Exploring the diverse functions of peptidoglycan hydrolases in the plant pathogen, Agrobacterium tumefaciens

The plant pathogen Agrobacterium tumefaciens displays an atypical form of unipolar elongation, followed by incipient pole synthesis during cell division and cell separation. Currently, how polar growing bacteria modulate cell wall hydrolysis during growth and division remains largely unknown. This work includes the comprehensive analysis and characterization of the role of cell wall hydrolyses involved in bacterial growth, division, recycling and beta-lactam resistance in A. tumefaciens. First, we performed bioinformatic analyses and used reverse genetics to better understand the role cell wall hydrolases in A. tumefaciens. Inactivation of most cell wall hydrolases, led to no phenotypic defects suggesting a high degree of redundancy. However, inactivation of the amidase, AmiD, and the lytic transglycosylase Atu3779, revealed significant changes in beta-lactam resistance suggesting that these proteins are involved in the activation beta-lactamases and outer-membrane integrity. Next, we developed a tool (Figueroa-Cuilan et al., 2016) to dissect the role of essential genes, which enabled characterization of the essential regulator of cell division, DipM, a LytM-containing factor. Absence of DipM causes severe cell division defects, including increased cell length, mid-cell width and lysis. A cell wall composition analysis of cells devoid of DipM shows an increase in the activity of the PG hydrolases, lytic transglycosylases, suggesting that DipM may inhibit the activity of these enzymes. Lastly, we find that deletion of individual lytic transglycolsylases (LTs) from the DipM depletion strain delays the onset of the DipM depletion phenotype. Overall, this research provides mechanistic insights about the roles of peptidoglycan hydrolases and their regulators in cell growth and division. Understanding how bacterial cell wall hydrolysis is spatiotemporally regulated and coordinated with cell wall synthesis and cell division (Figueroa-Cuilan and Brown, 2018), will be applicable to other closely related polar-growing bacteria.