@article {2624, title = {Chemically Induced Cell Wall Stapling in Bacteria.}, journal = {Cell Chem Biol}, volume = {28}, year = {2021}, month = {2021 Feb 18}, pages = {213-220.e4}, abstract = {

Transpeptidation reinforces the structure of cell-wall peptidoglycan, an extracellular heteropolymer that protects bacteria from osmotic lysis. The clinical success of transpeptidase-inhibiting \β-lactam antibiotics illustrates the essentiality of these cross-linkages for cell-wall integrity, but the presence of multiple, seemingly redundant transpeptidases in many species makes it challenging to determine cross-link function. Here, we present a technique to link peptide strands by chemical rather than enzymatic reaction. We employ biocompatible click chemistry to induce triazole formation between azido- and alkynyl-d-alanine residues that are metabolically installed in the peptidoglycan of Gram-positive or Gram-negative bacteria. Synthetic triazole cross-links can be visualized using azidocoumarin-d-alanine, an amino acid derivative that undergoes fluorescent enhancement upon reaction with terminal alkynes. Cell-wall stapling protects Escherichia coli from treatment with the broad-spectrum \β-lactams ampicillin and carbenicillin. Chemical control of cell-wall structure in live bacteria can provide functional insights that are orthogonal to those obtained by genetics.

}, issn = {2451-9448}, doi = {10.1016/j.chembiol.2020.11.006}, author = {Rivera, Sylvia L and Espaillat, Akbar and Aditham, Arjun K and Shieh, Peyton and Muriel-Mundo, Chris and Kim, Justin and Cava, Felipe and Siegrist, M Sloan} } @article {2626, title = {Membrane-partitioned cell wall synthesis in mycobacteria.}, journal = {Elife}, volume = {10}, year = {2021}, month = {2021 Feb 05}, abstract = {

Many antibiotics target the assembly of cell wall peptidoglycan, an essential, heteropolymeric mesh that encases most bacteria. In rod-shaped bacteria, cell wall elongation is spatially precise yet relies on limited pools of lipid-linked precursors that generate and are attracted to membrane disorder. By tracking enzymes, substrates, and products of peptidoglycan biosynthesis in , we show that precursors are made in plasma membrane domains that are laterally and biochemically distinct from sites of cell wall assembly. Membrane partitioning likely contributes to robust, orderly peptidoglycan synthesis, suggesting that these domains help template peptidoglycan synthesis. The cell wall-organizing protein DivIVA and the cell wall itself promote domain homeostasis. These data support a model in which the peptidoglycan polymer feeds back on its membrane template to maintain an environment conducive to directional synthesis. Our findings are applicable to rod-shaped bacteria that are phylogenetically distant from , indicating that horizontal compartmentalization of precursors may be a general feature of bacillary cell wall biogenesis.

}, issn = {2050-084X}, doi = {10.7554/eLife.60263}, author = {Garc{\'\i}a-Heredia, Alam and Kado, Takehiro and Sein, Caralyn E and Puffal, Julia and Osman, Sarah H and Judd, Julius and Gray, Todd A and Morita, Yasu S and Siegrist, M Sloan} } @article {2625, title = {Trehalose Recycling Promotes Energy-Efficient Biosynthesis of the Mycobacterial Cell Envelope.}, journal = {mBio}, volume = {12}, year = {2021}, month = {2021 01 19}, abstract = {

The mycomembrane layer of the mycobacterial cell envelope is a barrier to environmental, immune, and antibiotic insults. There is considerable evidence of mycomembrane plasticity during infection and in response to host-mimicking stresses. Since mycobacteria are resource and energy limited under these conditions, it is likely that remodeling has distinct requirements from those of the well-characterized biosynthetic program that operates during unrestricted growth. Unexpectedly, we found that mycomembrane remodeling in nutrient-starved, nonreplicating mycobacteria includes synthesis in addition to turnover. Mycomembrane synthesis under these conditions occurs along the cell periphery, in contrast to the polar assembly of actively growing cells, and both liberates and relies on the nonmammalian disaccharide trehalose. In the absence of trehalose recycling, trehalose synthesis fuels mycomembrane remodeling. However, mycobacteria experience ATP depletion, enhanced respiration, and redox stress, hallmarks of futile cycling and the collateral dysfunction elicited by some bactericidal antibiotics. Inefficient energy metabolism compromises the survival of trehalose recycling mutants in macrophages. Our data suggest that trehalose recycling alleviates the energetic burden of mycomembrane remodeling under stress. Cell envelope recycling pathways are emerging targets for sensitizing resource-limited bacterial pathogens to host and antibiotic pressure. The glucose-based disaccharide trehalose is a stress protectant and carbon source in many nonmammalian cells. Mycobacteria are relatively unique in that they use trehalose for an additional, extracytoplasmic purpose: to build their outer \"myco\" membrane. In these organisms, trehalose connects mycomembrane biosynthesis and turnover to central carbon metabolism. Key to this connection is the retrograde transporter LpqY-SugABC. Unexpectedly, we found that nongrowing mycobacteria synthesize mycomembrane under carbon limitation but do not require LpqY-SugABC. In the absence of trehalose recycling, compensatory anabolism allows mycomembrane biosynthesis to continue. However, this workaround comes at a cost, namely, ATP consumption, increased respiration, and oxidative stress. Strikingly, these phenotypes resemble those elicited by futile cycles and some bactericidal antibiotics. We demonstrate that inefficient energy metabolism attenuates trehalose recycling mutant in macrophages. Energy-expensive macromolecule biosynthesis triggered in the absence of recycling may be a new paradigm for boosting host activity against bacterial pathogens.

