- Email: [email protected]
Catalysing camouflage
COMMENT
teleologically, why the system is so well preserved in different branches of the bacterial taxons, and why conductive characteristics of the channels are so similar in remote species11.
Sergei Sukharev Dept of Biology, University of Maryland, College Park, MD 20742, USA References 1 Blount, P. and Moe, P.C. (1999) Bacterial mechanosensitive channels: integrating physiology, structure and function. Trends Microbiol. 7, 420–424 2 Martinac, B. et al. (1987) Pressuresensitive ion channel in Escherichia coli. Proc. Natl. Acad. Sci, U. S. A. 84, 2297–2301
3 Zoratti, M. and Szabo, I. (1991) Stretchactivated composite channels in Bacillus subtilis. Biochim. Biophys. Res. Commun. 168, 443–450 4 Sukharev, S.I. et al. (1993) Two types of mechanosensitive channels in the Escherichia coli cell envelope: solubilization and functional reconstitution. Biophys. J. 65, 177–183 5 Sukharev, S.I. et al. (1994) A largeconductance mechanosensitive channel in E. coli encoded by mscL alone. Nature 368, 265–268 6 Levina, N. et al. (1999) Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identification of genes required for MscS activity. EMBO J. 18, 1730–1737 7 Sukharev, S.I. et al. (1999) Energetic and spatial parameters for gating of the bacterial large conductance
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mechanosensitive channel, MscL. J. Gen. Physiol. 113, 525–540 Chang, G. et al. (1998) Structure of the MscL homolog from Mycobacterium tuberculosis: a gated mechanosensitive ion channel. Science 282, 2220–2226 Berrier, C. et al. (1996) Multiple mechanosensitive ion channels from Escherichia coli, activated at different thresholds of applied pressure. J. Membrane Biol. 151, 175–187 Lenski, R.E. and Travisano, M. (1994) Dynamics of adaptation and diversification: a 10 000-generation experiment with bacterial populations. Proc. Natl. Acad. Sci, U. S. A. 91, 6808–6814 Moe, P.C. et al. (1998) Functional and structural conservation in the mechanosensitive channel MscL implicates elements crucial for mechanosensation. Mol. Microbiol. 28, 583–592
Horizons Catalysing camouflage
G
ram-positive bacteria decorate their surfaces with proteins in order to evade a variety of host immune factors, as well as to assist in establishing infection. These surface proteins are anchored to the cell wall by a mechanism requiring a carboxyl-terminal sorting signal with a conserved LPXTG motif (where X is any amino acid). The sorting signal is cleaved between the Thr and Gly, and is subsequently amide-linked to the pentaglycine crossbridge of the peptidoglycan. The sorting of proteins to the cell wall has been extensively examined; however, until recently, the identity of the enzymatic activity associated with this process has remained elusive. Now, Schneewind and colleagues1 have used chemical mutagenesis to generate mutants of Staphylococcus aureus defective in anchoring surface proteins to the cell wall. Using genetic complementation to rescue mutations in sorting, the srtA gene (sortase) was identified. Overexpression of srtA rescues surfaceprotein anchoring in these mutants; it also increases sorting in a wild-type strain. Recombinant sortase catalyses the hydrolysis and hydroxylaminolysis of peptides containing the LPXTG motif2,3.
Analysis of enzyme activity in the presence of specific inhibitors confirms that sortase is a sulfhydrylcontaining enzyme that utilizes peptidoglycan precursors, but not an assembled cell wall, as a substrate for the anchoring of proteins. Mutation of the conserved cysteine of sortase abolished the enzyme activity. Thus, sortase catalyses the anchoring of surface proteins by a transpeptidation reaction that utilizes thioester enzyme intermediates. The identification of sortase as well as studies on inhibitors of this enzyme should allow the development of new therapies for infections caused by Gram-positive bacteria.
1 Mazmanian, S.K. et al. (1999) Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Science 285, 760–763 2 Ton-That, H. et al. (1999) Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif. Proc. Natl. Acad. Sci. U. S. A. 96, 12424–12429 3 Ton-That, H. and Schneewind, O. (1999) Anchor structure of staphylococcal surface proteins: inhibitors of the cell wall sorting reaction. J. Biol. Chem. 274, 24316–24320
Kirkwood M. Land [email protected]
In the other Trends journals A selection of recently published articles of interest to TIM readers. • Microbial genomes- the untapped resource, by D.A. Cowan – Trends in Biotechnology, 18, 14–16 • MIcrobial Biotechnology, by A. Demain – Trends in Biotechnology, 18, 26–31 • No longer an exclusive club: eukaryotic signalling domains in bacteria, by C.J. Bakal and J.E. Davies – Trends in Cell Biology, 10, 32–38 • Immunopathology of schistosomiasis: a cautionary tale of mice and men, by P.G. Fallon – Immunology Today, 21, 29–35
0966-842X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. TRENDS
IN
MICROBIOLOGY
13
VOL. 8
NO. 1
JANUARY 2000
teleologically, why the system is so well preserved in different branches of the bacterial taxons, and why conductive characteristics of the channels are so similar in remote species11.
