Pamela J. B. Brown

Bacterial cell division is a well-conserved, tightly regulated process that allows the separation of 2 viable daughter cells. In most bacteria, the proteins that drive division, termed the divisome, are recruited to mid-cell by FtsZ after it polymerizes to form the Z-ring. Interestingly, FtsZ has undergone several independent duplication events across the bacterial kingdom. In this work, we searched for FtsZ GTPase protein sequences within Alphaproteobacterial genomes and observed numerous ftsZ duplications in the order Hyphomicrobiales. Hidden Markov Modeling (HMM) supported the maintenance of 2 distinct lineages of FtsZ GTPase duplications among 3 families. The Nitrobacteraceae duplication, occurring in only the genus Bradyrhizobium, exhibits a different substitution pattern from that shared by the Phyllobacteriaceae and Rhizobiaceae families. Within the Rhizobiaceae lineage, Agrobacterium tumefaciens contains 3 paralogs of FtsZ including the essential FtsZAT and paralogs FtsZ1 and FtsZ3. We show that FtsZ1, but not FtsZ3, inhibits cell division when expressed continuously. A hyperactive allele of ftsW partially protected against continuous expression of ftsZ1, suggesting that FtsZ1 may function to negatively regulate septal peptidoglycan biosynthesis during cell division. Overall, these observations suggest that maintenance of FtsZ paralogs in some bacteria may help control the division process.

Brucella abortus is a globally significant zoonotic pathogen. To explore the functional relevance of its genes in acidic, oxidative, and antimicrobial stress conditions, we constructed high-density transposon sequencing libraries in the attenuated B. abortus S19 strain. Here, we present the results and analysis of transposon insertion frequencies and effective densities.

Bacterial cell division is a well conserved, tightly regulated process that allows the separation of two viable daughter cells. In most bacteria, the proteins that drive division, termed the divisome, are recruited to mid-cell by FtsZ after it polymerizes to form the Z- ring. Interestingly, FtsZ has undergone several independent duplication events across the bacterial kingdom. We identified FtsZ GTPase protein sequences across alphaproteobacterial genomes, from representative genera for each family, and observed numerous ftsZ duplications in the order Hyphomicrobiales. Hidden Markov Modeling (HMM) supported the maintenance of two distinct lineages of FtsZ GTPase duplications among three families. The Nitrobacteraceae duplication, occurring in only the genus Bradyrhizobium , exhibits a different substitution pattern from that shared by the Phyllobacteraceae and Rhizobiaceae families. Within the Rhizobiaceae lineage, Agrobacterium tumefaciens contains three paralogs of FtsZ including the essential FtsZ _AT, and paralogs FtsZ ₁ , and FtsZ ₃ . In A. tumefaciens, we show that FtsZ ₁ , but not FtsZ ₃ , inhibits cell division when overexpressed. A hyperactive allele of ftsW partially protected against overexpression of ftsZ ₁ suggesting that FtsZ ₁ may inhibit proper regulation of septal peptidoglycan biosynthesis during cell division. Overall, these observations suggest that maintenance of FtsZ paralogs in some bacteria which may fine tune the division process. Article Summary: Gene duplication drives evolutionary innovation by providing raw genetic material and allowing microbes to acquire novel functions. Here, we explore the duplication of a gene encoding the essential cell division protein FtsZ in the Hyphomicrobiales and find that the duplication is primarily conserved in genera that interact with plant hosts. In A. tumefaciens FtsZ ₁ is not essential for cell division; however, overexpression of FtsZ ₁ inhibited cell division suggesting it may have a regulatory role during this essential process. This observation highlights the key role for gene duplication in the modulation of complex processes in bacteria.

Agrobacterium tumefaciens shifts from a free-living soil bacterium to a plant-invading state upon encountering the plant root microenvironment. The acid-induced two-component sensor system ChvG–ChvI drives this shift and triggers a complex transcriptional program that promotes host invasion and survival against host immune defenses. Remarkably, ChvG–ChvI is also activated under cell wall stress conditions, suggesting that the transcriptional response may have a broader function. Here, we find that blocking cell wall synthesis either genetically or chemically leads to ChvG–ChvI activation. Mutations in key cell wall synthesis enzymes, such as penicillin-binding protein 1a and FtsW, suppress ChvG–ChvI activation in cell wall stress inducing conditions, suggesting that providing structural integrity is a primary function of the ChvG–ChvI regulon. Here, we investigated regulon components for this function. First, deletion of exoA, a gene required for production of the exopolysaccharide succinoglycan, confers resistance to multiple β-lactam antibiotics targeting different enzymes. Next, a class D β-lactamase is expressed that may contribute to the high level of β-lactam resistance in A. tumefaciens. Finally, outer membrane proteins are upregulated, suggesting that outer membrane remodeling may compensate for the accumulation of cell wall damage by providing structural integrity. Overall, we expand our understanding of mechanisms driving ChvG–ChvI activation and β-lactam resistance in a bacterial plant pathogen.

Agrobacterium tumefaciens shifts from a free-living soil bacterium to a plant-invading state upon encountering the plant root microenvironment. The acid-induced two- component sensor system ChvG-ChvI drives this shift and triggers a complex transcriptional program that promotes host invasion and survival against host immune defenses. Remarkably, ChvG-ChvI is also activated under cell wall stress conditions suggesting that the transcriptional response may have a broader function. Here, we find that blocking cell wall synthesis either genetically or chemically leads to ChvG-ChvI activation. Mutations in key cell wall synthesis or outer membrane proteins, such as PBP1a, FtsW, and AopA1, suppress ChvG-ChvI activation suggesting that providing structural integrity is a primary function of the ChvG-ChvI regulon. Here, we investigated regulon components for this function. First, the exopolysaccharide succinoglycan confers tolerance to multiple β-lactam antibiotics targeting different enzymes by forming a protective barrier around the cells. Next, a Class D β-lactamase is expressed which may contribute to the high level of β-lactam resistance in A. tumefaciens . Finally, outer membrane remodeling compensates for the accumulation of cell wall damage by providing structural integrity. Overall, we expand our understanding of mechanisms driving ChvG-ChvI activation and β-lactam resistance in a bacterial plant pathogen. Significance Statements.: Activation of the ChvG-ChvI two component system promotes survival when the bacterial cell walls are damaged by a variety of genetic or chemical approaches The ChvG-ChvI dependent production of the exopolysaccharide succinoglycan, β-lactamase Cbl activity, and outer membrane proteome remodeling all contribute to survival in the presence of β-lactam antibiotics Improved understanding of bacterial stress responses that promote antibiotic tolerance and resistance has the potential to inform development of novel drug targets

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 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 . Finally, 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. IMPORTANCE: Cell 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 that 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.

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 also 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 β-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.

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 β-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.

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. Importance: Cell 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.