Periodic Reporting for period 4 - PREDATOR (Revealing the cell biology of a predatory bacterium in space and time)
Periodo di rendicontazione: 2023-07-01 al 2024-12-31
Predatory bacteria such as Bdellovibrio bacteriovorus are remarkable microorganisms capable of penetrating and consuming other diderm bacteria, including antibiotic-resistant strains. After slipping inside the prey’s outer cell envelope, B. bacteriovorus embarks on an unusual developmental process. It grows as a long filament with multiple copies of its genetic material and then divides into a variable number of offspring—odd or even—releasing these new predators into the environment to continue the hunt.
Because of its ability to target and kill harmful bacteria, B. bacteriovorus has gained attention as a potential “living antibiotic.” However, its unique lifestyle and complex cell cycle still pose many unanswered questions. How does it organize and copy its genetic material while nestled inside the prey’s envelope? Which molecules help define the predator’s cell polarity, i.e. which side of the cell is the prey-invading “head”— a problem that is particularly intriguing as the mother cell has to produce more than two daughter cells ? And how do different features of the prey bacteria affect the outcome of this predatory interaction?
The PREDATOR project set out to answer these questions by combining advanced microscopic imaging, genetics, and molecular biology. Specifically, it aimed to:
(i) Understand how the predator’s genetic information is replicated and distributed in a cell that can grow into a long, multi-copy filament before a non-binary division.
(ii) Identify the molecular factors that establish and maintain the predator’s cell polarity.
(iii) Reveal key features of the prey that affect how the predator multiplies within its prey’s periplasmic space.
Throughout the project, we successfully provided key insights into these areas, revealing unique mechanisms that set B. bacteriovorus apart from many well-studied bacteria. Our findings contribute to a deeper understanding of bacterial cell biology
Because of its ability to target and kill harmful bacteria, B. bacteriovorus has gained attention as a potential “living antibiotic.” However, its unique lifestyle and complex cell cycle still pose many unanswered questions. How does it organize and copy its genetic material while nestled inside the prey’s envelope? Which molecules help define the predator’s cell polarity, i.e. which side of the cell is the prey-invading “head”— a problem that is particularly intriguing as the mother cell has to produce more than two daughter cells ? And how do different features of the prey bacteria affect the outcome of this predatory interaction?
The PREDATOR project set out to answer these questions by combining advanced microscopic imaging, genetics, and molecular biology. Specifically, it aimed to:
(i) Understand how the predator’s genetic information is replicated and distributed in a cell that can grow into a long, multi-copy filament before a non-binary division.
(ii) Identify the molecular factors that establish and maintain the predator’s cell polarity.
(iii) Reveal key features of the prey that affect how the predator multiplies within its prey’s periplasmic space.
Throughout the project, we successfully provided key insights into these areas, revealing unique mechanisms that set B. bacteriovorus apart from many well-studied bacteria. Our findings contribute to a deeper understanding of bacterial cell biology
From the outset, we dedicated substantial efforts to streamline protocols for cultivating and genetically engineering Bdellovibrio bacteriovorus (e.g. improved methods for precise gene knockouts, expression systems, inducible CRISPRi, and microplate-based predation assays). Early on, we also optimized live fluorescence microscopy tools to visualize B. bacteriovorus in real time at high spatial and temporal resolution.
A key finding was how chromosomal regions move and organize during the predator’s lifecycle. We confirmed that the chromosome is highly compacted in free-swimming attack-phase cells, which can exclude small soluble proteins from the nucleoid region. Once inside the prey, B. bacteriovorus grows as a filament containing multiple copies of its genome, and we discovered that replication initiates asynchronously, with multiple replication forks active at different times and starting from different ori copies. Meanwhile, chromosome segregation begins as soon as new copies are synthesized, decoupling it from the final multiple division step. These data led us to propose a concurrent replication–segregation model, compatible with the production of odd or even number of daughter cells.
In parallel, we investigated how cell polarity is established and maintained in a predatory bacterium. We identified and tracked the localization of pole-associated proteins (DivIVA and RomR) and uncovered their distinct, dynamic cell cycle–dependent distribution. RomR was established as early pole organizer, connecting prey-attacking nanomachines with signaling pathways and intracellular organization starting in the mother cell. We further advanced our understanding of predatory bacterial cell organization by developing a proximity-labeling approach to reveal in vivo protein networks throughout the predatory lifecycle.
Finally, we explored how prey features (such as size and envelope integrity) affect B. bacteriovorus proliferation. We showed that prey size and content differentially influence predator progeny number and growth rate, respectively, while typical envelope perturbations in E. coli do not prevent predation. Toward the end of the project, we introduced B. exovorus as a new model organism with a distinct predatory lifestyle, further broadening the scope of our findings. Overall, these achievements have already resulted in several peer-reviewed publications, specialized protocols, and multiple conference presentations, thereby disseminating our results across the bacterial cell biology community.
A key finding was how chromosomal regions move and organize during the predator’s lifecycle. We confirmed that the chromosome is highly compacted in free-swimming attack-phase cells, which can exclude small soluble proteins from the nucleoid region. Once inside the prey, B. bacteriovorus grows as a filament containing multiple copies of its genome, and we discovered that replication initiates asynchronously, with multiple replication forks active at different times and starting from different ori copies. Meanwhile, chromosome segregation begins as soon as new copies are synthesized, decoupling it from the final multiple division step. These data led us to propose a concurrent replication–segregation model, compatible with the production of odd or even number of daughter cells.
