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1.
Nat Commun ; 14(1): 7489, 2023 Nov 18.
Article in English | MEDLINE | ID: mdl-37980336

ABSTRACT

Bacterial chromosomes are dynamically and spatially organised within cells. In slow-growing Escherichia coli, the chromosomal terminus is initially located at the new pole and must therefore migrate to midcell during replication to reproduce the same pattern in the daughter cells. Here, we use high-throughput time-lapse microscopy to quantify this transition, its timing and its relationship to chromosome segregation. We find that terminus centralisation is a rapid discrete event that occurs ~25 min after initial separation of duplicated origins and ~50 min before the onset of bulk nucleoid segregation but with substantial variation between cells. Despite this variation, its movement is tightly coincident with the completion of origin segregation, even in the absence of its linkage to the divisome, suggesting a coupling between these two events. Indeed, we find that terminus centralisation does not occur if origin segregation away from mid-cell is disrupted, which results in daughter cells having an inverted chromosome organisation. Overall, our study quantifies the choreography of origin-terminus positioning and identifies an unexplored connection between these loci, furthering our understanding of chromosome segregation in this bacterium.


Subject(s)
Escherichia coli Proteins , Escherichia coli , Escherichia coli/genetics , Chromosomes , Escherichia coli Proteins/genetics , Chromosomes, Bacterial/genetics , Chromosome Segregation , Cell Movement , DNA Replication , Replication Origin/genetics
2.
Biophys J ; 122(9): 1577-1585, 2023 05 02.
Article in English | MEDLINE | ID: mdl-36966362

ABSTRACT

Fluorescent microscopy is the primary method to study DNA organization within cells. However, the variability and low signal/noise commonly associated with live-cell time-lapse imaging challenges quantitative measurements. In particular, obtaining quantitative or mechanistic insight often depends on the accurate tracking of fluorescent particles. Here, we present ★Track, an inference method that determines the most likely temporal tracking of replicating intracellular particles such DNA loci while accounting for missing, merged, and spurious detections. It allows the accurate prediction of particle copy numbers as well as the timing of replication events. We demonstrate ★Track's abilities and gain new insight into plasmid copy number control and the volume dependence of bacterial chromosome replication initiation. By enabling the accurate tracking of DNA loci, ★Track can help to uncover the mechanistic principles of chromosome organization and dynamics across a range of systems.


Subject(s)
DNA Replication , DNA , DNA/genetics , Microscopy , Chromosomes, Bacterial/genetics
3.
Elife ; 102021 04 15.
Article in English | MEDLINE | ID: mdl-33856342

ABSTRACT

Translesion synthesis (TLS) is a highly conserved mutagenic DNA lesion tolerance pathway, which employs specialized, low-fidelity DNA polymerases to synthesize across lesions. Current models suggest that activity of these polymerases is predominantly associated with ongoing replication, functioning either at or behind the replication fork. Here we provide evidence for DNA damage-dependent function of a specialized polymerase, DnaE2, in replication-independent conditions. We develop an assay to follow lesion repair in non-replicating Caulobacter and observe that components of the replication machinery localize on DNA in response to damage. These localizations persist in the absence of DnaE2 or if catalytic activity of this polymerase is mutated. Single-stranded DNA gaps for SSB binding and low-fidelity polymerase-mediated synthesis are generated by nucleotide excision repair (NER), as replisome components fail to localize in the absence of NER. This mechanism of gap-filling facilitates cell cycle restoration when cells are released into replication-permissive conditions. Thus, such cross-talk (between activity of NER and specialized polymerases in subsequent gap-filling) helps preserve genome integrity and enhances survival in a replication-independent manner.


Subject(s)
Bacterial Proteins/metabolism , Caulobacter crescentus/enzymology , DNA Breaks, Single-Stranded , DNA Repair , DNA Replication , DNA, Bacterial/biosynthesis , DNA-Directed DNA Polymerase/metabolism , Bacterial Proteins/genetics , Caulobacter crescentus/genetics , Caulobacter crescentus/growth & development , DNA, Bacterial/genetics , DNA-Directed DNA Polymerase/genetics , Gene Expression Regulation, Bacterial , Microbial Viability , Mutagenesis
4.
FEMS Microbiol Lett ; 365(22)2018 11 01.
Article in English | MEDLINE | ID: mdl-30304479

ABSTRACT

The factors contributing to antibiotic resistance in bacteria are an important area of study. Sodium salicylate (NaSal), a non-steroidal anti-inflammatory drug (NSAID), increases antibiotic resistance by inducing the expression of MarA, a transcription factor, which increases the AcrAB-TolC efflux pump. MarA is a substrate of Lon protease and the Δlon strain displays a high degree of antibiotic resistance. This study was initiated to identify commonly used NSAIDs that may induce antibiotic resistance and to compare their efficacies with NaSal and acetyl salicylic acid (ASA). Quantitative real-time expression analysis revealed induction of marA and acrB by NaSal, ASA, acetaminophen (APAP) and ibuprofen. Further, dose studies demonstrated that NaSal and ASA induce resistance at ∼2 mM while APAP and ibuprofen induce resistance at ∼5-10 mM. To dissect the roles of key molecules, atomic force microscopy and functional studies were performed using WT, Δlon, ΔmarA, ΔacrB, ΔlonΔmarA and ΔlonΔacrB strains. The induction of antibiotic resistance by NaSal, ASA and APAP is relatively higher and is partly dependent on marA, whereas ibuprofen which induces lower antibiotic resistance shows complete marA dependence. Notably, NaSal, ASA, APAP and ibuprofen induce antibiotic resistance in an acrB-dependent manner. The possible significance of some NSAIDs in inducing antibiotic resistance is discussed.


Subject(s)
Acetaminophen/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Anti-Inflammatory Agents/pharmacology , DNA-Binding Proteins/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/drug effects , Ibuprofen/pharmacology , Multidrug Resistance-Associated Proteins/metabolism , DNA-Binding Proteins/genetics , Drug Resistance, Bacterial/drug effects , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Microscopy, Atomic Force , Multidrug Resistance-Associated Proteins/genetics , Protease La/genetics , Protease La/metabolism , Sodium Salicylate , Transcription Factors/genetics , Transcription Factors/metabolism
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