Strains and growth conditions
In this study,E. coliK-12 MG1655 [F- lambda- ilvG rfb-50 rph-1 glpK(G184T)] was used. The E. coliderivatives were cultured in lysogeny broth (LB) or minimal M9 medium supplemented with 0.4% glucose as the carbon source at 37 °C with a shaking. The composition of the M9 solution was as follows: 33.9 g Na2HPO4, 15 g KH2PO4, 5 g NH4Cl, and 2.5 g NaCl (prepared in 1 L distilled water). The following components were sterilized and added to the M9 minimal medium: 200 mL of M9 solution, 2 mL of 1 M MgSO4 solution, 0.1 mL of 1 M CaCl2 solution, and 20 mL of glucose (20%) (for 1 L medium). The composition of the LB medium was as follows: 10 g tryptone, 5 g yeast extract, and 5 g NaCl (prepared in 1 L distilled water) and supplemented with antibiotics (100 μg/mL ampicillin or 50 μg/mL kanamycin). The antibiotics were used to select the E. coli MG1655 and its derivatives. Nutrient-rich media (LB, LB supplemented with 0.4% glucose, YT, and terrific broth (TB) medium) or minimal media (M9 medium supplemented with 0.4% glucose or 0.2% glycerol) were used to test the repeatability and reproducibility of the cell growth rate.
Selection of ectopic sites
The ectopic replication origin was introduced in the convergent intergenic region between dadX and cvrA and the coding region oflacZ in theE. coliMG1655 genome, which did not result in adverse effects on growth or physiology. Genomic properties, including the orientation of genes, location of promoters, and binding sites of transcription factors were elucidated using the EcoGene (http://www.ecogene.org/), EcoCyc (https://ecocyc.org/), and RegulonDB (http://regulondb.ccg.unam.mx/)[30-33].
Genetic manipulation of E. coli
oriC-mioCand oriCdeletion cassettes were used to integrate a native replication origin into the ectopic loci and delete the original replication origin. oriCand mioCwere PCR-amplified from the genomic DNA of E. coliMG1655. The kanamycin resistance gene with two flippase (FLP) recognition target(FRT) sequences was amplified from pKD4. The oriC-mioC cassette comprised the replication origin of chromosome (oriC), the mioC gene, a selectable kanamycin resistance gene, two FRTsites, and two homology extension sequences (35 bp). The oriCdeletion cassette comprised two homology extension sequences (50 bp) and a kanamycin resistance gene. The insertion of oriC-mioC or oriC deletion cassette into the target regions, such as lacZ, intergenic region, and native replication origin was performed using λ Red recombination with the temperature-sensitive plasmid pKD46 expressing the λ Red proteins(γ, β, and exo). The recombinant E. coli cells harboring pKD46 were cultured at 30 °C. The expression of λ Red proteins was induced using 10 mM L-arabinose. The target regions were directly disrupted by transforming the oriC-mioC or oriCdeletion cassettes. The recombinant E. coli cells were selected on L agar plates containing antibiotics and the genotype was confirmed using direct colony PCR. After the curing step of pKD46, kanamycin resistance genes of E. coliderivatives with multiple replication origins were eliminated using FLP/FRT recombination through the transformation of the FLP helper plasmid pCP20. The pCP20 plasmid expresses FLP recombinase that acts on the repeated FRT sites flanking the resistance gene. Next, pCP20 was eliminated by culturing the cells at 42 °C.
