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You searched for subject:(L D Transpeptidase). One record found.

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University of Rochester

1. Sanders, Akeisha Nichole. Genetic and phenotypic analysis of bacterial L,D-transpeptidases.

Degree: PhD, 2014, University of Rochester

L,D-transpeptidases (Ldts) are peptidoglycan (PG) synthesizing enzymes that bypass the classical penicillin-sensitive transpeptidation pathway by making 3-3 crosslinkages. Until recently, there has been little physiological data on the role of 3-3 crosslinks in cell envelope biology. However, organisms with complex cell walls, such as M. tuberculosis, use L,D transpeptidation as the predominant form of cross-linking, indicating their importance. In E. coli and other gram-negative bacteria, some of these enzymes crosslink Braun’s lipoprotein to the PG and incorporate non-canonical D-amino acids (NCDAA) into the PG, suggesting additional roles for these proteins in cell envelope stability. In this study, we constructed and characterized Ldt mutants in E. coli and M. smegmatis, as a model organism for the pathogenic M. tuberculosis, to investigate the physiological role these enzymes play in cell envelope biology. Of the five LDts in E. coli, we show that loss of the Ldts involved with coupling the PG to Braun's lipoprotein resulted in the loss of OM stability. The triple mutant strain was sensitive to the metal chelating agent EDTA, leaked periplasmic proteins, and was resistant to the toxic effects of NCDAA. These data are consistent with a role for these enzymes in coupling the PG to the OM. Loss of the Ldt enzymes involved with 3-3 crosslinkages had no discernable effect on the cell envelope. However, we detected an effect on cell growth only in the quintuple mutant bearing a DAP auxotrophic mutation. The genomes of M. tuberculosis and M. smegmatis encode at least five Ldts. We compared the translated sequences of the Ldt genes in 18 mycobacterial genomes and grouped them into five classes. Of the single mutants, only one, ldtC (Class 1), displayed an increased susceptibility to imipenem, which targets the Ldt enzymes. A triple mutant missing ldtC and the Class 2 genes exhibited a hypersusceptibility to imipenem, ampicillin, lysozyme, and NCDAA. Consistent with previous findings, we also show that the catalytic activity of LdtC was dependent upon an invariant cysteine in the active site. This data suggests that the enzymes represented by Class 1 and 2 may be key players in mycobacterial peptidoglycan physiology and that the phenotypic differences displayed by the mutant strains may reflect differences in function of the mycobacterial Ldts classes. In addition, we constructed an Ldt null strain in M. smegmatis. This strain displayed a variety of growth defects such as a decreased growth rate, and temperature sensitivity at 42°C. We also show the cell wall was affected in this mutant, as the strain was hypersensitivity to penicillins, cephalosporins, carbapenems, vancomycin, and lysozyme. Our data further suggests that the cell envelope is altered as the Ldt null strain was hypersusceptible to rifampin, and ethambutol antibiotics and demonstrated enhanced binding and uptake of Congo red. Collectively, we have shown the importance of Ldts in cell envelope biology. LDt mutant strains…

Subjects/Keywords: Peptidoglycan; Mycobacteria; L,D-Transpeptidase

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Sanders, A. N. (2014). Genetic and phenotypic analysis of bacterial L,D-transpeptidases. (Doctoral Dissertation). University of Rochester. Retrieved from http://hdl.handle.net/1802/28490

Chicago Manual of Style (16th Edition):

Sanders, Akeisha Nichole. “Genetic and phenotypic analysis of bacterial L,D-transpeptidases.” 2014. Doctoral Dissertation, University of Rochester. Accessed August 21, 2019. http://hdl.handle.net/1802/28490.

MLA Handbook (7th Edition):

Sanders, Akeisha Nichole. “Genetic and phenotypic analysis of bacterial L,D-transpeptidases.” 2014. Web. 21 Aug 2019.

Vancouver:

Sanders AN. Genetic and phenotypic analysis of bacterial L,D-transpeptidases. [Internet] [Doctoral dissertation]. University of Rochester; 2014. [cited 2019 Aug 21]. Available from: http://hdl.handle.net/1802/28490.

Council of Science Editors:

Sanders AN. Genetic and phenotypic analysis of bacterial L,D-transpeptidases. [Doctoral Dissertation]. University of Rochester; 2014. Available from: http://hdl.handle.net/1802/28490

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