DNA Replication in Engineered Escherichia coli Genomes with Extra

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DNA replication in engineered Escherichia coli genomes with extra replication origins Sarah Milbredt, Neda Farmani, Patrick Sobetzko, and Torsten Waldminghaus ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00064 • Publication Date (Web): 07 Jun 2016 Downloaded from http://pubs.acs.org on June 9, 2016

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DNA replication in engineered Escherichia coli genomes with extra replication origins Sarah Milbredt, Neda Farmani, Patrick Sobetzko and Torsten Waldminghaus LOEWE Center for Synthetic Microbiology, SYNMIKRO, Philipps-University Marburg, Hans-MeerweinStr. 6, D-35043 Marburg, Germany Abstract The standard outline of bacterial genomes is a single circular chromosome with a single replication origin. From the bioengineering perspective it appears attractive to extent this basic setup. Bacteria with split chromosomes or multiple replication origins have been successfully constructed in the last years. The characteristics of these engineered strains will largely depend on the respective DNA replication patterns. However, the DNA replication has not been investigated systematically in engineered bacteria with multiple origins or split replicons. Here we fill this gap by studying a set of strains consisting of (i) E. coli strains with an extra copy of the native replication origin (oriC), (ii) E. coli strains with an extra copy of the replication origin from the secondary chromosome of Vibrio cholerae (oriII) and (iii) a strain in which the E. coli chromosome is split in two linear replicons. A combination of flow cytometry, microarray-based comparative genomic hybridization (CGH) and modelling revealed silencing of extra oriC copies and differential timing of ectopic oriII copies compared to the native oriC. The results were used to derive construction rules for future multiorigin and multi-replicon projects. Keywords Genetic engineering, bacteria, synthetic chromosomes, synthetic genomes, genome design Introduction General rules derived from observations in naturally occurring organisms do increasingly motivate synthetic biology approaches to probe the limits of such rules. For example, bacteria usually possess a single circular chromosome, which is replicated from a single replication origin. By linearization of the circular E. coli chromosome it was shown that bacteria could in principle tolerate a linear chromosome very well (1). Previous studies also revealed that E. coli can deal with an additional replication origin, oriC, on the same chromosome (2). The ectopic origin, oriZ, was located 1 Mb apart from the original oriC and showed a similar timing of replication initiation. Notably, it was possible to delete oriC and completely run the chromosome based on oriZ. Although these cells were viable, they showed a strong growth defect and concomitantly an accumulation of suppressor mutants (3). Nevertheless, these results demonstrate a general robustness and adaptability of the bacterial chromosome. Such genome alterations do not only increase our understanding of basic biological processes on the molecular level, but could also facilitate genome engineering and potentially synthetic circuit design. It was for example argued that splitting of the chromosome into smaller replicons would allow editing in vitro or in yeast, which is not possible with large chromosomes (4-6). Splitting of a bacterial chromosome would certainly require a second replication origin. One option is to use the replication origin of a low copy number plasmid to drive replication of the secondary replicon as it was successfully done in Bacillus subtilis (7). Alternatively the bipartite genome of V. cholerae can be seen as ingenious template for chromosome splitting approaches (8). V. cholerae has two different sized chromosomes: the primary chromosome (ChrI) with 2.96 Mbp and the secondary 1 ACS Paragon Plus Environment


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chromosome (ChrII) with 1.07 Mbp (9). Initiation of replication starts on both chromosomes at different origins of replication. The origin of ChrI (oriI) is similar to oriC from E. coli and is initiated by the highly conserved DnaA protein (10, 11). In contrast, the origin of ChrII (oriII) is driven by the initiator protein RctB, which is unique to the group of Vibrionaceae (10-12). The timing of origin firing is different with oriII starting only after about 2/3 of the primary chromosome have been replicated from oriI (13-15). However, it is not well understood how the coordinated replication of both chromosomes during the cell cycle is regulated. It was proposed that oriI/ChrI acts as pace maker to command oriII (15). This would make the timing of replication initiation at oriII directly dependent on the timing of oriI initiation. The details of timing of oriI firing are not fully uncovered but might be similar to E. coli oriC. In E. coli timing is regulated by at least two main mechanisms. First, the regulatory inactivation of DnaA (RIDA) is based on a switch from active ATP-bound DnaA to inactive ADP-DnaA with regard to the binding activity to certain DnaA binding sites at the origin (16, 17). Also DnaA in V. cholerae was found to bind and hydrolyze ATP (12). The second timing mechanism is based on the SeqA protein binding to hemi-methylated GATC sites in the origin region to temporarily block DnaA from binding (18, 19). SeqA and methylation have also been shown to participate in oriI timing in V. cholerae (20-22). The Mazel group constructed V. cholerae strains with large scale genome rearrangements leading to strains with only a single chromosome replicated by oriI or two chromosomes both driven by oriI (23). Previous studies also showed that oriII from V. cholerae can replicate a small replicon in E. coli and this fact was actually used to define the V. cholerae ChrII replication origin, oriII (10). More recently we have constructed a synthetic, secondary chromosome in E. coli based on oriII from V. cholerae (5). The V. cholerae oriII was also used in a chromosome splitting approach by Liang et al. (6). They used two components of the bacteriophage N15 system: the telomerase occupancy site element (tos) and the protelomerase protein (TelN) (24). Pairs of tos sites were inserted at various positions on the E. coli chromosome and an additional replication origin between them (6). It was then tested if plasmidderived expression of the TelN protelomerase could produce viable cells, which should harbor a split chromosome. Interestingly, this was not possible with different arrangements of a secondary copy of E. coli oriC. If, however, oriII from V. cholerae was used as secondary origin, splitting of the chromosome was possible in one case (6). Replication characteristics of engineered multi-origin and multi-chromosome strains will determine the success and output of future genome-rearrangement projects but remain largely unexplored. In the present study we analyze a set of E. coli strains with additional ectopic insertions of replication origins (6). Our work suggests that oriII from V. cholerae is the replication origin of choice for multichromosome approaches in E. coli and functions fully orthogonal regarding replication timing. In addition, our results explain why it was not possible to split the E. coli chromosome based on some of the origin arrangements in a previous study (6). Results and Discussion Ectopic copies of oriC in engineered E. coli strains are not functional To characterize the DNA replication in engineered E. coli strains with extra replication origins we applied two main approaches throughout this study. First is the analysis of DNA content on single cell level by respective staining and analysis by flow cytometry. In fast growing wildtype cultures the corresponding histogram shows a distribution of cells with different DNA contents according to different stages of overlapping replication cycles (Fig. 1 A). A more detailed picture of the replication 2 ACS Paragon Plus Environment