In parallel, we investigated how cell polarity is established and maintained in a predatory bacterium. We identified and tracked the localization of pole-associated proteins (DivIVA and RomR) and uncovered their distinct, dynamic cell cycle–dependent distribution. RomR was established as early pole organizer, connecting prey-attacking nanomachines with signaling pathways and intracellular organization starting in the mother cell. We further advanced our understanding of predatory bacterial cell organization by developing a proximity-labeling approach to reveal in vivo protein networks throughout the predatory lifecycle.
Finally, we explored how prey features (such as size and envelope integrity) affect B. bacteriovorus proliferation. We showed that prey size and content differentially influence predator progeny number and growth rate, respectively, while typical envelope perturbations in E. coli do not prevent predation. Toward the end of the project, we introduced B. exovorus as a new model organism with a distinct predatory lifestyle, further broadening the scope of our findings. Overall, these achievements have already resulted in several peer-reviewed publications, specialized protocols, and multiple conference presentations, thereby disseminating our results across the bacterial cell biology community.
Our efforts substantially enriched the cell biology field by unveiling how a predatory bacterium executes a highly unconventional life cycle. Notably:
• The highly compacted chromosome of B. bacteriovorus introduced new questions about nucleoid architecture in bacteria with non-binary divisions. To address these, we collaborated on OMICs-based approaches (HiC, transcriptomics, ChipSeq), confirming that chromosome organization and expression profiles are dynamic and tightly coordinated with cell cycle transitions and physiological states.
• We identified essential regulatory factors (e.g. ParABS) operating under a novel multi-layered regulation, significantly refining the broader understanding of cell cycle control in bacteria and challenging standard textbook models of bacterial chromosome segregation and spatial organization.
• The miniTurbo proximity labeling technique, developed and applied in B. bacteriovorus, enabled the detection of transient protein–protein interactions at key sites like the cell poles. These data yielded deeper insights into polar organization in non-binary dividing bacteria and the coordination between the cell cycle and the predatory cycle. The method has already been adopted by other research groups.
• Our exploration of prey size and envelope properties demonstrated that fundamental aspects of predation efficiency depend on prey characteristics.
• Introducing B. exovorus enabled comparisons among related predatory bacteria, underscoring that different predation strategies might rely on similarly intricate regulatory networks.
In terms of exploitation and dissemination, we have:
• Published our findings in high-impact journals (Current Biology, PLOS Genetics, Journal of Bacteriology, Nature Communications) and contributed protocol chapters in recognized methods series, ensuring widespread access to our experimental workflows.
• Presented regularly at international conferences (including invited talks at EMBO workshops, GRC meetings, and specialized symposia) to share results and inspire collaborations.
• Provided open-source tools and data, such as specialized image analysis pipelines and predation assays, fostering an inclusive research environment that encourages follow-up studies in the wider bacterial community.
By the project’s conclusion, our work has firmly positioned B. bacteriovorus and related predatory bacteria as emerging model systems for dissecting unique bacterial cell-cycle processes, thus driving a new research frontier in bacterial cell biology and offering promising leads for future biotechnological applications.
• The highly compacted chromosome of B. bacteriovorus introduced new questions about nucleoid architecture in bacteria with non-binary divisions. To address these, we collaborated on OMICs-based approaches (HiC, transcriptomics, ChipSeq), confirming that chromosome organization and expression profiles are dynamic and tightly coordinated with cell cycle transitions and physiological states.
• We identified essential regulatory factors (e.g. ParABS) operating under a novel multi-layered regulation, significantly refining the broader understanding of cell cycle control in bacteria and challenging standard textbook models of bacterial chromosome segregation and spatial organization.
• The miniTurbo proximity labeling technique, developed and applied in B. bacteriovorus, enabled the detection of transient protein–protein interactions at key sites like the cell poles. These data yielded deeper insights into polar organization in non-binary dividing bacteria and the coordination between the cell cycle and the predatory cycle. The method has already been adopted by other research groups.
• Our exploration of prey size and envelope properties demonstrated that fundamental aspects of predation efficiency depend on prey characteristics.
• Introducing B. exovorus enabled comparisons among related predatory bacteria, underscoring that different predation strategies might rely on similarly intricate regulatory networks.
In terms of exploitation and dissemination, we have:
• Published our findings in high-impact journals (Current Biology, PLOS Genetics, Journal of Bacteriology, Nature Communications) and contributed protocol chapters in recognized methods series, ensuring widespread access to our experimental workflows.
• Presented regularly at international conferences (including invited talks at EMBO workshops, GRC meetings, and specialized symposia) to share results and inspire collaborations.
• Provided open-source tools and data, such as specialized image analysis pipelines and predation assays, fostering an inclusive research environment that encourages follow-up studies in the wider bacterial community.
By the project’s conclusion, our work has firmly positioned B. bacteriovorus and related predatory bacteria as emerging model systems for dissecting unique bacterial cell-cycle processes, thus driving a new research frontier in bacterial cell biology and offering promising leads for future biotechnological applications.