}, issn = {2150-7511}, doi = {10.1128/mBio.02801-20}, author = {Pohane, Amol Arunrao and Carr, Caleb R and Garhyan, Jaishree and Swarts, Benjamin M and Siegrist, M Sloan} } @article {2622, title = {Chemical Biology Tools for Examining the Bacterial Cell Wall.}, journal = {Cell Chem Biol}, volume = {27}, year = {2020}, month = {2020 08 20}, pages = {1052-1062}, abstract = {

Bacteria surround themselves with cell walls to maintain cell rigidity and protect against environmental insults. Here we review chemical and biochemical techniques employed to study bacterial cell wall biogenesis. Recent advances including the ability to isolate critical intermediates, metabolic approaches for probe incorporation, and isotopic labeling techniques have provided critical insight into the biochemistry of cell walls. Fundamental manuscripts that have used these techniques to discover cell wall-interacting proteins, flippases, and cell wall stoichiometry are discussed in detail. The review highlights that these powerful methods and techniques have exciting potential to identify and characterize new targets for antibiotic development.

}, issn = {2451-9448}, doi = {10.1016/j.chembiol.2020.07.024}, author = {Brown, Ashley R and Gordon, Rebecca A and Hyland, Stephen N and Siegrist, M Sloan and Grimes, Catherine L} } @article {2621, title = {Photoactivatable Glycolipid Probes for Identifying Mycolate-Protein Interactions in Live Mycobacteria.}, journal = {J Am Chem Soc}, volume = {142}, year = {2020}, month = {2020 04 29}, pages = {7725-7731}, abstract = {

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 photo-cross-linking and click-chemistry-mediated analysis of mycolate-interacting proteins. When deployed in 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.

}, issn = {1520-5126}, doi = {10.1021/jacs.0c01065}, author = {Kavunja, Herbert W and Biegas, Kyle J and Banahene, Nicholas and Stewart, Jessica A and Piligian, Brent F and Groenevelt, Jessica M and Sein, Caralyn E and Morita, Yasu S and Niederweis, Michael and Siegrist, M Sloan and Swarts, Benjamin M} } @article {2623, title = {Supramolecular antibiotics: a strategy for conversion of broad-spectrum to narrow-spectrum antibiotics for .}, journal = {Nanoscale}, volume = {12}, year = {2020}, month = {2020 Oct 22}, pages = {20693-20698}, abstract = {

The propensity of broad-spectrum antibiotics to indiscriminately kill both pathogenic and beneficial bacteria has a profound impact on the spread of resistance across multiple bacterial species. Alternative approaches that narrow antibacterial specificity towards desired pathogenic bacterial population are of great interest. Here, we report an enzyme-responsive antibiotic-loaded nanoassembly strategy for narrow delivery of otherwise broad-spectrum antibiotics. We specifically target Staphylococcus aureus (S. aureus), an important blood pathogen that secretes PC1 \β-lactamases. Our nanoassemblies selectively eradicate S. aureus grown in vitro with other bacteria, highlighting its potential capability in targeting the desired pathogenic bacterial population.

}, issn = {2040-3372}, doi = {10.1039/d0nr04886k}, author = {Koyasseril-Yehiya, Thameez M and Garc{\'\i}a-Heredia, Alam and Anson, Francesca and Rangadurai, Poornima and Siegrist, M Sloan and Thayumanavan, S} } @article {2620, title = {Engineering the Mycomembrane of Live Mycobacteria with an Expanded Set of Trehalose Monomycolate Analogues.}, journal = {Chembiochem}, volume = {20}, year = {2019}, month = {2019 05 15}, pages = {1282-1291}, abstract = {

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.