Growth rate analysis
For Erlenmeyer flask culture, each strain was inoculated into 3 mL of LB or M9 minimal medium supplemented with 0.4% glucose and cultured overnight at 37 °C. The optical density at 600 nm (OD600nm) was measured using an OPIZEN POP spectrophotometer (Mecasys, Korea). The OD600nm value of the stationary phase cultures of E. coli was diluted to less than 0.05 in 50 mL of fresh medium. The diluted samples were cultured to an OD600nmvalue of less than 0.5. The amount of inoculum for determining the initial OD600nmat time zero was calculated based on the OD600nm value of 0.05 in 50 mL fresh growth medium. The exponential phase cultures were then diluted in fresh growth medium and cultured to an OD600nm value of 0.3 for growth rate analysis. Therefore, E. colicells in the exponential phase were used to determine the growth rate. Growth curve analysis of E. coli MG1655 and its derivatives was performed by measuring the OD600nm of triplicate cultures. The OD600nm values of exponential phase E. colicultures were recorded at a range of 0.05–0.3 to calculate the specific growth rate. The specific growth rate (μ), whichwas defined as the linear slope in a plot of ln (OD600nm) versus time (h), was calculated using the following equation:
where N or N0is the optical density of E. coli cells in a flask at the time in the exponential phase or at time zero, respectively, and t is the cultivation time. Additionally, the specific growth rates of wild-type and O3lacZ dadXstrains from triplicate samples were verified using a plate reader (Epoch 2 microplate spectrophotometer, BioTek, USA). The following settings were employed for OD measurements: interval time, 20 min, shaking speed, 330 cpm, wavelength, 600 nm, temperature, 37 °C, temperature gradient, 1 °C, read speed, normal, read delay, 100 ms, number of measurements/data point, 8. The log phase E. coliculture was re-inoculated into 200μL of fresh culture medium in 96-well plates. TheOD600nmwas measured using a microplate reader to determine the specific growth rates using the sameprocessesas described above.
Cell morphology analysis
The stationary or exponential phase E. coli cultures were harvested and subjected to Gram staining. The stained E. coli cells were observed using a Nikon Eclipse Ci-E microscope under a 100X immersion oil lens (NA 1.45). The images were captured using NIS-Elements software. Cell length (l) and width (w) were analyzed using the image analysis program ImageJ with the ObjectJ plugin (Object-Image version 2.21). The length and width of 200 individual cells were measured under each growth condition. For cell volume (V) measurements, the cells were assumed to have a cylindrical structure capped by two hemispherical ends as the dividing cells only exhibited an "8" morphology. Cell volume was calculated by measuring the length and width of dividing cells using the following equation:
Cell viability analysis
Cell viability was determined using the Live/Dead BacLight bacterial viability kit (Molecular Probes, L7012). The E. colicells were cultivated in a 250 mL flask containing 50 mL of LB medium until the late log phase. The bacterial culture (25 mL) was centrifuged at 1,000 g for 15 min and the pellet was resuspended in 2 mL of 0.85% NaCl. Next, 1 mL of E. coli suspension was mixed with 20 mL of 0.85% NaCl (for live cells) or 70% isopropanol (for dead cells) in 50 mL Falcon tubes and incubated for 1 h. The samples were then centrifuged at 1000 g for 15 min. The pellet was resuspended and aliquoted into separate tubes containing 10 mL of 0.85% NaCl. The OD600nm was measured to determine the number of E. coli cells. The E. colisuspension was mixed in different ratios of live cells to dead cells (100:0, 50:50, and 0:100) to a volume of 3 mL and the number of cells was adjusted to 1×107 cells/mL (~0.01 OD600nm).The E. colisuspension (3 mL) containing live or dead cells was incubated with 6 μL of the SYTO9 (3.34 mM) and propidium iodide (20 mM) (1:1) mixture at room temperature in the dark for more than 15 min. The number of live or dead cells was calculated using flow cytometric analysis. The SYTO9 and propidium iodide fluorescence intensities were measured at emission wavelengths of were measured at 538 and 620 nm, respectively, and an excitation wavelength of 485 nm using a flow cytometer (BD LSR II cytometer, BD Biosciences). The relative bacterial cell viability was then obtained using the following equation:
Flow cytometry analysis
The number of origins per cell was determined using the replication run-out method. The fresh stationary phase cultures were diluted 200-fold to an OD600nm of 0.01 and cultured to an OD600nm of 0.4 in LB medium. For replication run-out, the exponential phase culture was incubated with rifampin (150μg/mL, Sigma-Aldrich, USA) and cephalexin (10μg/mL, Sigma-Aldrich, USA) to prevent replication initiation and cell division for at least 3 h and chilled on ice for 5 min. All steps were performed at 4 °C. The cultures (2 mL) of the wild type or its derivatives were centrifuged at 300 g for 15 min. The pellet was washed twice with 1 mL Tris-EDTA (TE) buffer (10 mM Tris HCl (pH 7.4) and 10 mM EDTA), resuspended in 0.1 mL TE buffer, and fixed with 0.9 mL of 77% ethanol at −20 °C overnight or longer. The fixed sample was washed twice with 1 mL of TM buffer (10 mM Tris HCl (pH 7.4) and 10 mM MgSO4). The OD600nm of the cell suspensions was then determined. The final volume of the cell suspension (1×106 cells) was adjusted to 0.5–1 mL in TM buffer. Next, the cells were incubated with Hoechst 33342 (Molecular Probes, USA) solution in TM buffer (final concentration of 0.5μg/mL) for at least 30 min in the dark. Flow cytometry was performed using a flow cytometer (BD LSR II cytometer, BD Biosciences, USA). A Coherent Sapphire 488 nm laser was used to generate forward and side scatter signals, while a Lightwave Xcite 355 nm (UV) laser was used to generate fluorescence signals. Additionally, a 505 LP dichroic mirror and a 440/40 band-pass filter were used to detect Hoechst 33342 fluorescence. Raw data (fcs3.0) were transformed and analyzed using FlowJo (version 10.2).
