Techniques for metabolic labeling of the bacterial cell surface. Methods are organized by the cell envelope layer containing the target biomolecule and subdivided by the chemical class of the probe. Route indicates whether the probe is incorporated via a cytosolic pathway (internal) or via biosynthetic pathways outside the plasma membrane (external). Target indicates the biomolecule into which the probe is incorporated. Labeling strategy indicates whether the label is attached before or after probe incorporation into the target. Probe source indicates if probe is available commercially (COMM), or must be made by multi-step organic synthesis (MSOS) or solid-phase peptide synthesis (SPPS). Abbreviations: AAA, artificial amino acid; AEK, alanine-glutamate-lysine tripeptide; Alk, alkynyl chemical reporter; Az, azido chemical reporter; FITC, fluorescein fluorophore; FucAl, 6-alkynyl fucose; GlcNAc, N-acetyl glucosamine; KDO, 3-deoxy-D-manno-octulosonic acid; LPS, lipopolysaccharide; NAG, N-acetyl gluosamine; NAM, N-acetyl muramic acid; NBD, nitrobenzofurazan fluorophore; NHS, N-hydroxy succinimide ester; T3SS, type 3 secretion system; TAMRA, carboxy tetramethylrhodamine fluorophore; TMM, trehalose monomycolate; * - likely found in the plasma membrane (Bansal-Mutalik and Nikaido 2014); TDM, trehalose dimycolate; PG, peptidoglycan; PP, peptidoglycan pentapeptide.
| Labeled . | . | . | . | Labeling . | . | . |
|---|---|---|---|---|---|---|
| metabolite . | Route . | Target . | Probe . | strategy . | Probe source . | Selected references . |
| Peptidoglycan . | ||||||
| Amino acid, chemical handle | External, internal | PG stem peptide | D-cysteine | Post | COMM | de Pedro et al. (1997) |
| External, internal | PG stem peptide | Alk/Az-D-ala | Post | COMM | Kuru et al. (2012), Siegrist et al. (2013) | |
| External, Internal | PG stem peptide | D-ala D-ala with Alk or Az at N or C terminus | Post | COMM, MSOS (3–6 steps) | Liechti et al. (2013) | |
| External | PG stem peptide | Az-LPETG peptide | Post | SPPS | Nelson et al. (2010) | |
| Amino acid, fluorescent | External | PG stem peptide | FITC-LPETG peptide | Pre | SPPS | Nelson et al. (2010) |
| External, Internal | PG stem peptide | NBD-AEK | Pre | SPPS | Olrichs et al. (2011) | |
| External, Internal | PG stem peptide | NBD-D-ala, Coumarin-D-ala, FITC-D-lys, TAMRA-D-lys | Pre | MSOS (1–4 steps) | Kuru et al. (2012) | |
| External | PG stem peptide | FITC-D-lys carboxamide | Pre | MSOS (1–4 steps) | Lebar et al. (2014) | |
| Outer membrane and mycomembrane | ||||||
| Glycan, chemical handle | External, Internal | Fucose-containing bacterial polysaccharides | C6 modified fucoses | Post | MSOS | Yi et al. (2009) |
| Internal | E. coli LPS inner core | 8-azido-8-deoxy-KDO | Post | MSOS | Dumont et al. (2012) | |
| Internal | Fucosylated glycoproteins | Alk-Fuc | Post | MSOS | Besanceney-Webler et al. (2011) | |
| Internal | H. pylori glycoproteins | Ac4GlcNAz | Post | COMM | Koenigs, Richardson and Dube (2009) | |
| Internal | C. jejuni flaggelin | Per-acetylated Az-pseudaminic acid | Post | MSOS | Liu et al. (2009) | |