}, keywords = {Acyltransferases, Alkynes, Azides, Bacillus subtilis, Cell Engineering, Cell Membrane, Click Chemistry, Cord Factors, Corynebacterium, Escherichia coli, Fluorescent Dyes, Molecular Structure, Mycobacterium smegmatis, Mycobacterium tuberculosis}, issn = {1439-7633}, doi = {10.1002/cbic.201800687}, author = {Fiolek, Taylor J and Banahene, Nicholas and Kavunja, Herbert W and Holmes, Nathan J and Rylski, Adrian K and Pohane, Amol Arunrao and Siegrist, M Sloan and Swarts, Benjamin M} } @article {1503, title = {Host Actin Polymerization Tunes the Cell Division Cycle of an Intracellular Pathogen.}, journal = {Cell Rep}, year = {2015}, month = {2015 Apr 15}, abstract = {

Growth and division are two of the most fundamental capabilities of a bacterial cell. While they are well described for model organisms growing in broth culture, very little is known about the cell division cycle of bacteria replicating in more complex environments. Using a D-alanine reporter strategy, we found that intracellular Listeria monocytogenes (Lm) spend a smaller proportion of their cell cycle dividing compared to Lm growing in broth culture. This alteration to the cell division cycle is independent of bacterial doubling time. Instead, polymerization of host-derived actin at the bacterial cell surface extends the non-dividing elongation period and compresses the division period. By decreasing the relative proportion of dividing Lm, actin polymerization biases the population toward cells with the highest propensity to form actin tails. Thus, there is a positive-feedback loop between the Lm cell division\ cycle and a physical interaction with the host cytoskeleton.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2015.03.046}, author = {Siegrist, M Sloan and Aditham, Arjun K and Espaillat, Akbar and Cameron, Todd A and Whiteside, Sarah A and Cava, Felipe and Portnoy, Daniel A and Bertozzi, Carolyn R} } @article {1437, title = {Illumination of growth, division and secretion by metabolic labeling of the bacterial cell surface.}, journal = {FEMS Microbiol Rev}, volume = {39}, year = {2015}, month = {2015 Mar}, pages = {184-202}, abstract = {

The cell surface is the essential interface between a bacterium and its surroundings. Composed primarily of molecules that are not directly genetically encoded, this highly dynamic structure accommodates the basic cellular processes of growth and division as well as the transport of molecules between the cytoplasm and the extracellular milieu. In this review, we describe aspects of bacterial growth, division and secretion that have recently been uncovered by metabolic labeling of the cell envelope. Metabolite derivatives can be used to label a variety of macromolecules, from proteins to non-genetically-encoded glycans and lipids. The embedded metabolite enables precise tracking in time and space, and the versatility of newer chemoselective detection methods offers the ability to execute multiple experiments concurrently. In addition to reviewing the discoveries enabled by metabolic labeling of the bacterial cell envelope, we also discuss the potential of these techniques for translational applications. Finally, we offer some guidelines for implementing this emerging technology.

}, issn = {1574-6976}, doi = {10.1093/femsre/fuu012}, author = {Siegrist, M Sloan and Swarts, Benjamin M and Fox, Douglas M and Lim, Shion An and Bertozzi, Carolyn R} } @article {1622, title = {Regulation of Clostridium difficile Spore Formation by the SpoIIQ and SpoIIIA Proteins.}, journal = {PLoS Genet}, volume = {11}, year = {2015}, month = {2015 Oct}, pages = {e1005562}, abstract = {

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.

}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1005562}, author = {Fimlaid, Kelly A and Jensen, Owen and Donnelly, M Lauren and Siegrist, M Sloan and Shen, Aimee} } @article {1442, title = {Imaging bacterial peptidoglycan with near-infrared fluorogenic azide probes.}, journal = {Proc Natl Acad Sci U S A}, volume = {111}, year = {2014}, month = {2014 Apr 15}, pages = {5456-61}, abstract = {

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.

