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Identification of YfhD from Escherichia coli as membrane-bound lytic transglycosylase F, the archetype for family 1B lytic transglycosylases

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Title: Identification of YfhD from Escherichia coli as membrane-bound lytic transglycosylase F, the archetype for family 1B lytic transglycosylases
Author: Scheurwater, Edie Marie
Department: Department of Molecular and Cellular Biology
Advisor: Clarke, Anthony J.
Abstract: This thesis is an investigation of the activity of the hypothetical protein product of the open reading frame from 'Escherichia coli' identified as 'yfhD'. We demonstrated that YfhD functions as a lytic transglycosylase (LT) and renamed it membrane-bound lytic transglycosylase F (MltF). LTs act on the glycan backbone of the bacterial heteropolymer peptidoglycan (PG), cleaving the [beta]-1, 4 glycosidic bond between 'N'-acetylmuramic acid (MurNAc) and 'N'-acetylglucosamine (GlcNAc) with the concomitant formation of a 1, 6-anhydromuramoyl product. This action allows for space to be made within the PG sacculus to allow for biosynthesis, cell division, and the insertion of cell-envelope spanning structures, and is also important in PG recycling. This research is the first demonstration of activity for a family 1B LT, for which MltF is the prototype. MltF is a bimodular protein with a C-terminal LT module that is independently responsible for carrying out PG degradation and an N-terminal non-LT module that does not appear to bind or act on PG. MltF degrades PG 'in vitro 'and 'in vivo' as demonstrated with zymography and turbidometric analysis of PG digests and its ability to replace the bacteriophage [lambda] LT and cause cellular lysis. All PG degrading activity is eliminated with the replacement of the catalytic glutamate with an alanine residue. Amino sugar analysis and MALDI-TOF MS of MltF-produced digestion products showed that they contained 1, 6-anhydromuramic acid, indicative of an LT-catalyzed reaction. Analysis of reaction products suggested the release of oligomers from an insoluble PG substrate. Glycan strands isolated from cells overexpressing 'mltF 'were enriched with oligomers containing 3 or 6 disaccharide units, indicating that MltF may be endo-acting. A chromosomal disruption of 'yfhD 'in ' E. coli' does not present any obvious phenotype while attempts to disrupt the ORF in 'E. coli' MHD79, a strain lacking functional copies of the other 6 LTs encoded by 'E. coli', were unsuccessful. This suggests that in this strain, MltF is needed to perform the necessary functions of the other LTs in order to keep the cells viable. More importantly, this suggests that LT activity is essential and that the redundancy in LT production assures continued cell viability.
URI: https://hdl.handle.net/10214/25020
Date: 2008
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