| External, internal | TMM*, TDM | Az-trehalose | Post | MSOS | Swarts et al. (2012), Urbanek et al. (2014) | |
| Glycan, fluorescent | External | TMM*, TDM | FITC-trehalose | Pre | MSOS | Backus et al. (2011) |
| Artificial amino acid, chemical handle | Internal | Protein, not site-specific | Met replaced by Az-alanine, Az-homoalanine, Az-norvaline, Az-norleucine | Post | COMM, MSOS | Link and Tirrell (2003), Link, Vink and Tirrell (2004) |
| Internal | Protein, site-specific | Tyr replaced by m- or p-acetyl-L-phenylalanine | Post | MSOS | Zhang et al. (2003) | |
| Internal | Protein, site-specific | Met replaced by Az-norleucine AAA | Post | COMM | Link et al. (2006), Tanrikulu et al. (2009) | |
| Internal | Protein, site-specific | Several Alk/Az AAAs as Met surrogates or UAG suppressors | Post | COMM, MSOS | Ngo and Tirrell (2011) | |
| Secretion system components and substrates | ||||||
| Artificial amino acid, cross-linking | Internal | Sec system | SecY–p-benzoyl-phenylalanine AAA | Cross-link | COMM | Mori and Ito (2006) |
| Internal | Translocating polysaccharides | Wza–p-benzoyl-phenylalanine AAA | Cross-link | COMM | Nickerson et al. (2014) | |
| Internal | T3SS secreted proteins | Met replaced by Azidonorleucine AAA | Post (biotin) | COMM | Mahdavi et al. (2014) | |
| Lipid, chemical handle | Internal | Legionella effector proteins | Alkynyl-farnesol-1 | Post | MSOS (3 steps) | Ivanov et al. (2010) |
| Internal | Salmonella T3SS effector proteins | Alkynyl-16 (palmitate analog) | Post | MSOS (6 steps) | Hicks et al. (2011) | |
| Labeled . | . | . | . | Labeling . | . | . |
|---|---|---|---|---|---|---|
| metabolite . | Route . | Target . | Probe . | strategy . | Probe source . | Selected references . |
| Peptidoglycan . | ||||||
| Amino acid, chemical handle | External, internal | PG stem peptide | D-cysteine | Post | COMM | de Pedro et al. (1997) |
| External, internal | PG stem peptide | Alk/Az-D-ala | Post | COMM | Kuru et al. (2012), Siegrist et al. (2013) | |
| External, Internal | PG stem peptide | D-ala D-ala with Alk or Az at N or C terminus | Post | COMM, MSOS (3–6 steps) | Liechti et al. (2013) | |
| External | PG stem peptide | Az-LPETG peptide | Post | SPPS | Nelson et al. (2010) | |
| Amino acid, fluorescent | External | PG stem peptide | FITC-LPETG peptide | Pre | SPPS | Nelson et al. (2010) |
| External, Internal | PG stem peptide | NBD-AEK | Pre | SPPS | Olrichs et al. (2011) | |
| External, Internal | PG stem peptide | NBD-D-ala, Coumarin-D-ala, FITC-D-lys, TAMRA-D-lys | Pre | MSOS (1–4 steps) | Kuru et al. (2012) | |
| External | PG stem peptide | FITC-D-lys carboxamide | Pre | MSOS (1–4 steps) | Lebar et al. (2014) | |
| Outer membrane and mycomembrane | ||||||
| Glycan, chemical handle | External, Internal | Fucose-containing bacterial polysaccharides | C6 modified fucoses | Post | MSOS | Yi et al. (2009) |
| Internal | E. coli LPS inner core | 8-azido-8-deoxy-KDO | Post | MSOS | Dumont et al. (2012) | |
| Internal | Fucosylated glycoproteins | Alk-Fuc | Post | MSOS | Besanceney-Webler et al. (2011) | |