}, keywords = {Azides, Fluorescent Dyes, Molecular Imaging, Molecular Probe Techniques, Molecular Structure, Peptidoglycan}, issn = {1091-6490}, doi = {10.1073/pnas.1322727111}, author = {Shieh, Peyton and Siegrist, M Sloan and Cullen, Andrew J and Bertozzi, Carolyn R} } @article {1441, title = {Mycobacterial Esx-3 requires multiple components for iron acquisition.}, journal = {MBio}, volume = {5}, year = {2014}, month = {2014}, pages = {e01073-14}, abstract = {

ABSTRACT The type VII secretion systems are conserved across mycobacterial species and in many Gram-positive bacteria. While the well-characterized Esx-1 pathway is required for the virulence of pathogenic mycobacteria and conjugation in the model organism Mycobacterium smegmatis, Esx-3 contributes to mycobactin-mediated iron acquisition in these bacteria. Here we show that several Esx-3 components are individually required for function under low-iron conditions but that at least one, the membrane-bound protease MycP3 of M. smegmatis, is partially expendable. All of the esx-3 mutants tested, including the ΔmycP3ms mutant, failed to export the native Esx-3 substrates EsxHms and EsxGms to quantifiable levels, as determined by targeted mass spectrometry. Although we were able to restore low-iron growth to the esx-3 mutants by genetic complementation, we found a wide range of complementation levels for protein export. Indeed, minute quantities of extracellular EsxHms and EsxGms were sufficient for iron acquisition under our experimental conditions. The apparent separation of Esx-3 function in iron acquisition from robust EsxGms and EsxHms secretion in the ΔmycP3ms mutant and in some of the complemented esx-3 mutants compels reexamination of the structure-function relationships for type VII secretion systems. IMPORTANCE Mycobacteria have several paralogous type VII secretion systems, Esx-1 through Esx-5. Whereas Esx-1 is required for pathogenic mycobacteria to grow within an infected host, Esx-3 is essential for growth in vitro. We and others have shown that Esx-3 is required for siderophore-mediated iron acquisition. In this work, we identify individual Esx-3 components that contribute to this process. As in the Esx-1 system, most mutations that abolish Esx-3 protein export also disrupt its function. Unexpectedly, however, ultrasensitive quantitation of Esx-3 secretion by multiple-reaction-monitoring mass spectrometry (MRM-MS) revealed that very low levels of export were sufficient for iron acquisition under similar conditions. Although protein export clearly contributes to type VII function, the relationship is not absolute.

}, keywords = {Bacterial Secretion Systems, Gene Order, Genetic Loci, Iron, Mycobacterium, Oxazoles}, issn = {2150-7511}, doi = {10.1128/mBio.01073-14}, author = {Siegrist, M Sloan and Steigedal, Magnus and Ahmad, Rushdy and Mehra, Alka and Dragset, Marte S and Schuster, Brian M and Philips, Jennifer A and Carr, Steven A and Rubin, Eric J} } @article {1443, title = {Mycobacterial lipid logic.}, journal = {Cell Host Microbe}, volume = {15}, year = {2014}, month = {2014 Jan 15}, pages = {1-2}, abstract = {

During infection of the lung epithelium, Mycobacterium tuberculosis must infect and survive within macrophages long enough to be transported into deeper lung tissues. Cambier et~al. (2013) show that pathogenic mycobacteria use the coordinated action of two cell wall glycolipids to regulate macrophage recruitment to initial infection sites.

}, keywords = {Animals, Female, Immune Evasion, Macrophages, Membrane Lipids, Mycobacterium}, issn = {1934-6069}, doi = {10.1016/j.chom.2013.12.005}, author = {Siegrist, M Sloan and Bertozzi, Carolyn R} } @article {1440, title = {Subpolar addition of new cell wall is directed by DivIVA in mycobacteria.}, journal = {Proc Natl Acad Sci U S A}, volume = {111}, year = {2014}, month = {2014 Aug 5}, pages = {E3243-51}, abstract = {

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.

}, keywords = {Bacterial Proteins, Cell Cycle Proteins, Cell Membrane, Cell Polarity, Cell Wall, Models, Biological, Mycobacterium smegmatis, Mycolic Acids, Protein Binding}, issn = {1091-6490}, doi = {10.1073/pnas.1402158111}, author = {Meniche, Xavier and Otten, Renee and Siegrist, M Sloan and Baer, Christina E and Murphy, Kenan C and Bertozzi, Carolyn R and Sassetti, Christopher M} } @article {1445, title = {(D)-Amino acid chemical reporters reveal peptidoglycan dynamics of an intracellular pathogen.}, journal = {ACS Chem Biol}, volume = {8}, year = {2013}, month = {2013 Mar 15}, pages = {500-5}, abstract = {

Peptidoglycan (PG) is an essential component of the bacterial cell wall. Although experiments with organisms in vitro have yielded a wealth of information on PG synthesis and maturation, it is unclear how these studies translate to bacteria replicating within host cells. We report a chemical approach for probing PG in vivo via metabolic labeling and bioorthogonal chemistry. A wide variety of bacterial species incorporated azide and alkyne-functionalized d-alanine into their cell walls, which we visualized by covalent reaction with click chemistry probes. The d-alanine analogues were specifically incorporated into nascent PG of the intracellular pathogen Listeria monocytogenes both in vitro and during macrophage infection. Metabolic incorporation of d-alanine derivatives and click chemistry detection constitute a facile, modular platform that facilitates unprecedented spatial and temporal resolution of PG dynamics in vivo.

