An optimized workflow to measure bacterial predation in microplates

Remy, Ophélie; Lamot, Thomas; Santin, Yoann G.; Kaljević, Jovana; Pierpont, Charles de; Laloux, Géraldine

doi:10.1016/j.xpro.2021.101104

Cited by 15 publications

(32 citation statements)

References 14 publications

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“…C. crescentus prey cells were co-incubated with B. exovorus in a microplate for 16h at 30°C. Optical density at 660 nm (here represented as the percentage of the initial population) was monitored over time and metrics were extracted using CuRveR 40 . r max corresponds to the killing rate, and s is the time point at which r max reaches its maximum value.…”

Section: Resultsmentioning

confidence: 99%

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Lifecycle of a predatory bacterium vampirizing its prey through the cell envelope and S-layer

Santin,

Sogues,

Bourigault

et al. 2023

Preprint

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Predatory bacteria feed upon and kill other bacteria in various natural environments. Obligate epibiotic predators like Bdellovibrio exovorus consume their prey whilst remaining attached to the outside of the prey. How these predators achieve epibiotic feeding through the prey cell envelope has not been explored previously. Whereas the S-layer is the only proposed defensive structure against predatory bacteria, it remains unclear how this thin outer layer of the envelope might prevent epibiotic attacks. Similarly, the lifecycle of B. exovorus during the predator-prey interaction is poorly understood, with current models suggesting a binary division. Here we imaged the entire predatory lifecycle of B. exovorus and the fate of its Caulobacter crescentus prey by time-lapse microscopy and cryo-electron microscopy to monitor predator attack, growth and division and assess the impact of the S-layer on epibiotic predation. Our data reveal that B. exovorus uses non-binary division in a novel proliferation pattern that mainly generates three progenies. Moreover, we found that B. exovorus predates regardless of the presence of an S-layer, calling for revisiting its protective role against predators. Finally, our results indicate that epibiotic predation relies on the establishment of a secured junction between the prey and predator outer membranes, which must be resolved unilaterally to maintain cellular integrity of the predator departing from the prey surface.

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Section: Resultsmentioning

confidence: 99%

“…Decrease of OD 660 indicates prey lysis, as B. exovorus cells do not affect absorbance. Predation kinetics metrics were extracted using CuRveR as previously described 40 .…”

Section: Methodsmentioning

confidence: 99%

Lifecycle of a predatory bacterium vampirizing its prey through the cell envelope and S-layer

Santin,

Sogues,

Bourigault

et al. 2023

Preprint

Self Cite

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“…Plasmids were maintained by adding kanamycin (50 μg.mL −1 ) or chloramphenicol (15 μg.mL −1 ). B. bacteriovorus strains were routinely grown in DNB medium (Dilute Nutrient Broth, Becton, Dickinson and Company, supplemented with 2 mM CaCl 2 and 3 mM MgCl 2 salts) with E. coli MG1655 as prey at 30°C and constant shaking as previously described [37].…”

Section: Methodsmentioning

confidence: 99%

“…B. bacteriovorus strains were first grown overnight on WT MG1655 (as described [37]) before the start of the imaging experiment. For time-lapse imaging of synchronous predation cycles, E. coli prey cells were grown in LB medium (or supplemented M9 when indicated) to late exponential phase (OD 600 = 0.8), harvested at 2600 x g at room temperature (RT) for 5 minutes, washed twice and resuspended in DNB-salts medium.…”

Section: Methodsmentioning

confidence: 99%

Modulation of prey size reveals adaptability and robustness in the cell cycle of an intracellular predator

Santin

Lamot

Kaljević

et al. 2022

Preprint

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Despite the remarkable diversity of bacterial lifestyles, the sophisticated regulatory networks underlying bacterial replication have only been investigated in a limited number of model species so far. In bacteria that do not rely on canonical binary division for proliferation, the coordination of major cellular processes is still mysterious. Moreover, bacterial growth and division remain largely unexplored within spatially confined niches where nutrients are limited. This includes the lifecycle of the model endobiotic predatory bacterium Bdellovibrio bacteriovorus, which grows by filamentation within its host or prey cells and produces a variable number of daughter cells. Here, we examined how the size of the micro-compartment in which predators replicate (i.e., the prey bacterium) impacts their cell cycle progression at the single-cell level. Using Escherichia coli with genetically encoded size differences, we show that the duration of the predator cell cycle scales with prey size. As a result, the size of the prey determines the number of predator offspring, through a relationship that is maintained across prey species. Strikingly, the nutritional quality of the prey cell determined the specific growth rate of predators regardless of prey size, reminiscent of the effect of medium composition on the growth of other bacteria. Tuning the predatory cell cycle by modulating prey dimensions also allowed us to reveal invariable temporal connections between key cellular processes. Altogether, our data uncover adaptability and robustness shaping the enclosed replication of B. bacteriovorus. Consequently, predators optimally exploit the finite prey resources and space while ensuring a strict cell cycle progression. This study opens the way to explore diverse cell cycle control strategies by extending their characterization to intracellular lifestyles, beyond canonical models and growth conditions.

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“…This is an undeniable bottleneck as it delays interpreting experimental results. To address this, researchers have adopted a variety of protocols and genetic tools, including the use of fluorescent predatory strains ( 8 , 9 ), bioluminescent prey ( 10 ), SYBR green ( 11 ), or flow cytometry ( 12 ). While each offers some benefits, they also have their own limitations, including the need for genetic engineering of the predator or prey, which may not always be possible, or problems differentiating between live and dead predators.…”

Section: Observationmentioning

confidence: 99%

Use of Resazurin To Rapidly Enumerate Bdellovibrio and Like Organisms and Evaluate Their Activities

et al. 2022

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Bdellovibrio and like organisms are predatory bacteria that are capable of attacking, killing, and consuming many bacterial pathogens, including multidrug-resistant strains. These qualities have led to them being labeled as “living antibiotics.” Research work with these remarkable strains, however, has been hampered by long growth times needed to quantify the predatory populations through plaque assays, which typically take 4 days to develop.

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An optimized workflow to measure bacterial predation in microplates

Cited by 15 publications

References 14 publications

Lifecycle of a predatory bacterium vampirizing its prey through the cell envelope and S-layer

Lifecycle of a predatory bacterium vampirizing its prey through the cell envelope and S-layer

Modulation of prey size reveals adaptability and robustness in the cell cycle of an intracellular predator

Use of Resazurin To Rapidly Enumerate Bdellovibrio and Like Organisms and Evaluate Their Activities

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