Office: SCIE 3522
Lab: SCIE 4204
My interest in microbiology began as an undergraduate student at the University of Waterloo. I was pursuing a double honours B.Sc. in chemistry and biology and during the third and fourth years of my program I focused my attention on microbiological systems. My fourth year honours research project concerned a study on the inhibition of ß-lactamase, the enzyme that renders the ß-lactam antibiotics (penicillins, cephalosporins) ineffective and thus representing a major factor of antibiotic resistance. Through this experience, I developed a passion for research in enzymology and so I continued my studies at Waterloo for my doctorate in biochemistry. I was introduced to carbohydrate-active enzymes as a post-doctoral fellow at the Carlsberg Research Centre in Copenhagen, and then as a research associate at the National Research Council in Ottawa. On joining the University of Guelph, I established a research program that integrates biochemical, biophysical, microbiological, and molecular biological techniques to investigate the structure-function relationship of the enzymes that synthesize and lyse the essential bacterial cell wall polymer peptidoglycan and the plant cell wall polymer cellulose, with the major goals of identifying new targets for antibiotic development and the efficient production of cellulosic ethanol, respectively. This multi-disciplinary approach involves collaborations with colleagues at Guelph, across the country, and around the World.
Research in my laboratory concerns investigating the structure and function relationship of enzymes involved in the metabolism of the bacterial cell wall polymer peptidoglycan, and the biodegradation of cellulose. Peptidoglycan is a heteropolymer of both aminosugars and short peptides that completely surrounds bacterial cells as a single macromolecule to provide their rigidity and structure. Its biosynthesis occurs in three stages involving the cytoplasm, the cytoplasmic membrane, and the cell wall. Specific reactions in each of these stages have proven to be effective targets for antibiotic therapy, including the inhibition of peptide crosslinking within the cell wall by the ß-lactam class of drugs (penicillins and celphalosporins). Our studies focus on the events within the cell wall with the aim to identify new potential targets for antibiotic development.
The biodegradation of the plant cell wall polymer cellulose involves the concerted action of three distinct enzymes. One field of biofuel production involves the application of these enzymes in the generation of cellulosic ethanol and other value-added products from waste plant matter. To be economically viable, the efficiency of the cellulolytic enzymes needs to be enhanced and to this end, we combine protein engineering with the biophysical analyses of cellulose degradation.
Both the search for new antibiotic targets and enhancing the production of biofuels provide fascinating challenges for experimental research involving a wide range of experimental approaches that span the biochemistry, biophysics, microbiology, molecular biology, and structural biology.
Current areas of emphasis include:
- Structure and function of the lytic transglycosylases, endogenous ‘autolysins’ that are involved in the biosynthesis and turnover of peptidoglycan, and the insertion of multi-protein complexes through the cell wall, such as flagella, pili, and secretion systems.
- Characterization of the enzyme systems required for the O-acetylation of peptidoglycan in both Gram positive and Gram negative bacteria, using primarily Bacillus anthracis and Neisseria gonorrhoeae as model systems, respectively.
- Structural basis for substrate recognition and inhibition of O-acetylpeptidoglycan esterases by ABC transporters involved in the export of bacterial cell-surface polysaccharides.
- Localization and characterization of peptidoglycan metabolizing enzyme systems within bacterial cell walls.
- Engineering of cellulolytic enzymes to enhance their physico-chemical and catalytic properties.
Wang, J., A. Quirk, J. Lipkowski, J. R. Dutcher, C. Hill, A. Mark, and A. J. Clarke. (2012) Real-time observation of swelling and hydrolysis of a single crystalline cellulose fiber catalyzed by cellulase 7B from Trichoderma reesei. Langmuir 28: 9664-9672.
Pfeffer, J. M., and A. J. Clarke (2012) Identification of first-known inhibitors of O-acetylpeptidoglycan esterase: A potential new antibacterial target. ChemBioChem. 13: 722-731
Laaberki, M.-H., J. Pfeffer, A. J. Clarke, and J. Dworkin (2011) O-Acetylation of peptidoglycan is required for proper cell separation and S-layer anchoring in Bacillus anthracis. J. Biol. Chem. 286: 5278-5288.
Clarke, C.A., E. M. Scheurwater and A. J. Clarke (2010) The vertebrate lysozyme inhibitor Ivy functions to inhibit the activity of lytic transglycosylase . J. Biol. Chem. 285: 14843-14847.
