Thank you!!

While it’s still unclear how C diff PBP1 evolved this specific function, we have found several key features of this enzyme and identified a new C diff divisome protein (PBP3). Most excitingly, we now have some clues to latch onto as we continue trying to unravel this mystery! 😄

Tracking down the aberrant septa phenotype, we discovered that a non-essential orphan class B PBP called PBP3 plays a non-catalytic regulatory role in promoting PBP1 function (and causes TPase* PBP1 to synthesize too many septa) likely by over-activating PBP1’s glycosyltransferase activity

Through our structure-function analysis, we surprisingly discovered that while the transpeptidase activity of PBP1 is required for C diff growth, the bacteria could still form septa and divide. They actually formed too many septa! And often in the wrong location 🧐

We found a domain that we call the GI domain that is critical to PBP1 function and shared with orthologs in some G+ anaerobes. Species with a GI domain in their PBP1 orthologs occupy a range of niches from the gut, oral cavity, to the vaginal tract. Some even live in marine mud and deep-sea vents!

Class A PBPs like PBP1 are not known to drive cell division in other bacteria. So we set out to determine how PBP1 evolved to take on this very important job. To begin to tackle this question, we used CRISPRi-compatible trans-complementation to conditionally express mutant variants of PBP1 in C diff