Marker frequency analysis
The copy numbers of oriCand specific regions from oriCto terC were determined using qRT-PCR analysis. The delta delta CT (2-ΔΔCT) method was used for relative quantification[41,42]. The E. coli cells for analyzing replicating genomic DNA samples were cultured to an OD600nm of 0.4, treated with sodium azide (30μg/mL, Fluka BioChemika, Switzerland), and incubated for 3 h at 37 °C. Genomic DNA was extracted using the Wizard® genomic DNA purification kit (Promega) and amplified using qRT-PCR system (ABI 7300, Applied Biosystems, USA) with premix (iTaq™ Universal SYBR® Green Supermix, Bio-Rad, USA). Twelve loci that included proximal regions for both oriCand terC,as well as specific priming regions per 500 kb from the origin (oriC) to terminus (terC) along the right and left replichores were used to profile the growth-dependent differences in the copy numbers in each priming region (see Additional file 1: Table S2). Cycle threshold (Ct) obtained from wild-type E. coli or its derivatives was analyzed using the 2−ΔΔCt method. Marker frequency ratios were normalized to the oriC to terC ratio of each reference genomic DNA sample from E. coli cells treated with rifampicin (150μg/mL, Sigma-Aldrich, USA) and cephalexin (10μg/mL, Sigma-Aldrich, USA).
Replication profile analysis
The read counts across the genome of E. colistrains were determined using replication profile analysis. Genomic DNA from cultures of E. colistrains was extracted as described in the marker frequency analysis and subjected to next-generation sequencing. The genomic DNA was sequenced using Illumina HiSeqTM (50 bp or 100 bp single reads) or NovaSeq at Macrogen, Korea. The sequencing reads were imported into the CLC Genomics Workbench 9.0 (QIAGEN Bioinformatics, Germany) and assembled to the reference chromosome. The assembly was performed with the following parameters: 0.01 trim using quality scores, 15 discard reads below length, 2 mismatch cost, 3 insertion cost, 3 deletion cost, 0.5 length fraction, and 0.8 similarity fraction. Assembly data were mapped to the reference chromosome. The CSV files of the resulting mapping data were exported, and the average coverage per 1,000 bp was calculated.
Measurement of cell cycle parameters
The ratio of oriCto terC (oriC/terC) was determined using the marker frequency analysis described above. The C period was calculated using the following equation:
whereis the generation time of each E. coli strain in the exponential phase. Furthermore, the chromosome number at the exponential phase was determined using flow cytometry analysis as described above. The exponential phase of E. coliculture in LB medium was assumed to contain a mixture of 4N cells that have not initiated DNA replication and 8N cells that have completed DNA replication. Each fraction of the DNA contents was multiplied by chromosome equivalents (4 or 8) and added to determine the oriCper cell. The D period was calculated using the following formula:
The initial age (ai) was measured using the following equation:
where Fwas the fraction of E. colicells that have not initiated DNA replication.