A conserved hub protein for coordinating peptidoglycan turnover that activates cell division amidases in...

Gram-negative bacteria produce a multilayered cell envelope in which their peptidoglycan is sandwiched between two membranes, an inner membrane made of glycerophospholipids and an asymmetric outer membrane with glycerophospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. The Acinetobacter baumannii outer membrane contains lipooligosaccharide (LOS), a variant of LPS lacking O-antigen. LPS/LOS is typically essential, but A. baumannii can survive without LOS. Previously, we found that the peptidoglycan biogenesis protein NlpD becomes essential during LOS-deficiency. NlpD is typically redundant and is one of the cell's amidase activators for regulating peptidoglycan degradation, a process critical for cell division. We found that NlpD is essential under these conditions because a second putative amidase activator, termed WthA (cell w all turnover h ub protein A ), no longer functions in LOS-deficient cells. Mutants lacking WthA had severe cell division defects and were synthetically sick with loss of NlpD. Both Acinetobacter WthA and NlpD were found to activate an amidase activity of Oxa51, a chromosomally encoded β -lactamase. Further, WthA is homologous to Pseudomonas LbcA that impacts two other classes of peptidoglycan degradation enzymes, endopeptidases and lytic transglycosylases. WthA/LbcA homologs were identified across Proteobacteria, Bacteroidota, and Chlorobiota, suggesting they belong to a conserved family involved in regulation of peptidoglycan turnover. While Acinetobacter WthA may share functions of Pseudomonas LbcA, we found no evidence that LbcA is an amidase activator. Altogether, we have identified a missing player in Acinetobacter peptidoglycan biogenesis, a conserved hub protein that regulates multiple peptidoglycan turnover enzymes including cell division amidases.

Peptidoglycan is a rigid layer that provides structural support to bacterial cells. Peptidoglycan must be degraded to make room for new synthesis and for cells to divide, a process termed turnover. Turnover enzymes are tightly regulated to prevent their activities from lysing the cell. The critical pathogen Acinetobacter baumannii was missing known peptidoglycan amidases, a class of turnover enzymes, and the key activator that controls their activity during cell division. We have identified WthA as having a role in cell division most likely as an amidase activator. WthA homologs were widely distributed in bacteria and the closely related LbcA in Pseudomonas impacts two other types of turnover enzymes. We explore the possible functions of this new family of proteins that serves as a hub for impacting peptidoglycan turnover.