Molecular dissection of Class A PBP function uncovers novel features of the non-canonical Clostridioides difficile divisome complex

Abstract

Cell division in bacteria is mediated by the "divisome," a multiprotein complex that synthesizes the septal peptidoglycan needed to divide one cell into two. We recently showed that the major nosocomial pathogen Clostridioides difficile assembles a divisome that is fundamentally distinct from previously studied bacteria because it lacks functional orthologs of the septal peptidoglycan-synthesizing enzymes, FtsW and FtsI. While these enzymes were previously thought to mediate cell division in all walled bacteria, C. difficile instead uses the bifunctional Class A Penicillin Binding Protein PBP1 to mediate cell division. Here, we optimized a CRISPRi-based conditional expression system to define features within PBP1 that are critical for its essential functions. Our analyses identify a novel accessory domain that is required for PBP1 function and is conserved across Peptostreptococcaceae family PBP1 homologs. We further show that PBP1s glycosyltransferase and transpeptidase activities are both strictly required for bacterial growth. While PBP1 glycosyltransferase activity is required for septum synthesis during cell division, PBP1s transpeptidase activity is surprisingly dispensable for cell division, although TPase-deficient (PBP1TPase*) cells produce multiple aberrant septa. We demonstrate that the uncontrolled septum synthesis observed in PBP1TPase* cells depends on the non-essential Class B PBP, PBP3, but PBP3s catalytic activity is dispensable for this function. Since we also show that PBP3 is recruited to the divisome complex and forms a complex with PBP1, our analyses reveal a cryptic but important regulatory function for PBP3 in promoting C. difficile cell division. Author SummaryBacterial cell division is an ancient and essential process, but our molecular understanding of this process is primarily based on studies in a select few model systems. Recent work found that the major nosocomial pathogen Clostridioides difficile divides by a fundamentally distinct mechanism because homologs of the canonical septum synthesis complex used by virtually all walled bacteria are either missing from the C. difficile genome or have lost their function during vegetative division. This prior work revealed that septum synthesis in C. difficile is instead driven by the catalytic activity of the Class A Penicillin Binding Protein called PBP1, but the features of PBP1 that enable it to carry out this unusual function were unknown. In the current study, we perform detailed structure-function analyses to determine how C. difficile PBP1 has become specialized for this role. We find that PBP1 carries an unusual regulatory domain that is critical for its function. Our analyses also uncover an unexpected function for the non-essential enzyme PBP3 in cell division, identifying a new component of the C. difficile cell division complex. These analyses provide new insight into how bacteria can repurpose cell wall synthesis enzymes to fulfill essential functions in novel ways.