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Revealing the cell biology of a predatory bacterium in space and time

Periodic Reporting for period 2 - PREDATOR (Revealing the cell biology of a predatory bacterium in space and time)

Reporting period: 2020-07-01 to 2021-12-31

The model predatory bacterium Bdellovibrio bacteriovorus feeds upon other Gram-negative bacteria, including pathogenic strains. Upon entry inside the periplasmic space of the prey envelope, B. bacteriovorus initiates an exquisite developmental program in which it digests the host resources while ensuring the osmotic stability of its niche. In the periplasm, the predator cell grows as a polyploid filament, before releasing a variable, odd or even number of daughter cells upon a non-binary division event. The progeny is then liberated to hunt for new prey. B. bacteriovorus is now attracting a revived attention as several in vivo models of infection established its promising “living antibiotic” potential. Despite this remarkable lifestyle, the fields of bacterial cell biology and antibiotics research still lacked a comprehensive understanding of how this micro-predator thrives inside the envelope of other bacteria, at the beginning of this project. Indeed, the molecular factors behind the non-canonical cell biology of B. bacteriovorus are still largely mysterious.
The goal of the PREDATOR project is to tackle this question by unraveling the novel mechanisms that control key processes of the fascinating cell cycle of B. bacteriovorus, using a unique combination of quantitative live imaging of predation at the single-cell level, bacterial genetics and molecular biology. Specifically, I aim to (i) uncover how the genetic information is organized, copied and partitioned in a polyploid cell before non-binary division, (i) shed light on factors that polarize the predator cell, and (iii) discover prey features that influence the predation cycle. Because the biology of B. bacteriovorus stands beyond textbook standards, our results will provide mechanistic insight into important biological questions that remained unexplored using “classical” model species. If successful, this project will advance bacterial cell biology, while offering an innovative contribution to the fight against antibiotics-resistant pathogens.
We could for the first time follow key chromosomal regions inside the predatory cell during the distinct steps of its lifecycle. We also discovered that the chromosome of B. bacteriovorus is strongly compacted when predators are freely swimming outside their prey. The dense packing of the chromosome likely explains the unique exclusion of small soluble proteins from the nucleoid. We also revealed the dynamics of DNA replication and segregation by monitoring the machineries in charge of these processes in B. bacteriovorus. This led us to highlight intriguing features related to the timing and coordination of these major cell cycle events, which cannot be grasped with canonical model species. For instance, we found that several rounds of replication take place simultaneously but asynchronously as the predator elongates in its prey, and that chromosome segregation is initiated as soon as new copies are being synthesized, thus uncoupled from the final multiple division steps (at odds with textbook models). Monitoring the chromosome choreography throughout the predatory cell cycle allowed us to propose a model, whereby concomitant replication of chromosomal copies and their progressive segregation ensures that the mother predator cell is always ready to divide into progenies equipped with a correct copy of the genetic information. Importantly, this model is compatible with the generation of variable, odd or even numbers of daughter cells. From the start of the project, we have put a lot of effort into streamlining previously existing and new protocols for handling and genetically engineering B. bacteriovorus, and for measuring its population density and predation capacities. Besides, progress was made towards understanding how B. bacteriovorus polarizes its cellular content, as our approach starts to reveal the physical partners and unexpected localization dynamics of a potential cell-pole organizing protein, which will contribute to understanding its function during the rest of the project.
Our results so far constitute a major step forward in the study of the cell biology of bacterial predators. Our recent article (Current Biology, in press) provides an unprecedented view of the entire cell cycle of Bdellovibrio bacteriovorus, by the use of live fluorescence microscopy with unmatched spatiotemporal resolution and quantitative analysis at the single-cell level. Our finding of the strong chromosome compaction opens new avenues of investigation, which we have started to pursue using high-end methodologies including chromosome conformation capture. This will allow us to decipher the inner architecture of the nucleoid in predatory bacteria, at different stages of their cell cycle. Performing such analyses in different genetic backgrounds will also help us identifying the molecular factor(s) involved in chromosome compaction. Our work on the DNA replication and segregation dynamics in B. bacteriovorus provides important insights into the non-binary proliferation in bacteria, which had remained largely unexplored. Following-up this study, we were invited to write a protocol manuscript that will be submitted before the end of 2021, to describe our methodological advances related to B. bacteriovorus handling and predation assessment. Our work until the end of the project will also go deeper in the molecular and mechanistic understanding of cell cycle coordination and in chromosome dynamics in particular. Preliminary data indicate novel regulation mechanisms for the highly conserved segregation protein ParB and we have started the functional dissection of this protein. We also aim at providing a mechanistic study of how cell polarity is established in bacteria that proliferate by non-binary division. Finally, the molecular reporters that we generated during the first part of this project will be exploited to quantitatively assess the impact of prey features (envelope integrity, surface content, cellular physiology, cell size…) on the predation cycle, e.g. prey invasion efficiency, predator growth rate and duration, progeny numbers and size.
Predatory lifecycle of Bdellovibrio bacteriovorus, from the perspective of chromosome dynamics