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Microfluidic time-lapse analysis and reevaluation of the Bacillus subtilis cell cycle.

ABSTRACT: Recent studies taking advantage of automated single-cell time-lapse analysis have reignited interest in the bacterial cell cycle. Several studies have highlighted alternative models, such as Sizer and Adder, which differ essentially in relation to whether cells can measure their present size or their amount of growth since birth. Most of the recent work has been done with Escherichia coli. We set out to study the well-characterized Gram-positive bacterium, Bacillus subtilis, at the single-cell level, using an accurate fluorescent marker for division as well as a marker for completion of chromosome replication. Our results are consistent with the Adder model in both fast and slow growth conditions tested, and with Sizer but only at the slower growth rate. We also find that cell size variation arises not only from the expected variation in size at division but also that division site offset from mid-cell contributes to a significant degree. Finally, although traditional cell cycle models imply a strong connection between the termination of a round of replication and subsequent division, we find that at the single-cell level these events are largely disconnected.

SUBMITTER: Lee S

PROVIDER: S-EPMC6813450 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Microfluidic time-lapse analysis and reevaluation of the Bacillus subtilis cell cycle.

Lee Seoungjun S Wu Ling Juan LJ Errington Jeff J

MicrobiologyOpen 20190613 10

Recent studies taking advantage of automated single-cell time-lapse analysis have reignited interest in the bacterial cell cycle. Several studies have highlighted alternative models, such as Sizer and Adder, which differ essentially in relation to whether cells can measure their present size or their amount of growth since birth. Most of the recent work has been done with Escherichia coli. We set out to study the well-characterized Gram-positive bacterium, Bacillus subtilis, at the single-cell l ...[more]

PMID: 31197963

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Project description:The physiological heterogeneity of cells within a microbial population imparts resilience to stresses such as antimicrobial treatments and nutrient limitation. This resilience is partially due to a subpopulation of cells that can survive such stresses and regenerate the community. Microfluidic approaches now provide a means to study microbial physiology and bacterial heterogeneity at the single cell level, improving our ability to isolate and examine these subpopulations. Drop-based microfluidics provides a high-throughput approach to study individual cell physiology within bacterial populations. Using this approach, single cells are isolated from the population and encapsulated in growth medium dispersed in oil using a 15 ?m diameter drop making microfluidic device. The drops are arranged as a packed monolayer inside a polydimethylsiloxane (PDMS) microfluidic device. Growth of thousands of individual cells in identical microenvironments can then be imaged using confocal laser scanning microscopy (CLSM). A challenge for this approach has been the maintenance of drop stability during extended time-lapse imaging. In particular, the drops do not maintain their volume over time during incubation in PDMS devices, due to fluid transport into the porous PDMS surroundings. Here, we present a strategy for PDMS device preparation that stabilizes drop position and volume within a drop array on a microfluidic chip for over 20 h. The stability of water-in-oil drops is maintained by soaking the device in a reservoir containing both water and oil in thermodynamic equilibrium. This ensures that phase equilibrium of the drop emulsion fluids within the porous PDMS material is maintained during drop incubation and imaging. We demonstrate the utility of this approach, which we label DropSOAC (Drop Stabilization On A Chip), for time-lapse studies of bacterial growth. We characterize growth of Pseudomonas aeruginosa and its ?hpf mutant derivative during resuscitation and growth following starvation. We demonstrate that growth rate and lag time heterogeneity of hundreds of individual bacterial cells can be determined starting from single isolated cells. The results show that the DropSOAC capsule provides a high-throughput approach toward studies of microbial physiology at the single cell level, and can be used to characterize physiological differences of cells from within a larger population.

Project description:BackgroundChlamydia trachomatis is a major human pathogen with a unique obligate intracellular developmental cycle that takes place inside a modified cytoplasmic structure known as an inclusion. Following entry into a cell, the infectious elementary body (EB) differentiates into a non-infectious replicative form known as a reticulate body (RB). RBs divide by binary fission and at the end of the cycle they redifferentiate into EBs. Treatment of C.trachomatis with penicillin prevents maturation of RBs which survive and enlarge to become aberrant RBs within the inclusion in a non-infective persistent state. Persistently infected individuals may be a reservoir for chlamydial infection. The C.trachomatis genome encodes the enzymes for peptidoglycan (PG) biosynthesis but a PG sacculus has never been detected. This coupled to the action of penicillin is known as the chlamydial anomaly. We have applied video microscopy and quantitative DNA assays to the chlamydial developmental cycle to assess the effects of penicillin treatment and establish a framework for investigating penicillin induced chlamydial persistence.Principal findingsAddition of penicillin at the time of cell infection does not prevent uptake and the establishment of an inclusion. EB to RB transition occurs but bacterial cytokinesis is arrested by the second binary fission. RBs continue to enlarge but not divide in the presence of penicillin. The normal developmental cycle can be recovered by the removal of penicillin although the large, aberrant RBs do not revert to the normal smaller size but remain present to the completion of the developmental cycle. Chromosomal and plasmid DNA replication is unaffected by the addition of penicillin but the arrest of bacterial cytokinesis under these conditions results in RBs accumulating multiple copies of the genome.ConclusionsWe have applied video time lapse microscopy to the study of the chlamydial developmental cycle. Linked with accurate measures of genome replication this provides a defined framework to analyse the developmental cycle and to investigate and provide new insights into the effects of antibiotic treatments. Removal of penicillin allows recovery of the normal developmental cycle by 10-20 hrs and the process occurs by budding from aberrant RBs.

Dataset Information

Microfluidic time-lapse analysis and reevaluation of the Bacillus subtilis cell cycle.

Publications

Microfluidic time-lapse analysis and reevaluation of the Bacillus subtilis cell cycle.

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