findings identify an osmosensory pathway orchestrated by PknD, Rv0516c, and SigF that enables adaptation to osmotic stress through cell wall remodeling and virulence factor production. Given the widespread occurrence of eukaryotic-like Ser/Thr protein kinases in bacteria, these proteins may play a broad role in bacterial osmosensing.

Measurement of Small Molecule Accumulation into Diderm Bacteria.

Some of the most dangerous bacterial pathogens (Gram-negative and mycobacterial) deploy a formidable secondary membrane barrier to reduce the influx of exogenous molecules. For Gram-negative bacteria, this second exterior membrane is known as the outer membrane (OM), while for the Gram-indeterminate Mycobacteria, it is known as the "myco" membrane. Although different in composition, both the OM and mycomembrane are key structures that restrict the passive permeation of small molecules into bacterial cells. Although it is well-appreciated that such structures are principal determinants of small molecule permeation, it has proven to be challenging to assess this feature in a robust and quantitative way or in complex, infection-relevant settings. Herein, we describe the development of the bacterial chloro-alkane penetration assay (BaCAPA), which employs the use of a genetically encoded protein called HaloTag, to measure the uptake and accumulation of molecules into model Gram-negative and mycobacterial species, Escherichia coli and Mycobacterium smegmatis, respectively, and into the human pathogen Mycobacterium tuberculosis. The HaloTag protein can be directed to either the cytoplasm or the periplasm of bacteria. This offers the possibility of compartmental analysis of permeation across individual cell membranes. Significantly, we also showed that BaCAPA can be used to analyze the permeation of molecules into host cell-internalized E. coli and M. tuberculosis, a critical capability for analyzing intracellular pathogens. Together, our results show that BaCAPA affords facile measurement of permeability across four barriers: the host plasma and phagosomal membranes and the diderm bacterial cell envelope.

A cell wall synthase accelerates plasma membrane partitioning in mycobacteria.

Lateral partitioning of proteins and lipids shapes membrane function. In model membranes, partitioning can be influenced both by bilayer-intrinsic factors like molecular composition and by bilayer-extrinsic factors such as interactions with other membranes and solid supports. While cellular membranes can departition in response to bilayer-intrinsic or -extrinsic disruptions, the mechanisms by which they partition de novo are largely unknown. The plasma membrane of Mycobacterium smegmatis spatially and biochemically departitions in response to the fluidizing agent benzyl alcohol, then repartitions upon fluidizer washout. By screening for mutants that are sensitive to benzyl alcohol, we show that the bifunctional cell wall synthase PonA2 promotes membrane partitioning and cell growth during recovery from benzyl alcohol exposure. PonA2's role in membrane repartitioning and regrowth depends solely on its conserved transglycosylase domain. Active cell wall polymerization promotes de novo membrane partitioning and the completed cell wall polymer helps to maintain membrane partitioning. Our work highlights the complexity of membrane-cell wall interactions and establishes a facile model system for departitioning and repartitioning cellular membranes.

Tuberculostearic Acid Controls Mycobacterial Membrane Compartmentalization.

