Defining the order of assembly of the <i>Clostridioides difficile</i> 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 <i>Clostridioides difficile</i> is missing FtsW-FtsI and instead relies on the activity of the bifunctional Class A PBP called PBP1 to synthesize the septal peptidoglycan. Furthermore, <i>C. difficile</i> 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 <i>C. difficile</i>, suggesting that they have evolved to have a critical function in cell division without the redundant mechanisms present in model organisms. Thus, <i>C. difficile</i> uses a fundamentally different division mechanism compared to previously studied bacteria. To understand how this unusual complex is assembled in <i>C. difficile</i>, 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 <i>C. difficile</i> and validates genetic and cytological tools that can be used to mechanistically dissect this pathway in the future.