| Internal | H. pylori glycoproteins | Ac4GlcNAz | Post | COMM | Koenigs, Richardson and Dube (2009) | |
| Internal | C. jejuni flaggelin | Per-acetylated Az-pseudaminic acid | Post | MSOS | Liu et al. (2009) | |
| External, internal | TMM*, TDM | Az-trehalose | Post | MSOS | Swarts et al. (2012), Urbanek et al. (2014) | |
| Glycan, fluorescent | External | TMM*, TDM | FITC-trehalose | Pre | MSOS | Backus et al. (2011) |
| Artificial amino acid, chemical handle | Internal | Protein, not site-specific | Met replaced by Az-alanine, Az-homoalanine, Az-norvaline, Az-norleucine | Post | COMM, MSOS | Link and Tirrell (2003), Link, Vink and Tirrell (2004) |
| Internal | Protein, site-specific | Tyr replaced by m- or p-acetyl-L-phenylalanine | Post | MSOS | Zhang et al. (2003) | |
| Internal | Protein, site-specific | Met replaced by Az-norleucine AAA | Post | COMM | Link et al. (2006), Tanrikulu et al. (2009) | |
| Internal | Protein, site-specific | Several Alk/Az AAAs as Met surrogates or UAG suppressors | Post | COMM, MSOS | Ngo and Tirrell (2011) | |
| Secretion system components and substrates | ||||||
| Artificial amino acid, cross-linking | Internal | Sec system | SecY–p-benzoyl-phenylalanine AAA | Cross-link | COMM | Mori and Ito (2006) |
| Internal | Translocating polysaccharides | Wza–p-benzoyl-phenylalanine AAA | Cross-link | COMM | Nickerson et al. (2014) | |
| Internal | T3SS secreted proteins | Met replaced by Azidonorleucine AAA | Post (biotin) | COMM | Mahdavi et al. (2014) | |
| Lipid, chemical handle | Internal | Legionella effector proteins | Alkynyl-farnesol-1 | Post | MSOS (3 steps) | Ivanov et al. (2010) |
| Internal | Salmonella T3SS effector proteins | Alkynyl-16 (palmitate analog) | Post | MSOS (6 steps) | Hicks et al. (2011) | |
Techniques for metabolic labeling of the bacterial cell surface. Methods are organized by the cell envelope layer containing the target biomolecule and subdivided by the chemical class of the probe. Route indicates whether the probe is incorporated via a cytosolic pathway (internal) or via biosynthetic pathways outside the plasma membrane (external). Target indicates the biomolecule into which the probe is incorporated. Labeling strategy indicates whether the label is attached before or after probe incorporation into the target. Probe source indicates if probe is available commercially (COMM), or must be made by multi-step organic synthesis (MSOS) or solid-phase peptide synthesis (SPPS). Abbreviations: AAA, artificial amino acid; AEK, alanine-glutamate-lysine tripeptide; Alk, alkynyl chemical reporter; Az, azido chemical reporter; FITC, fluorescein fluorophore; FucAl, 6-alkynyl fucose; GlcNAc, N-acetyl glucosamine; KDO, 3-deoxy-D-manno-octulosonic acid; LPS, lipopolysaccharide; NAG, N-acetyl gluosamine; NAM, N-acetyl muramic acid; NBD, nitrobenzofurazan fluorophore; NHS, N-hydroxy succinimide ester; T3SS, type 3 secretion system; TAMRA, carboxy tetramethylrhodamine fluorophore; TMM, trehalose monomycolate; * - likely found in the plasma membrane (Bansal-Mutalik and Nikaido 2014); TDM, trehalose dimycolate; PG, peptidoglycan; PP, peptidoglycan pentapeptide.