}, keywords = {Alanine, Click Chemistry, Listeria monocytogenes, Molecular Conformation, Molecular Dynamics Simulation, Peptidoglycan}, issn = {1554-8937}, doi = {10.1021/cb3004995}, author = {Siegrist, M Sloan and Whiteside, Sarah and Jewett, John C and Aditham, Arjun and Cava, Felipe and Bertozzi, Carolyn R} } @article {1444, title = {Osmosensory signaling in Mycobacterium tuberculosis mediated by a eukaryotic-like Ser/Thr protein kinase.}, journal = {Proc Natl Acad Sci U S A}, volume = {110}, year = {2013}, month = {2013 Dec 24}, pages = {E5069-77}, abstract = {

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.

}, keywords = {Adaptation, Biological, Blotting, Western, Gene Expression Regulation, Bacterial, Green Fluorescent Proteins, Microarray Analysis, Mycobacterium tuberculosis, Osmolar Concentration, Osmotic Pressure, Phosphorylation, Protein Kinases, Signal Transduction}, issn = {1091-6490}, doi = {10.1073/pnas.1321205110}, author = {Hatzios, Stavroula K and Baer, Christina E and Rustad, Tige R and Siegrist, M Sloan and Pang, Jennifer M and Ortega, Corrie and Alber, Tom and Grundner, Christoph and Sherman, David R and Bertozzi, Carolyn R} } @article {1446, title = {Probing the mycobacterial trehalome with bioorthogonal chemistry.}, journal = {J Am Chem Soc}, volume = {134}, year = {2012}, month = {2012 Oct 3}, pages = {16123-6}, abstract = {

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.

}, keywords = {Alkynes, Azides, Fluorescent Dyes, Glycolipids, Mycobacterium, Trehalose}, issn = {1520-5126}, doi = {10.1021/ja3062419}, author = {Swarts, Benjamin M and Holsclaw, Cynthia M and Jewett, John C and Alber, Marina and Fox, Douglas M and Siegrist, M Sloan and Leary, Julie A and Kalscheuer, Rainer and Bertozzi, Carolyn R} } @article {1447, title = {Mycobacterial Esx-3 is required for mycobactin-mediated iron acquisition.}, journal = {Proc Natl Acad Sci U S A}, volume = {106}, year = {2009}, month = {2009 Nov 3}, pages = {18792-7}, abstract = {

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.

}, keywords = {Animals, Bacterial Proteins, Genome, Bacterial, Iron, Macrophages, Mice, Mutation, Mycobacterium, Mycobacterium Infections, Oxazoles, Protein Binding, Secretory Pathway, Siderophores, Transcription, Genetic, Up-Regulation}, issn = {1091-6490}, doi = {10.1073/pnas.0900589106}, author = {Siegrist, M Sloan and Unnikrishnan, Meera and McConnell, Matthew J and Borowsky, Mark and Cheng, Tan-Yun and Siddiqi, Noman and Fortune, Sarah M and Moody, D Branch and Rubin, Eric J} } @article {1448, title = {Phage transposon mutagenesis.}, journal = {Methods Mol Biol}, volume = {465}, year = {2009}, month = {2009}, pages = {311-23}, abstract = {

Phage transduction is an attractive method of genetic manipulation in mycobacteria. PhiMycoMarT7 is well suited for transposon mutagenesis as it is temperature sensitive for replication and contains T7 promoters that promote transcription, a highly active transposase gene, and an Escherichia coli oriR6 K origin of replication. Mycobacterial transposon mutant libraries produced by PhiMycoMarT7 transduction are amenable to both forward and reverse genetic studies. In this protocol, we detail the preparation of PhiMycoMarT7, including a description of the phage, reconstitution of the phage, purification of plaques, preparation of phage stock, and titering of phage stock. We then describe the transduction procedure and finally outline the isolation of individual transposon mutants.

}, keywords = {Bacteriophages, DNA Transposable Elements, Mutagenesis, Mutation, Mycobacterium, Transduction, Genetic}, issn = {1940-6029}, doi = {10.1007/978-1-59745-207-6_21}, author = {Siegrist, M Sloan and Rubin, Eric J} }