Activation of ChvG-ChvI regulon by cell wall stress confers resistance to β-lactam antibiotics and initiates surface spreading in Agrobacterium tumefaciens

A core component of nearly all bacteria, the cell wall is an ideal target for broad spectrum antibiotics. Many bacteria have evolved strategies to sense and respond to antibiotics targeting cell wall synthesis, especially in the soil where antibiotic-producing bacteria compete with one another. Here we show that cell wall stress caused by both chemical and genetic inhibition of the essential, bifunctional penicillin-binding protein PBP1a prevents microcolony formation and activates the canonical host-invasion two-component system ChvG-ChvI in Agrobacterium tumefaciens . Using RNA-seq, we show that depletion of PBP1a for 6 hours results in a downregulation in transcription of flagellum-dependent motility genes and an upregulation in transcription of type VI secretion and succinoglycan biosynthesis genes, a hallmark of the ChvG-ChvI regulon. Depletion of PBP1a for 16 hours, results in differential expression of many additional genes and may promote a general stress response. Remarkably, the overproduction of succinoglycan causes cell spreading and deletion of the succinoglycan biosynthesis gene exoA restores microcolony formation. Treatment with cefsulodin phenocopies depletion of PBP1a and we correspondingly find that chvG and chvI mutants are hypersensitive to cefsulodin. This hypersensitivity only occurs in response to treatment with β-lactam antibiotics and moenomycin, suggesting that the ChvG-ChvI pathway may play a key role in resistance to antibiotics targeting cell wall synthesis. Finally, we provide evidence that ChvG-ChvI likely has a conserved role in conferring resistance to cell wall stress within the Alphaproteobacteria. AUTHOR SUMMARY Soil dwelling bacteria reside in changing environments requiring them to frequently adapt to stressful conditions to ensure survival. The bacterial envelope provides structural integrity and protection against osmotic stress and turgor pressure imposed by the environment. While the mechanisms of cell membrane and cell wall biogenesis have been extensively studied, our understanding of how diverse microbes respond to cell envelope and cell wall stress to increase their fitness remains limited. In this work, we identify ChvG-ChvI regulon as an envelope stress response system that confers protection under cell wall stress conditions in the bacterial plant pathogen Agrobacterium tumefaciens . This is a new function for the well-characterized ChvG-ChvI pathway which is also acid induced and promotes plant host invasion. Our results suggest that the ChvG-ChvI pathway has a broadly conserved role in protecting Alphaproteobacterial cells from extracellular stress and a more specific role in response to acid stress and promoting plant-microbe interactions. ### Competing Interest Statement The authors have declared no competing interest.