Iorga, B. | bioRxiv Science

bioRxiv2025

A novel peptidoglycan deacetylase modulates daughter cell separation in E. coli

Peptidoglycan hydrolases facilitate bacterial cell wall growth by creating space for insertion of new material and allowing physical separation of daughter cells. In Escherichia coli, three peptidoglycan amidases, AmiA, AmiB and AmiC, cleave septal peptidoglycan during cell division. The LytM-domain proteins EnvC and NlpD activate these amidases either from inside the cell or the outer membrane: EnvC binds to the cytoplasmic membrane-anchored divisome components FtsEX, and NlpD and ActS are outer membrane lipoproteins. Here we report the identification of a novel periplasmic deacetylase called SddA that removes acetyl groups from denuded peptidoglycan glycan strands, the products of amidases. SddA is a substrate for the periplasmic protease Prc, suggesting regulation via protein degradation. The sddA gene is co-expressed with the gene encoding EnvC, linking SddA function to amidase activation. Consistent with this link, the deletion of sddA alleviates phenotypes associated with lack of amidase activation, while overexpression of sddA alleviates phenotypes related to a defective Tol-Pal system and causes cell chaining due to reduced septum peptidoglycan cleavage unless envC is co-expressed. We present a model according to which SddA modulates the activation of the septum-splitting amidases during cell division. AUTHOR SUMMARYBacteria surround their cell membrane by the essential peptidoglycan (cell wall) layer to prevent bursting open due to their turgor. During cell division, bacteria produce a septum at midcell, which must be cleaved for daughter cells to separate. Here, we report the identification of a new enzyme, SddA, that modifies a particular type of peptidoglycan material, denuded glycan chains released during the splitting of septal peptidoglycan for daughter cell separation, in the Gram-negative Escherichia coli. We propose a model in which SddA modulates a switch in the septal peptidoglycan splitting, ensuring splitting is activated from the cell membrane in the early stages of cell division and from the outer membrane in the late stages.

microbiology↗

bioRxiv2025

Identification of receptor-binding domains of Bacteroidales antibacterial pore-forming toxins

Bacteroidales are abundant Gram-negative bacteria present in the gut microbiota of most animals, including humans, where they carry out vital functions for host health. To thrive in this competitive environment, Bacteroidales use sophisticated weapons to outmatch competitors. Among these, BSAPs (Bacteroidales Secreted Antimicrobial Proteins) represent a novel class of bactericidal pore-forming toxins that are highly specific to their receptor, typically targeting only a single membrane protein or lipopolysaccharide. The molecular determinants conferring this high selectivity remain unknown. In this study, we therefore investigated the model protein BSAP-1 and determined which of its domains is involved in providing receptor specificity. We clearly demonstrate that receptor recognition is entirely driven by the C-terminal domain (CTD) of BSAP-1 using a combination of in vivo competition assays and in vitro protein binding studies. Specifically, we show that deletion of the CTD abrogates BSAP-1 bactericidal activity by preventing receptor binding, while grafting the CTD to unrelated carrier proteins enables CTD-driven interaction with the BSAP-1 receptor. Building upon this discovery, we show that BSAPs can be categorized according to the structure of their CTD and that BSAPs within the same cluster are likely to target the same type of receptor. Additionally, we show that the CTD of BSAP-1 can be repurposed to generate probes for fluorescent labelling of membrane proteins in live cells. In summary, our research demonstrates that BSAP receptor recognition is driven by their CTD and that these can be engineered to develop novel tools for the investigation of Bacteroidales biology.

microbiology↗