# Streptococcus pneumoniae synchronizes the states of cell wall peptidoglycan acetylation and genome methylation by programmed DNA inversions

## TL;DR

Streptococcus pneumoniae uses cell wall acetylation to control genome methylation and colony phase, linking metabolism to epigenetic changes.

## Contribution

Discovery of a novel epigenetic mechanism linking cell wall acetylation to DNA methylation and phase variation in S. pneumoniae.

## Key findings

- NAM O-acetylation controls colony phase by modulating DNA methyltransferase gene inversions.
- O-acetylation correlates with acetyl-CoA and glucose levels, linking nutrient availability to epigenetic changes.
- Multiple proteins form a signaling circuit connecting NAM O-acetylation to genome methylation patterns.

## Abstract

Bacterial cell wall peptidoglycan (PG) consists of alternating β-(1,4) linked N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG). The C-6 hydroxyl group of NAM is acetylated by transmembrane O-acetyltransferases post PG biosynthesis in many pathogenic bacteria. This modification is important for bacterial resistance to lysozyme. It is also known that the extent of NAM O-acetylation varies greatly, depending on genetic background and growth phase. However, it remains unclear if the fluctuation of NAM O-acetylation has any function. In this study, we show that NAM O-acetylation functions as a potential extracellular signal of cellular metabolism for epigenetic response to nutrient conditions in human pathogen Streptococcus pneumoniae (pneumococcus). The O-acetylation was found to control reversible switch between opaque and transparent colony phases by modulating inversion reactions of DNA methyltransferase hsdS genes in the colony opacity determinant (cod) locus, and thereby phase-defining genome methylation pattern. The NAM O-acetylation made S. pneumoniae adopt the HsdSA1 methylome and opaque colony phase, whereas the lack of this modification favored the HsdSA3 methylome and transparent colony phenotype. Further analysis revealed that the major autolysin LytA and multiple other proteins are required for the O-acetylation-dependent control of epigenetic machinery. Lastly, the extent of NAM O-acetylation was found to correlate with the cellular level of the acetyl donor acetyl-CoA and glucose. These data support the postulation that S. pneumoniae uses NAM O-acetylation as an extracellular marker of cellular acetyl-CoA to synchronize nutrient availability with bacterial lifestyle by epigenetic modulation of cellular metabolism.

Bacterial cell wall peptidoglycan (PG) consists of two basic sugars: N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG). The C-6 hydroxyl group of NAM is acetylated in many pathogenic bacteria after the PG is synthesized, which is important for bacterial resistance to lysozyme. The extent of NAM O-acetylation varies greatly, depending on genetic background and growth phase. However, it is unknown whether the fluctuation of NAM O-acetylation has any role in bacterial biology. Here, we show that human pathogen Streptococcus pneumoniae uses the level of NAM O-acetylation as an extracellular signal to epigenetically regulate cellular metabolism and phase variation in colony opacity. This is accomplished by O-acetylation-dependent modulation of inversion reactions of DNA methyltransferase hsdS genes in the colony opacity determinant (cod) locus, which leads to reversible switch among multiple methylation patterns of bacterial genome. Several extracellular, transmembrane and intracellular proteins are found to constitute a signaling circuit to connect the NAM O-acetylation and hsdS gene inversions. This work has thus uncovered a previously uncharacterized epigenetic mechanism of bacterial adaptation to nutrient availability.