The principal objectives of this proposal are to elucidate the pathway used by E. coli for recycling muropeptides, to determine the function recycling serves in the life of the cell and to characterize the role of muropeptides in the beta-lactamase induction process. These objectives follow from our preliminary results which support the following tentative conclusions: (1) AmpG, which is required for beta-lactamase induciton, is a permease required for recycling cell wall components. (2) AmpG most likely transports N-acetylglucosaminyl-beta 1-4, 1,6 anhydro-N-acetylmuramyl-L- alanyl-D-glutamyl-(L)-meso-diaminopimelic acid (G1cNAc-anhMurNAc-L-ala-D- glu-DAP) into the cytoplasm. (3) AmpD, required for beta-lactamase inducibility is also essential for recycling. (4) ampD mutants accumulate huge amounts of anhMurNAc-L-ala-D-glu-DAP indicating that it is an inducing ligand. (5) AmpD is an anhMurNAc-1-ala amidase. Presumably the tripeptide released by the AmpD amidase is added to UDPMurNAc by a yet to be discovered tripeptide-adding enzyme thereby reintroducing L-ala-D-glu- DAP into the biosynthetic pathway. (6) G1cNAc-anhMurNAC-L-ala-D-glu-DAP is probably another inducer of beta-lactamase. Thus, based on these new results of the past 16 months, the specific aims are: 1. To study and characterize the 4 predicted steps in the recycling pathway, namely: AmpG permease, beta-N-acetylglucosaminidase, AmpD amidase, and the hypothetical tripeptide-adding enzyme. 2. To identify the muropeptides involved in beta-lactamase induction and to further characterize their role in beta-lactamase induction. 3. To search for a role of recycling in the life of the cell.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM051610-02
Application #
2415268
Study Section
Special Emphasis Panel (ZRG5-MBC-1 (02))
Project Start
1996-05-01
Project End
1999-04-30
Budget Start
1997-05-01
Budget End
1998-04-30
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Tufts University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02111
Uehara, Tsuyoshi; Park, James T (2008) Growth of Escherichia coli: significance of peptidoglycan degradation during elongation and septation. J Bacteriol 190:3914-22
Uehara, Tsuyoshi; Park, James T (2007) An anhydro-N-acetylmuramyl-L-alanine amidase with broad specificity tethered to the outer membrane of Escherichia coli. J Bacteriol 189:5634-41
Uehara, Tsuyoshi; Suefuji, Kyoko; Jaeger, Tina et al. (2006) MurQ Etherase is required by Escherichia coli in order to metabolize anhydro-N-acetylmuramic acid obtained either from the environment or from its own cell wall. J Bacteriol 188:1660-2
Uehara, Tsuyoshi; Suefuji, Kyoko; Valbuena, Noelia et al. (2005) Recycling of the anhydro-N-acetylmuramic acid derived from cell wall murein involves a two-step conversion to N-acetylglucosamine-phosphate. J Bacteriol 187:3643-9
Uehara, Tsuyoshi; Park, James T (2004) The N-acetyl-D-glucosamine kinase of Escherichia coli and its role in murein recycling. J Bacteriol 186:7273-9
Uehara, Tsuyoshi; Park, James T (2003) Identification of MpaA, an amidase in Escherichia coli that hydrolyzes the gamma-D-glutamyl-meso-diaminopimelate bond in murein peptides. J Bacteriol 185:679-82
Uehara, Tsuyoshi; Park, James T (2002) Role of the murein precursor UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-diaminopimelic acid-D-Ala-D-Ala in repression of beta-lactamase induction in cell division mutants. J Bacteriol 184:4233-9
Cheng, Qiaomei; Park, James T (2002) Substrate specificity of the AmpG permease required for recycling of cell wall anhydro-muropeptides. J Bacteriol 184:6434-6
Park, J T (2001) Identification of a dedicated recycling pathway for anhydro-N-acetylmuramic acid and N-acetylglucosamine derived from Escherichia coli cell wall murein. J Bacteriol 183:3842-7
Cheng, Q; Li, H; Merdek, K et al. (2000) Molecular characterization of the beta-N-acetylglucosaminidase of Escherichia coli and its role in cell wall recycling. J Bacteriol 182:4836-40

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