@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 {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 {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 {597, title = {Application of the 5{\textquoteright} fluorogenic exonuclease assay (TaqMan) for quantitative ribosomal DNA and rRNA analysis in sediments.}, journal = {Appl Environ Microbiol}, volume = {67}, year = {2001}, month = {2001 Jun}, pages = {2781-9}, abstract = {In this study, we report on the development of quantitative PCR and reverse transcriptase PCR assays for the 16S rRNA of Geobacter spp. and identify key issues related to fluorogenic reporter systems for nucleic acid analyses of sediments. The lower detection limit of each assay was 5 to 50 fg of genomic DNA or < or =2 pg of 16S rRNA. TaqMan PCR spectral traces from uncontaminated, amended aquifer sediments were significantly lower (P < 0.0002) than traces for the external standard curve. We also observed a similar, significant decrease in mean quencher emissions for undiluted extracts relative to those for diluted extracts (P < 0.0001). If PCR enumerations were based solely upon the undiluted sample eluant, the TaqMan assay generated an inaccurate result even though the threshold cycle (C(t)) measurements were precise and reproducible in the sediment extracts. Assay accuracy was significantly improved by employing a system of replicate dilutions and replicate analyses for both DNA and rRNA quantitation. Our results clearly demonstrate that fluorescence quenching and autofluorescence can significantly affect TaqMan PCR enumeration accuracy, with subsequent implications for the design and implementation of TaqMan PCR to sediments and related environmental samples.}, keywords = {Deltaproteobacteria, DNA, Ribosomal, Fluorescent Dyes, Geologic Sediments, Polymerase Chain Reaction, Reproducibility of Results, Reverse Transcriptase Polymerase Chain Reaction, RNA, Ribosomal, 16S, Sensitivity and Specificity, Taq Polymerase}, issn = {0099-2240}, doi = {10.1128/AEM.67.6.2781-2789.2001}, author = {Stults, J R and Snoeyenbos-West, O and Methe, B and Lovley, D R and Chandler, D P} }