Title page for ETD etd-10012008-210035

Type of Document Master's Thesis
Author Xue, Yong
Author's Email Address xue@vt.edu
URN etd-10012008-210035
Title Effects of Protein Domains on Localization of Penicillin-Binding Proteins 2a and 2b in Bacillus Subtilis
Degree Master of Science
Department Biology
Advisory Committee
Advisor Name Title
Popham, David L. Committee Chair
Stevens, Ann M. Committee Member
Walker, Richard A. Committee Member
Yang, Zhaomin Committee Member
  • Bacillus subtilis
  • localization
  • immunofluorescence
  • penicillin-binding proteins (PBPs)
Date of Defense 2008-09-24
Availability unrestricted
Peptidoglycan not only protects bacterial cells against intracellular pressure but also provides the cells with a defined morphology. Penicillin-binding proteins (PBPs) catalyze the polymerization of the peptidoglycan in Bacillus subtilis. PBP2a and PBP2b are class B PBPs which have been known to have transpeptidase activities and they localize at different positions on the cell membrane. PBP2a spreads around the cylindrical wall as well as some at the septum, and PPB2b localizes exclusively to the septum and some at the cell poles. Both PBP2a and PBP2b are composed of four domains: S, N, P, and C domains from the N- to C- terminus. A FLAG epitope was tagged to the C-terminal ends of PBP2a and PBP2b. Cells with FLAG tagged PBP2a or PBP2b grow as well as wild type strain. Expression of PBP2a-FLAG and PBP2b-FLAG can be detected by western blotting using anti FLAG antibody. The expression of wild type PBP2a/PBP2b in these strains was tightly controlled by a xylose promoter. The FLAG fusion didn’t influence the normal membrane localizations of PBP2a or PBP2b.

PBP2a/2b mutant strains with the S and/or N domains switched between PBP2a and PBP2b were constructed. All these domain-switch proteins were tagged with a FLAG at the C-terminus. The expression of these recombinant proteins can be detected by western blotting. None of these domain-switch proteins was able to complement the wild type PBP2a and PBP2b and cells with only these recombinant proteins but no wild type proteins were non-viable. Cellular localization of these domain switch proteins were visualized using immunofluorescence microscopy. Proteins containing the PBP2a S domain had the same localization patterns as wild type PBP2a. Proteins that have the PBP2b S domain localized specifically at the septum and cell poles, which is similar to the wild type PBP2b. These results indicate that the S domain is the determinant to direct PBP2a and PBP2b to their cellular destinations.

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