Biology subjects

Brown, P. J. B.

Publications and source records attributed to Brown, P. J. B..

3 recordsLinked to original sources

bioRxiv2024

Essentiality of LD-Transpeptidation in Agrobacterium tumefaciens

Peptidoglycan (PG), a mesh-like structure which is the primary component of the bacterial cell wall, is crucial to maintain cell integrity and shape. While most bacteria rely on penicillin binding proteins (PBPs) for crosslinking, some species employ LD-transpeptidases (LDTs). Unlike PBPs, the essentiality and biological functions of LDTs remain largely unclear. The Hyphomicrobiales order of the Alphaproteobacteria, known for their polar growth, have PG which is unusually rich in LD-crosslinks, suggesting that LDTs may play a more significant role in PG synthesis in these bacteria. Here, we investigated LDTs in the plant pathogen Agrobacterium tumefaciens and found that LD-transpeptidation, resulting from at least one of 14 putative LDTs present in this bacterium, is essential for its survival. Notably, a mutant lacking a distinctive group of 7 LDTs which are broadly conserved among the Hyphomicrobiales exhibited reduced LD-crosslinking and tethering of PG to outer membrane {beta}-barrel proteins. Consequently, this mutant suffered severe fitness loss and cell shape rounding, underscoring the critical role played by these Hyphomicrobiales-specific LDTs in maintaining cell wall integrity and promoting elongation. Tn-sequencing screens further revealed non-redundant functions for A. tumefaciens LDTs. Specifically, Hyphomicrobiales-specific LDTs exhibited synthetic genetic interactions with division and cell cycle proteins, and a single LDT from another group. Additionally, our findings demonstrate that strains lacking all LDTs except one displayed distinctive phenotypic profiles and genetic interactions. Collectively, our work emphasizes the critical role of LD-crosslinking in A. tumefaciens cell wall integrity and growth and provides insights into the functional specialization of these crosslinking activities.

microbiology↗

bioRxiv2024

The Cell Division Protein FzlA Performs a Conserved Function in Diverse Alphaproteobacteria

In almost all bacteria, the tubulin-like GTPase FtsZ polymerizes to form a "Z-ring" that marks the site of division. FtsZ recruits other proteins, collectively known as the divisome, that together remodel and constrict the envelope. Constriction is driven by peptidoglycan (PG) cell wall synthesis by the glycosyltransferase FtsW and the transpeptidase FtsI (FtsWI), but these enzymes require activation to function. How recruitment of FtsZ to the division site leads to FtsWI activation and constriction remains largely unknown. Previous work in our laboratory demonstrated that an FtsZ-binding protein, FzlA, is essential for activation of FtsWI in the alphaproteobacterium Caulobacter crescentus. Additionally, we found that FzlA also binds to a DNA translocase called FtsK, suggesting that it may link constriction activation to chromosome segregation. FzlA is conserved throughout alphaproteobacteria but has only been examined in detail in C. crescentus. Here, we explored whether FzlA function is conserved in diverse alphaproteobacteria. We assessed FzlA homologs from Rickettsia parkeri and Agrobacterium tumefaciens, and found that, similar to C. crescentus FzlA, they bind directly to FtsZ and localize to midcell. The FtsZ-FzlA interaction interface is conserved, as we demonstrated that FzlA from each of the three species examined can bind to FtsZ from any of the three in vitro. Additionally, we determined that A. tumefaciens FzlA can fulfill the essential function of FzlA when produced in C. crescentus, indicating conservation of function. These results suggest that FzlA serves as an important regulator that coordinates chromosome segregation with envelope constriction across diverse alphaproteobacteria. ImportanceCell division is essential for bacterial replication and must be highly regulated to ensure robust remodeling of the cell wall in coordination with segregation of the genome to daughter cells. In Caulobacter crescentus, FzlA plays a major role in regulating this process by activating cell wall synthesis in a manner that couples constriction to chromosome segregation. FzlA is broadly conserved in alphaproteobacteria suggesting it plays a similar function across this class of bacteria. Here we have shown that, indeed, FzlA biochemical interactions and function are conserved in diverse alphaproteobacteria. Because FzlA is conserved in alphaproteobacterial human pathogens, understanding this protein and its interactome could present therapeutic benefits by identifying potential antibiotic targets to treat infections.

microbiology↗

bioRxiv2020

Induction of AmpC-mediated-βlactam resistance requires a single lytic transglycosylase in Agrobacterium tumefaciens

The remarkable ability of Agrobacterium tumefaciens to transfer DNA to plant cells has allowed the generation of important transgenic crops. One challenge of A. tumefaciens-mediated transformation is eliminating the bacteria after plant transformation to prevent detrimental effects to plants and the release of engineered bacteria to the environment. Here we use a reverse genetics approach to identify genes involved in ampicillin resistance with the goal of utilizing these antibiotic-sensitive strains for plant transformations. We show that treating A. tumefaciens C58 with ampicillin led to increased {beta}-lactamase production, a response dependent on the broad-spectrum {beta}-lactamase AmpC and its transcription factor AmpR. Loss of the putative ampD orthologue, atu2113, led to constitutive production of AmpC-dependent {beta}-lactamase activity and ampicillin resistance. Finally, one cell wall remodeling enzyme, MltB3, was necessary for the AmpC-dependent {beta}-lactamase activity and its loss elicited ampicillin and carbenicillin sensitivity in the A. tumefaciens C58 and GV3101 strains. Furthermore, GV3101{Delta} mltB3 transforms plants with comparable efficiency to wildtype but can be cleared with sub-lethal concentrations of ampicillin. The functional characterization of the genes involved in the inducible ampicillin resistance pathway of A. tumefaciens constitutes a major step forward in efforts to reduce the intrinsic antibiotic resistance of this bacterium. IMPORTANCEAgrobacterium tumefaciens, a significant biotechnological tool for production of transgenic plant lines, is highly resistant to a wide variety of antibiotics, posing challenges for various applications. One challenge is the efficient elimination of A. tumefaciens from transformed plant tissue without using levels of antibiotics that are toxic to the plants. Here, we present the functional characterization of genes involved in {beta}-lactam resistance in A. tumefaciens. Knowledge about proteins that promote or inhibit {beta}-lactam resistance will enable the development of strains to improve the efficiency of Agrobacterium-mediated plant genetic transformations. Effective removal of Agrobacterium from transformed plant tissue has the potential to maximize crop yield and food production, improving the outlook for global food security.

microbiology↗