# Dynamics and Regulation of Peptidoglycan Cell Wall Synthesis in the Superbug Streptococcus pneumoniae

> **NIH NIH R35** · TRUSTEES OF INDIANA UNIVERSITY · 2024 · $666,807

## Abstract

Peptidoglycan (PG) is the mesh-like scaffolding that determines the shape and size of bacterial cells and protects
them from osmotic shock. In Gram-positive bacteria, thick PG provides the outer cellular layer to which wall-
teichoic acids, capsule, and extracellular proteins are covalently attached. The human respiratory pathogen
Streptococcus pneumoniae (pneumococcus) has emerged as a leading model for PG synthesis and its
regulation in ovoid-shaped Gram-positive bacteria. Pneumococcal PG synthesis shows fundamental differences
from PG synthesis in rod-shaped and spherical bacteria. Most notably, PG synthesis is zonal and carried out by
two separable PG synthase nanomachines confined to the midcell of dividing pneumococcal cells. The septal
PG synthase (bPBP2x:FtsW) locates to the leading edge of the closing septal annulus, while the elongasome
PG synthase (bPBP2b:RodA with RodZ, MreCD) locates to the outer rim of the septal annulus and pushes
peripheral PG outward. Both the septal and elongasome PG synthases move circumferentially at midcell driven
by PG synthesis, but not by FtsZ treadmilling. This project builds on previous findings to address the most
important current questions about PG synthesis in S. pneumoniae. One set of questions centers on the functions
and regulation of the three Class A PBP PG synthases (aPBP1a, aPBP1b, aPBP2a) in exponentially growing
and stressed pneumococcal cells, about which relatively little is known. By using a comprehensive approach that
includes innovative assays of septal and elongation PG synthesis rates in live cells, single-molecule (sm) motion
dynamics, and high-resolution microscopy, we will determine the contributions of the aPBPs to PG synthesis
and how their dynamics and localization are altered by mutational changes and cell-wall stress. Unbiased and
directed approaches will be used to identify aPBP interactors at different stages of division and how these
interactions regulate aPBP functions. A second set of questions center on the mechanisms that organize and
regulate the septal and elongasome PG synthase nanomachines. We will determine mechanisms that organize
circumferential motion and nodal distributions of PG synthases at midcell. We will also determine the assembly
pathway of the septal divisome in early-divisional cells and the mechanisms by which the septal and elongasome
PG synthesis machines separate and are regulated later in division. We will continue studies of newly discovered
mechanisms that link PG synthase functions to the availability of PG precursor metabolites and to second
messengers. A third set of questions concerns the roles, interactions, and regulation of the FtsEX:PcsB
hydrolase in PG remodeling and the MpgB and MpgA muramidases in PG-chain release in septal and
elongasome PG synthesis, respectively. Altogether, this project will fill in major knowledge gaps about the
dynamics, functions, and regulation of aPBPs, about the organization and regulation of septal and elon...

## Key facts

- **NIH application ID:** 10840677
- **Project number:** 2R35GM131767-06
- **Recipient organization:** TRUSTEES OF INDIANA UNIVERSITY
- **Principal Investigator:** MALCOLM E. WINKLER
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $666,807
- **Award type:** 2
- **Project period:** 2019-09-05 → 2029-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10840677

## Citation

> US National Institutes of Health, RePORTER application 10840677, Dynamics and Regulation of Peptidoglycan Cell Wall Synthesis in the Superbug Streptococcus pneumoniae (2R35GM131767-06). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10840677. Licensed CC0.

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