Class A Penicillin-Binding Protein C Is Responsible for Stress Response by Regulation of Peptidoglycan Assembly in Clavibacter Michiganensis

The cell wall peptidoglycan of bacteria is essential for their survival and shape development. The penicillin-binding proteins (PBPs) are responsible for the terminal stage of peptidoglycan assembly. It has been shown that PBPC, a member of class A high-molecular-weight PBP, played an important role in morphology maintenance and stress response in Clavibacter michiganensis. Here, we reported the stress response strategies under viable but nonculturable (VBNC) state and revealed the regulation of peptidoglycan assembly by PBPC in C. michiganensis cells. Using atomic force microscopy imaging, we found that peptidoglycan of C. michiganensis cells displayed a relatively smooth and dense surface, whereas was characterized by a "ridge-and-groove" surface, which was more distinctive after Cu treatment. The peptidoglycan layer of wild type cells exhibited a significant increase in thickness and slight increase in cross-linkage following Cu treatment. Compared with wild type, the thickness and cross-linkage of peptidoglycan decreased during log phase in cells, but the peptidoglycan cross-linkage increased significantly under Cu stress, while the thickness did not change. It is noteworthy that the above changes in the peptidoglycan layer resulted in a significant increase in the accumulation of amylase and exopolysaccharide in . This study elucidates the role of PBPC in Gram-positive rod-shaped plant pathogenic bacterium in response to environmental stimuli by regulating the assembling of cell wall peptidoglycan, which is significant in understanding the survival of C. michiganensis under stress and the field epidemiology of tomato bacterial canker disease. Peptidoglycan of cell walls in bacteria is a cross-linked and meshlike scaffold that provides strength to withstand the external pressure. The increased cross-linkage in peptidoglycan and altered structure in VBNC cells endowed the cell wall more resistant to adversities. Here we systematically evaluated the stress response strategies in Gram-positive rod-shaped bacterium C. michiganensis log phase cells and revealed a significant increase of peptidoglycan thickness and slight increase of cross-linkage after Cu treatment. Most strikingly, knocking-out of PBPC leads to a significant increase in cross-linking of peptidoglycan in response to Cu treatment. Understanding the stress resistance mechanism and survival strategy of phytopathogenic bacteria is the basis of exploring bacterial physiology and disease epidemiology.