| Labeled . | . | . | . | Labeling . | . | . |
|---|---|---|---|---|---|---|
| metabolite . | Route . | Target . | Probe . | strategy . | Probe source . | Selected references . |
| Peptidoglycan . | ||||||
| Amino acid, chemical handle | External, internal | PG stem peptide | D-cysteine | Post | COMM | de Pedro et al. (1997) |
| External, internal | PG stem peptide | Alk/Az-D-ala | Post | COMM | Kuru et al. (2012), Siegrist et al. (2013) | |
| External, Internal | PG stem peptide | D-ala D-ala with Alk or Az at N or C terminus | Post | COMM, MSOS (3–6 steps) | Liechti et al. (2013) | |
| External | PG stem peptide | Az-LPETG peptide | Post | SPPS | Nelson et al. (2010) | |
| Amino acid, fluorescent | External | PG stem peptide | FITC-LPETG peptide | Pre | SPPS | Nelson et al. (2010) |
| External, Internal | PG stem peptide | NBD-AEK | Pre | SPPS | Olrichs et al. (2011) | |
| External, Internal | PG stem peptide | NBD-D-ala, Coumarin-D-ala, FITC-D-lys, TAMRA-D-lys | Pre | MSOS (1–4 steps) | Kuru et al. (2012) | |
| External | PG stem peptide | FITC-D-lys carboxamide | Pre | MSOS (1–4 steps) | Lebar et al. (2014) | |
| Outer membrane and mycomembrane | ||||||
| Glycan, chemical handle | External, Internal | Fucose-containing bacterial polysaccharides | C6 modified fucoses | Post | MSOS | Yi et al. (2009) |
| Internal | E. coli LPS inner core | 8-azido-8-deoxy-KDO | Post | MSOS | Dumont et al. (2012) | |
| Internal | Fucosylated glycoproteins | Alk-Fuc | Post | MSOS | Besanceney-Webler et al. (2011) | |
| Internal | H. pylori glycoproteins | Ac4GlcNAz | Post | COMM | Koenigs, Richardson and Dube (2009) | |
| Internal | C. jejuni flaggelin | Per-acetylated Az-pseudaminic acid | Post | MSOS | Liu et al. (2009) | |
| External, internal | TMM*, TDM | Az-trehalose | Post | MSOS | Swarts et al. (2012), Urbanek et al. (2014) | |
| Glycan, fluorescent | External | TMM*, TDM | FITC-trehalose | Pre | MSOS | Backus et al. (2011) |
| Artificial amino acid, chemical handle | Internal | Protein, not site-specific | Met replaced by Az-alanine, Az-homoalanine, Az-norvaline, Az-norleucine | Post | COMM, MSOS | Link and Tirrell (2003), Link, Vink and Tirrell (2004) |
| Internal | Protein, site-specific | Tyr replaced by m- or p-acetyl-L-phenylalanine | Post | MSOS | Zhang et al. (2003) | |
| Internal | Protein, site-specific | Met replaced by Az-norleucine AAA | Post | COMM | Link et al. (2006), Tanrikulu et al. (2009) | |
| Internal | Protein, site-specific | Several Alk/Az AAAs as Met surrogates or UAG suppressors | Post | COMM, MSOS | Ngo and Tirrell (2011) | |
| Secretion system components and substrates | ||||||
| Artificial amino acid, cross-linking | Internal | Sec system | SecY–p-benzoyl-phenylalanine AAA | Cross-link | COMM | Mori and Ito (2006) |
| Internal | Translocating polysaccharides | Wza–p-benzoyl-phenylalanine AAA | Cross-link | COMM | Nickerson et al. (2014) | |
| Internal | T3SS secreted proteins | Met replaced by Azidonorleucine AAA | Post (biotin) | COMM | Mahdavi et al. (2014) | |
| Lipid, chemical handle | Internal | Legionella effector proteins | Alkynyl-farnesol-1 | Post | MSOS (3 steps) | Ivanov et al. (2010) |
| Internal | Salmonella T3SS effector proteins | Alkynyl-16 (palmitate analog) | Post | MSOS (6 steps) | Hicks et al. (2011) | |
| Labeled . | . | . | . | Labeling . | . | . |