The intracellular membrane domain (IMD) is a laterally discrete region of the mycobacterial plasma membrane, enriched in the subpolar region of the rod-shaped cell. Here, we report genome-wide transposon sequencing to discover the controllers of membrane compartmentalization in Mycobacterium smegmatis. The putative gene cfa showed the most significant effect on recovery from membrane compartment disruption by dibucaine. Enzymatic analysis of Cfa and lipidomic analysis of a cfa deletion mutant (Δcfa) demonstrated that Cfa is an essential methyltransferase for the synthesis of major membrane phospholipids containing a C19:0 monomethyl-branched stearic acid, also known as tuberculostearic acid (TBSA). TBSA has been intensively studied due to its abundant and genus-specific production in mycobacteria, but its biosynthetic enzymes had remained elusive. Cfa catalyzed the S-adenosyl-l-methionine-dependent methyltransferase reaction using oleic acid-containing lipid as a substrate, and Δcfa accumulated C18:1 oleic acid, suggesting that Cfa commits oleic acid to TBSA biosynthesis, likely contributing directly to lateral membrane partitioning. Consistent with this model, Δcfa displayed delayed restoration of subpolar IMD and delayed outgrowth after bacteriostatic dibucaine treatment. These results reveal the physiological significance of TBSA in controlling lateral membrane partitioning in mycobacteria. IMPORTANCE As its common name implies, tuberculostearic acid is an abundant and genus-specific branched-chain fatty acid in mycobacterial membranes. This fatty acid, 10-methyl octadecanoic acid, has been an intense focus of research, particularly as a diagnostic marker for tuberculosis. It was discovered in 1934, and yet the enzymes that mediate the biosynthesis of this fatty acid and the functions of this unusual fatty acid in cells have remained elusive. Through a genome-wide transposon sequencing screen, enzyme assay, and global lipidomic analysis, we show that Cfa is the long-sought enzyme that is specifically involved in the first step of generating tuberculostearic acid. By characterizing a cfa deletion mutant, we further demonstrate that tuberculostearic acid actively regulates lateral membrane heterogeneity in mycobacteria. These findings indicate the role of branched fatty acids in controlling the functions of the plasma membrane, a critical barrier for the pathogen to survive in its human host.

Probing the mycobacterial trehalome with bioorthogonal chemistry.

Mycobacteria, including the pathogen Mycobacterium tuberculosis, use the non-mammalian disaccharide trehalose as a precursor for essential cell-wall glycolipids and other metabolites. Here we describe a strategy for exploiting trehalose metabolic pathways to label glycolipids in mycobacteria with azide-modified trehalose (TreAz) analogues. Subsequent bioorthogonal ligation with alkyne-functionalized probes enabled detection and visualization of cell-surface glycolipids. Characterization of the metabolic fates of four TreAz analogues revealed unique labeling routes that can be harnessed for pathway-targeted investigation of the mycobacterial trehalome.

Mycobacterial Esx-3 is required for mycobactin-mediated iron acquisition.

The Esx secretion pathway is conserved across Gram-positive bacteria. Esx-1, the best-characterized system, is required for virulence of Mycobacterium tuberculosis, although its precise function during infection remains unclear. Esx-3, a paralogous system present in all mycobacterial species, is required for growth in vitro. Here, we demonstrate that mycobacteria lacking Esx-3 are defective in acquiring iron. To compete for the limited iron available in the host and the environment, these organisms use mycobactin, high-affinity iron-binding molecules. In the absence of Esx-3, mycobacteria synthesize mycobactin but are unable to use the bound iron and are impaired severely for growth during macrophage infection. Mycobacteria thus require a specialized secretion system for acquiring iron from siderophores.

A Bifunctional Chemical Reporter for in Situ Analysis of Cell Envelope Glycan Recycling in Mycobacteria.

In mycobacteria, the glucose-based disaccharide trehalose cycles between the cytoplasm, where it is a stress protectant and carbon source, and the cell envelope, where it is released as a byproduct of outer mycomembrane glycan biosynthesis and turnover. Trehalose recycling via the LpqY-SugABC transporter promotes virulence, antibiotic recalcitrance, and efficient adaptation to nutrient deprivation. The source(s) of trehalose and the regulation of recycling under these and other stressors are unclear. A key technical gap in addressing these questions has been the inability to trace trehalose recycling in situ, directly from its site of liberation from the cell envelope. Here we describe a bifunctional chemical reporter that simultaneously marks mycomembrane biosynthesis and subsequent trehalose recycling with alkyne and azide groups. Using this probe, we discovered that the recycling efficiency for trehalose increases upon carbon starvation, concomitant with an increase in LpqY-SugABC expression. The ability of the bifunctional reporter to probe multiple, linked steps provides a more nuanced understanding of mycobacterial cell envelope metabolism and its plasticity under stress.