Role of Penicillin-Binding Proteins in the Viability, Morphology, Stress Tolerance, and Pathogenicity of Clavibacter michiganensis

Abstract

Previous research has shown that penicillin-binding proteins (PBPs), enzymes involved in peptidoglycan (PG) assembly, could play an important role during the induction of viable but non-culturable (VBNC) state, which allows non-spore forming bacteria to survive adverse environmental conditions. The current study found that C. michiganensis has a total of seven PBP proteins. Mutant analysis indicated that deletion of either of the class B PBPs was lethal, and that the class A PBP, PBPC, had an important role in PG synthesis, with the ΔpbpC mutant having an altered cellular morphology that resulted in longer cells that were swollen at one end, and had thinner cell walls. The ΔpbpC mutant was also found to produce mucoid colonies in solid culture and a lower final cell titer in liquid medium, as well as having increased sensitivity to osmotic stress and lysozyme treatment, and surprisingly increased pathogenicity. The double mutant, ΔdacB/ΔpbpE also had a slightly altered phenotype resulting in longer cells. Further analysis revealed that both mutants had increased sensitivity to copper, which resulted in quicker induction into the VBNC state. However, only the ΔpbpC mutant had significantly reduced survivorship in the VBNC state. The study also confirmed the VBNC state significantly improved the survivorship of wild-type C. michiganensis cells in response to environmental stresses, and systemically demonstrated the protective role of the VBNC state in C. michiganensis, which is an important finding regarding its epidemiology, and has serious implications for disease management.

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

“…S2I and J). These results were consistent with the observation by scanning electron microscopy ( 30 ), and confirmed that the lack of pbpC reduced the bacterial resistance to Cu 2+ in C. michiganensis .…”

“…It has been proved that loss of pbpC had significantly reduced survivorship of VBNC cells induced by Cu 2+ and also increased the sensitivity to a series of stress treatments in C. michiganensis ( 30 ). To evaluate whether pbpC is required for stress resistance in VBNC state, the mutant ΔpbpC was induced into VBNC state, followed by ultrasonication treatment.…”

“…It has been shown that peptidoglycan assembly relies on PBPs biosynthetic machinery ( 35 ). What is more, previous PBP evaluation in C. michiganensis has clearly indicated that the bifunctional enzyme, PBPC, played an important role in abiotic stresses resistance ( 30 ). Given the similar structure between the d-Ala - d-Ala and β-lactam antibiotic, the mutant ΔpbpC also exhibited great sensitivity to β-lactam antibiotic cefotaxime (Fig.…”

“…It has been reported that VBNC cells of Vibrio vulnificus are more resistant to heat and oxidative stress ( 29 ). Our previous study reported that VBNC cells of C. michiganensis were more tolerant to a wide variety of stresses than culturable cells, and the lack of class A HMW PBP ( pbpC ) significantly reduced the resistance of log phase cells to stresses ( 30 ). However, the functions of PBPC on peptidoglycan assembly and architecture, and the mechanism on stress response of VBNC C. michiganensis cells are still unclear.…”

A genetic toolkit for investigating Clavibacter: markerless deletion, permissive site identification and an integrative plasmid

“…The utility of two-step systems has been demonstrated in other actinobacteria 108 including Rhodococcus and Streptomyces bacteria as well as other Gram-positive 109 bacteria (Dubeau et al 2008;Kostner et al 2017;Chen et al 2020;Savory et al 2020).…”