Peptidoglycan (PG), the primary structural component of the bacterial cell wall, is essential for maintaining cell integrity and shape. While most bacteria rely on penicillin-binding proteins (PBPs) to generate crosslinks in PG, some species also use ld-transpeptidation (LDTs). Members of the Hyphomicrobiales order within the Alphaproteobacteria, which are characterized by polar growth, possess PG enriched in LD-crosslinks—suggesting an expanded role for LDTs. This work investigates the roles of LDTs and their interaction with outer membrane proteins (OMPs) in maintaining envelope integrity and supporting growth, particularly under stress conditions in the plant pathogen Agrobacterium tumefaciens.

In Chapter 2, a structural and phenotypic analysis identified a conserved set of Hyphomicrobiales-specific LDTs (Group 3) that play essential roles in cell elongation and polar growth. Despite a high degree of functional redundancy among these enzymes phenotypic differences suggest some LDTs have specialized functions, such as tethering OMPs to the cell wall. 

Chapter 3 builds on this by showing that a subset of these LDTs, along with the β-barrel OMP AopB, are transcriptionally regulated by the ChvG-ChvI two component system—a regulatory pathway activated by cell envelope stress. LDT-mediated crosslinking of AopB to the peptidoglycan enhances envelope stability under heat, osmotic, and cell wall stress, with both ΔaopB and Δgr3 mutants exhibiting similar stress sensitivity profiles. These findings highlight an expanded role for the ChvG-ChvI regulon in envelope remodeling during stress.

Finally, Chapter 4 explores the potential modulation of the outer membrane proteome by the ChvG-ChvI regulatory network response to envelope damage Activation of ChvG-ChvI likely promotes cell envelope integrity by enhancing structural reinforcement of the envelope through β-barrel OMP-PG crosslinking mediated by LDTs and increased production of EipA a periplasmic, β-barrel protein. Together, these studies define a critical role for the LDTs and their substrates in the adaptive envelope remodeling of A. tumefaciens and position the ChvG-ChvI pathway as a central regulator of envelope integrity and environmental resilience. 

Publications

Matthes MS, Darnell Z, Best NB, Guthrie K, Robil JM, Amstutz J, Durbak A, McSteen P (2022) Defects in meristem maintenance, cell division, and cytokinin signaling are early responses in the boron deficient maize mutant tassel‐less1. Physiologia Plantarum 174 (2), e13670. 

Amstutz J, Krol E., Verhaeghe A., De Bolle X., Becker A., Brown, PJB (2024) Getting to the point: unipolar growth of Hyphomicrobiales. Current Opinion in Microbiology, 79, 102470.

Aliashkevich A, Guest T, Alvarez L, Gilmore MC, Rea D, Amstutz J, et al. (2024) LD-transpeptidation is crucial for fitness and polar growth in Agrobacterium tumefaciens. PLoS Genetics 20 (10), e1011449. 

Bouchier JM, Knebel E, Amstutz J, Torrens G, Santiago Collazo G, McCurry C, Weisburg AJ, Cava F, Brown PJB. (2024) Activation of the ChvG-ChvI pathway promotes multiple survival strategies during cell wall stress in Agrobacterium tumefaciens. bioRxiv, 2024-12.

Doctoral Program Committee

• Dr. Pamela Brown, Chair
• Dr. Lauren Sullivan
• Dr. Paula McSteen
• Dr. Michael Calcutt