|---|---|---|---|---|---|---|
| metabolite . | Route . | Target . | Probe . | strategy . | Probe source . | Selected references . |
| Peptidoglycan . | ||||||
| Amino acid, chemical handle | External, internal | PG stem peptide | D-cysteine | Post | COMM | de Pedro et al. (1997) |
| External, internal | PG stem peptide | Alk/Az-D-ala | Post | COMM | Kuru et al. (2012), Siegrist et al. (2013) | |
| External, Internal | PG stem peptide | D-ala D-ala with Alk or Az at N or C terminus | Post | COMM, MSOS (3–6 steps) | Liechti et al. (2013) | |
| External | PG stem peptide | Az-LPETG peptide | Post | SPPS | Nelson et al. (2010) | |
| Amino acid, fluorescent | External | PG stem peptide | FITC-LPETG peptide | Pre | SPPS | Nelson et al. (2010) |
| External, Internal | PG stem peptide | NBD-AEK | Pre | SPPS | Olrichs et al. (2011) | |
| External, Internal | PG stem peptide | NBD-D-ala, Coumarin-D-ala, FITC-D-lys, TAMRA-D-lys | Pre | MSOS (1–4 steps) | Kuru et al. (2012) | |
| External | PG stem peptide | FITC-D-lys carboxamide | Pre | MSOS (1–4 steps) | Lebar et al. (2014) | |
| Outer membrane and mycomembrane | ||||||
| Glycan, chemical handle | External, Internal | Fucose-containing bacterial polysaccharides | C6 modified fucoses | Post | MSOS | Yi et al. (2009) |
| Internal | E. coli LPS inner core | 8-azido-8-deoxy-KDO | Post | MSOS | Dumont et al. (2012) | |
| Internal | Fucosylated glycoproteins | Alk-Fuc | Post | MSOS | Besanceney-Webler et al. (2011) | |
| Internal | H. pylori glycoproteins | Ac4GlcNAz | Post | COMM | Koenigs, Richardson and Dube (2009) | |
| Internal | C. jejuni flaggelin | Per-acetylated Az-pseudaminic acid | Post | MSOS | Liu et al. (2009) | |
| External, internal | TMM*, TDM | Az-trehalose | Post | MSOS | Swarts et al. (2012), Urbanek et al. (2014) | |
| Glycan, fluorescent | External | TMM*, TDM | FITC-trehalose | Pre | MSOS | Backus et al. (2011) |
| Artificial amino acid, chemical handle | Internal | Protein, not site-specific | Met replaced by Az-alanine, Az-homoalanine, Az-norvaline, Az-norleucine | Post | COMM, MSOS | Link and Tirrell (2003), Link, Vink and Tirrell (2004) |
| Internal | Protein, site-specific | Tyr replaced by m- or p-acetyl-L-phenylalanine | Post | MSOS | Zhang et al. (2003) | |
| Internal | Protein, site-specific | Met replaced by Az-norleucine AAA | Post | COMM | Link et al. (2006), Tanrikulu et al. (2009) | |
| Internal | Protein, site-specific | Several Alk/Az AAAs as Met surrogates or UAG suppressors | Post | COMM, MSOS | Ngo and Tirrell (2011) | |
| Secretion system components and substrates | ||||||
| Artificial amino acid, cross-linking | Internal | Sec system | SecY–p-benzoyl-phenylalanine AAA | Cross-link | COMM | Mori and Ito (2006) |
| Internal | Translocating polysaccharides | Wza–p-benzoyl-phenylalanine AAA | Cross-link | COMM | Nickerson et al. (2014) | |
| Internal | T3SS secreted proteins | Met replaced by Azidonorleucine AAA | Post (biotin) | COMM | Mahdavi et al. (2014) | |
| Lipid, chemical handle | Internal | Legionella effector proteins | Alkynyl-farnesol-1 | Post | MSOS (3 steps) | Ivanov et al. (2010) |
| Internal | Salmonella T3SS effector proteins | Alkynyl-16 (palmitate analog) | Post | MSOS (6 steps) | Hicks et al. (2011) | |
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