Defining the order of assembly of the Clostridioides difficile divisome complex

Cell division is the ancient pathway by which bacteria synthesize a septum of peptidoglycan, dividing the cell into two. Where all walled bacteria were previously thought to use FtsW-FtsI orthologs to synthesize septal peptidoglycan during division, we recently discovered that the major pathogen Clostridioides difficile is missing FtsW-FtsI and instead relies on the activity of the bifunctional Class A PBP called PBP1 to synthesize the septal peptidoglycan. Furthermore, C. difficile either does not encode or require the majority of canonical divisome proteins described in model bacteria aside from the divisome protein orthologs FtsZ, SepF, and ZapA. Indeed, unlike model systems, SepF and ZapA are essential in C. difficile, suggesting that they have evolved to have a critical function in cell division without the redundant mechanisms present in model organisms. Thus, C. difficile uses a fundamentally different division mechanism compared to previously studied bacteria. To understand how this unusual complex is assembled in C. difficile, we combine CRISPR interference (CRISPRi)-based knock-downs with fluorescent fusions to determine that the hierarchical order of assembly occurs in three phases: (i) FtsZ/ZapA, (ii) SepF, and (iii) PBP1. We further investigate the order of assembly of several non-essential mid-cell localizing proteins and discover that MldA, MldC, DivIVA, FtsK, and PBP3 depend on FtsZ, SepF, and PBP1 for localization, whereas MldB localizes independently of SepF and PBP1. Our work provides a model for divisome assembly in C. difficile and validates genetic and cytological tools that can be used to mechanistically dissect this pathway in the future. IMPORTANCEBacterial cell division has been extensively studied in model systems, but little is known about how this essential process occurs in the clinically important pathogen Clostridioides difficile. Studies in model systems have shown that cell division is carried out by a large multi-protein complex called the "divisome." While components of the divisome are widely conserved and can be traced back to the last bacterial common ancestor billions of years ago, C. difficile uses a unique mechanism of division that is independent of the majority of these canonical divisome genes. In the current study, we characterize the core, essential divisome comprised of FtsZ, ZapA, SepF, and PBP1, and build a model for the order of assembly of this unusual divisome complex.