Abstract
ABSTRACT
In most bacteria, peptidoglycan contains mainly 4-3 crosslinks formed by penicillin-binding proteins (PBPs). However, in the opportunistic pathogen
Clostridioides difficile
, 70% of the crosslinks are 3-3 crosslinks formed by L,D-transpeptidases (LDTs), and LDTs are essential for viability. PBPs and LDTs use different acyl donors for crosslinking; PBPs require a pentapeptide, while LDTs require a tetrapeptide. Here, we determined the source of the tetrapeptides in
C. difficile
and investigated the consequences of reengineering PG crosslinking from predominantly 3-3 to exclusively 4-3. We found that two D-alanyl-D-alanine carboxypeptidases (DD-CPase), DacA and DacC, supply LDTs with tetrapeptides during vegetative growth. Deleting these enzymes was sufficient to bypass the normal requirement for LDTs. The resulting mutant (Δ
dacAC
Δ
ldt
) was remarkably healthy despite the absence of 3-3 crosslinks. Its only major phenotypic defect was a 3- to 4-log decrease in sporulation, which could, however, be overcome by deleting a third DD-CPase,
dacB
. These findings fill gaps in our understanding of the pathway for LD-transpeptidation in
C. difficile
and imply that LDTs are not essential components of the elongasome or divisome, both of which function well in the complete absence of LDTs, provided there is sufficient pentapeptide to sustain crosslinking by PBPs. Thus, LDTs are essential for viability because
C. difficile
has intrinsically high levels of DD-CPase activity. Finally, we propose a model for how PBPs and LDTs work together during PG synthesis. In this model, PBPs construct a sparsely crosslinked PG sacculus that is subsequently strengthened with crosslinks introduced by LDTs.
IMPORTANCE
Synthesis of peptidoglycan (PG) in the opportunistic gut pathogen
Clostridioides difficile
depends mostly on 3-3 crosslinks formed by L,D-transpeptidases (LDTs). Here, we converted
C. difficile
from an organism that relies primarily on 3-3 crosslinks made by LDTs to one that relies exclusively on 4-3 crosslinks made by an alternative family of crosslinking enzymes, the penicillin-binding proteins (PBPs). Our findings explain why 3-3 crosslinking predominates over 4-3 crosslinking, clarify why
C. difficile
does not compensate for the loss of LDTs by increasing PBP activity, and suggest a model for how PBPs and LDTs work together in this problematic pathogen.