Title:
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Functional analysis of PBP2b in Bacillus subtilis
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The major structural component of the eubacterial cell wall is a net like matrix of
peptidoglycan (PG). The final steps of PG assembly are carried out by a large family of
penicillin-binding proteins (PBPs). Some PBPs are redundant, but most bacteria have an
essential PBP specialised for cell division. PBP2b of B. sllbtilis is essential for cell
division and it targets division sites by interaction with one or more components of the
division machinery. The aim of the work described in this thesis was to understand the
function ofPBP2b in the cell cycle ofB. sllbtilis.
Remarkably, substitution of the catalytic serine of PBP2b had almost no effect on
cell division in B. sllbtilis, even though depletion of the entire protein is lethal. Further
analysis of the catalytically inactive form of PBP2b revealed that PBP3, previously
shown to be dispensable, took on an essential role. This study also revealed that the
essential function of PBP3, in the absence of biochemically active PBP2b, lies in its
transpeptidase (TPase) activity. Overall, this research suggests that the division
machinery is constructed in such a way that if the primary TPase activity of the
architectural PBP2b enzyme is inactivated, a secondary, non-architectural PBP (PBP3)
can access the site and provide a back-up TPase that will allow the septum to be
completed. This has interesting parallels with the mechanism of methicillin-resistance in
Staphylococcus aureus (MRSA). In further studies, the combination of PBP2b Ts mutations with the divIB, divIC,
andftsL mutation backgrounds suggested that different parts of the N-terrninal domain of
PBP2b may be involved in interactions with specific division proteins. In addition,
although the TPase function is not essential, the TPase domain may be involved in the
interaction with other division proteins. The C-terminal domain of PBP2b was shown to
be inessential for vegetative cell growth and sporulation at 'permissive temperatures. Its
function is probably to stabilize PBP2b at elevated temperatures. Previous research
suggested that that PBP2b may take part in the early stages of sporulation. In this thesis,
its function has been further investigated and the results suggest that PBP2b may be also
involved in the late stages of sporulation.
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