Sergei Sukharev Dept of Biology, University of Maryland, College Park, MD 20742, USA References 1 Blount, P. and Moe, P.C. (1999) Bacterial mechanosensitive channels: integrating physiology, structure and function. Trends Microbiol. 7, 420–424 2 Martinac, B. et al. (1987) Pressuresensitive ion channel in Escherichia coli. Proc. Natl. Acad. Sci, U. S. A. 84, 2297–2301
3 Zoratti, M. and Szabo, I. (1991) Stretchactivated composite channels in Bacillus subtilis. Biochim. Biophys. Res. Commun. 168, 443–450 4 Sukharev, S.I. et al. (1993) Two types of mechanosensitive channels in the Escherichia coli cell envelope: solubilization and functional reconstitution. Biophys. J. 65, 177–183 5 Sukharev, S.I. et al. (1994) A largeconductance mechanosensitive channel in E. coli encoded by mscL alone. Nature 368, 265–268 6 Levina, N. et al. (1999) Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identification of genes required for MscS activity. EMBO J. 18, 1730–1737 7 Sukharev, S.I. et al. (1999) Energetic and spatial parameters for gating of the bacterial large conductance
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9
10
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mechanosensitive channel, MscL. J. Gen. Physiol. 113, 525–540 Chang, G. et al. (1998) Structure of the MscL homolog from Mycobacterium tuberculosis: a gated mechanosensitive ion channel. Science 282, 2220–2226 Berrier, C. et al. (1996) Multiple mechanosensitive ion channels from Escherichia coli, activated at different thresholds of applied pressure. J. Membrane Biol. 151, 175–187 Lenski, R.E. and Travisano, M. (1994) Dynamics of adaptation and diversification: a 10 000-generation experiment with bacterial populations. Proc. Natl. Acad. Sci, U. S. A. 91, 6808–6814 Moe, P.C. et al. (1998) Functional and structural conservation in the mechanosensitive channel MscL implicates elements crucial for mechanosensation. Mol. Microbiol. 28, 583–592
Horizons Catalysing camouflage
G
ram-positive bacteria decorate their surfaces with proteins in order to evade a variety of host immune factors, as well as to assist in establishing infection. These surface proteins are anchored to the cell wall by a mechanism requiring a carboxyl-terminal sorting signal with a conserved LPXTG motif (where X is any amino acid). The sorting signal is cleaved between the Thr and Gly, and is subsequently amide-linked to the pentaglycine crossbridge of the peptidoglycan. The sorting of proteins to the cell wall has been extensively examined; however, until recently, the identity of the enzymatic activity associated with this process has remained elusive. Now, Schneewind and colleagues1 have used chemical mutagenesis to generate mutants of Staphylococcus aureus defective in anchoring surface proteins to the cell wall. Using genetic complementation to rescue mutations in sorting, the srtA gene (sortase) was identified. Overexpression of srtA rescues surfaceprotein anchoring in these mutants; it also increases sorting in a wild-type strain. Recombinant sortase catalyses the hydrolysis and hydroxylaminolysis of peptides containing the LPXTG motif2,3.
Analysis of enzyme activity in the presence of specific inhibitors confirms that sortase is a sulfhydrylcontaining enzyme that utilizes peptidoglycan precursors, but not an assembled cell wall, as a substrate for the anchoring of proteins. Mutation of the conserved cysteine of sortase abolished the enzyme activity. Thus, sortase catalyses the anchoring of surface proteins by a transpeptidation reaction that utilizes thioester enzyme intermediates. The identification of sortase as well as studies on inhibitors of this enzyme should allow the development of new therapies for infections caused by Gram-positive bacteria.
1 Mazmanian, S.K. et al. (1999) Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Science 285, 760–763 2 Ton-That, H. et al. (1999) Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif. Proc. Natl. Acad. Sci. U. S. A. 96, 12424–12429 3 Ton-That, H. and Schneewind, O. (1999) Anchor structure of staphylococcal surface proteins: inhibitors of the cell wall sorting reaction. J. Biol. Chem. 274, 24316–24320
Kirkwood M. Land [email protected]
In the other Trends journals A selection of recently published articles of interest to TIM readers. • Microbial genomes- the untapped resource, by D.A. Cowan – Trends in Biotechnology, 18, 14–16 • MIcrobial Biotechnology, by A. Demain – Trends in Biotechnology, 18, 26–31 • No longer an exclusive club: eukaryotic signalling domains in bacteria, by C.J. Bakal and J.E. Davies – Trends in Cell Biology, 10, 32–38 • Immunopathology of schistosomiasis: a cautionary tale of mice and men, by P.G. Fallon – Immunology Today, 21, 29–35
0966-842X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. TRENDS
IN
MICROBIOLOGY
13
VOL. 8
NO. 1
JANUARY 2000