Moynihan, P. J., and A. J. Clarke (2010) O-Acetylation of peptidoglycan in Gram-negative bacteria: Identification and characterization of peptidoglycan O-acetyltransferase in Neisseria gonorrhoeae. J. Biol. Chem. 285: 13264-13273.
Ruiloba de León, S., K. Daniels, and A. J. Clarke (2010) Production and purification of the penicillin-binding protein 3 from Pseudomonas aeruginosa. Prot. Express. Purif. 73: 177-183.
Quirk, A., Lipkowski, J., Vandenende, C., Cockburn, D., Clarke, A. J., Dutcher, J., Roscoe, S.G. (2010) Direct visualization of enzymatic digestion of a single fiber of native cellulose in aqueous environment by atomic force microscopy. Langmuir 26: 5007-5013.
Cockburn, D. W., Vandenende, C., and A. J. Clarke (2010) Modulating the pH-activity profile of cellulase by substitution: replacing the general base catalyst aspartate with cysteinesulfinate in cellulase A from Cellulomonas fimi. Biochemistry 49: 2042-2050.
Jing, H., D. Cockburn, and A.J. Clarke (2009) Production and purification of the isolated family 2a carbohydrate binding module from Cellulomonas fimi. Protein Expr. Purif. 64: 63-68.
Legaree, B.A., and A.J. Clarke (2008) Interaction of penicillin-binding protein 2 with soluble
lytic transglycosylase B1 in Pseudomonas aeruginosa. J. Bacteriol. 190: 6922-6926.
Scheurwater, E.M., and A.J. Clarke (2008) The C-terminal domain of Escherichia coli YfhD functions as a lytic transglycosylase. J. Biol. Chem. 283: 8363-8373.
Reid, C.W., B.A. Legaree, and A.J. Clarke (2007) Role of Ser216 in the mechanism of action of membrane-bound lyti transglycosyalse B: Further evidence for substrate-assisted catalysis. FEBS Lett. 581: 4988-4992.
Legaree, B.A., C. Adams, and A.J. Clarke (2007) Overproduction of penicillin-binding protein 2 and its inactive variants causes morphological changes and lysis in Escherichia coli. J. Bacteriol. 189: 4975-4983.
Weadge, J.T. and A.J. Clarke (2007) Neisseria gonorrhoeae O-acetylpeptidoglycan esterase, a serine esterase with a Ser-His-Asp catalytic triad. Biochemistry 46: 4932-4941.
Legaree, B.A, K.E. Daniels, J.T. Weadge, D.Cockburn, and A.J. Clarke (2007) Function of penicillin-binding protein 2 in viability and morphology of Pseudomonas aeruginosa. J. Antimicrob. Chemother. 59: 411-424.
Reid, C. W., N. T. Blackburn, and A. J. Clarke (2006) Role of arginine residues in the active site
of membrane-bound lytic transglycosylase B from Pseudomonas aeruginosa. Biochemistry,
Pfeffer, J.M., H. Strating, J.T. Weadge, and A.J. Clarke (2006) Peptidoglcyan O-acetylation and autolysin profile of Enterococcus faecalis in the viable but non-culturable state. J. Bacteriol. 188:902-908.
Weadge, J.T., and A.J. Clarke (2006) Identification and characterization of O-acetylpeptidoglycan esterase: a novel enzyme discovered in Neisseria gonorrhoeae. Biochemistry 45:839-851
Weadge, J.T., J.M. Pfeffer, and A.J. Clarke (2005) Identification of a new family of enzymes with potential O-acetylpeptidoglycan esterase activity in both Gram-positive and Gram- negative bacteria. BMC Microbiology 5: 49.
Reid, C.W., N.T. Blackburn, B.A. Legaree, F.-I. Auzanneau, and A.J. Clarke (2004) Inhibition of membrane-bound lytic transglysosylase by NAG-thiazoline. FEBS Letters 574: 73-79.
Reid, C.W., D. Brewer, and A.J. Clarke (2004) Substrate binding affinity of Pseudomonas aeruginosa membrane-bound lytic transglycosylase B by hydrogen-deuterium exchange MALDI-MS. Biochemistry 43: 11275-11282.
MICR*2030 Microbial Growth
Chris Vandenende - Technician
Herlihey, Frankie (M.Sc.)
Holland, Katie (M.Sc.)
Mark, Adam (Ph.D.)
Moynihan, Patrick (Ph.D.)
Pfeffer, John (Ph.D.)
Selander, Iris (M.Sc.)
Sychantha, Dave (M.Sc.)
Witney, Rebecca (M.Sc.)
Zia, Asad (M.Sc.)