Selective Penicillin-Binding
Protein Imaging Probes
Reveal Substructure in Bacterial Cell Division
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Abstract
The peptidoglycan cell wall is a common target for antibiotic
therapy,
but its structure and assembly are only partially understood. Peptidoglycan
synthesis requires a suite of penicillin-binding proteins (PBPs),
the individual roles of which are difficult to determine because each
enzyme is often dispensable for growth perhaps due to functional redundancy.
To address this challenge, we sought to generate tools that would
enable selective examination of a subset of PBPs. We designed and
synthesized fluorescent and biotin derivatives of the β-lactam-containing
antibiotic cephalosporin C. These probes facilitated specific <i>in vivo</i> labeling of active PBPs in both <i>Bacillus
subtilis</i> PY79 and an unencapsulated derivative of D39 <i>Streptococcus pneumoniae</i>. Microscopy and gel-based analysis
indicated that the cephalosporin C-based probes are more selective
than BOCILLIN-FL, a commercially available penicillin V analogue,
which labels all PBPs. Dual labeling of live cells performed by saturation
of cephalosporin C-susceptible PBPs followed by tagging of the remaining
PBP population with BOCILLIN-FL demonstrated that the two sets of
PBPs are not co-localized. This suggests that even PBPs that are located
at a particular site (<i>e.g.</i>, septum) are not all intermixed,
but rather that PBP subpopulations are discretely localized. Accordingly,
the Ceph C probes represent new tools to explore a subset of PBPs
and have the potential to facilitate a deeper understand of the roles
of this critical class of proteins