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state is generated with so called run-out experiments in which cells are treated with rifampicin that stops the initiation of DNA replication and cephalexin, which inhibits cell division (25). Ongoing replication rounds will be finished to produce cells with integer numbers of chromosomes corresponding to the number of replication origins in the cell at the time point of drug treatment. In E. coli wildtype cells, chromosome copy numbers will be 1, 2, 4, 8 or 16 after rifampicin/cephalexin treatment. Under the conditions used here, most wildtype cells have four replication origins and some cells have 2 and 8 as indicated by the chromosome copy number after rifampicin/cephalexin treatment (Fig. 1 B). The second approach that was used to analyze DNA replication is the profiling of genome wide copy numbers by comparative genomic hybridization (CGH). In exponentially growing cultures the copy number of a replication origin will always be higher compared to the replication terminus with an exponential distribution for the regions in-between as seen for wildtype E. coli (Fig. 1 C+D). In conclusion, active replication origins are detectable as maxima in a respective copy number plot. A computer program was implemented to determine exact positions of maxima and minima based on the CGH analysis (see Method section for details). For the wildtype E. coli the resulting maximum was close to the actual oriC position at a about 32 kbp to the left of oriC corresponding to only 0.7 % deviation relative to genome size (Tab. 1). Growth curves for all strains used in this study are shown in supplementary figure S1. One approach to split the E. coli chromosome in two linear replicons would be to insert a second copy of the native replication origin oriC at an ectopic location and generate telomere-like sites at two inter-origin sites. Liang and colleagues followed this idea and constructed strains with an extra copy of oriC in different distances to the native oriC (6). We analyzed two of these strains (#2 and #6) regarding their replication behavior by flow cytometry (Fig. 1 E+I). The distribution of cells according to their DNA content was very similar for the engineered strains compared to the wildtype (Fig. 1 A). This indicates that the extra replication origin does not increase the amount of DNA per cell. Also the run-out experiments showed a pattern very similar to the wildtype indicating that the over-all replication is not perturbed by the secondary replication origins (Fig. 1 F+J). Genome-wide DNA copy numbers were determined by CGH to analyze the activity of the two replication origins (Fig. 1 G+K). Interestingly, the pattern of both engineered strains, each with an extra copy of oriC, are very similar to the wildtype genomic pattern (Fig. 1 C). Only one maximum was detected in the copy number pattern corresponding to the position of the native oriC (Fig. 1 H+L). These results clearly indicate that the ectopic oriC copies are not functional in the engineered strains #2 and #6. To check if the strains actually contain the extra oriC copy we performed a PCR using the DNA of the respective strains as template. Results clearly show that the extra oriC copies are inserted correctly (Fig. S2). One reason for the non-functionality of the ectopic replication origins could be mutations. However, sequencing of the extra replication origins revealed an identical sequence of the wildtype and ectopic oriC (Suppl. fig. S3+4). Liang and colleagues assessed the functionality of the ectopic oriC copies by constructing strains in which the native oriC was deleted. The viability of these strains was taken as indication that the ectopic origin must be functional since the cells could not replicate their chromosome otherwise. To see if the ectopic origins are only functional in the absence of the native oriC we analyzed the two strains #3 and #7, which are supposed to encode the extra origins only. To our surprise the strains gave very similar flow cytometry histograms of cellular DNA contents as well as a CGH pattern very similar to strains #2, #6 and wildtype (compare Fig. 1 A with Fig. 2). The maximal DNA copy number was not detected at the position of the ectopic origin but instead at the position of the native oriC. These results clearly indicate that the native oriC is actually not deleted as described by Liang et al. A PCR-based analysis also shows that the native oriC region is not deleted (Fig. S2). Taken together, our results clearly show that the ectopic copies of the E. coli replication 3 ACS Paragon Plus Environment


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origin are not used to initiate DNA replication in the engineered E. coli strains. Notably, Liang and colleagues did not succeed in splitting the E. coli chromosome of the two engineered strains #2 and #6 analyzed here. We can now present the non-functionality of the ectopic replication origins as reason for the impossibility of the strains to live with a split chromosome. Splitting the chromosome would lead to a loss of the chromosome halve encoding the non-functional origin and subsequently to cell death. Why are the ectopic replication origins not functional? One reason could be that bacterial chromosomes tolerate one functional replication origin only. Indeed, no natural occurring bacterial chromosome has been found to date with more than one functional replication origin, although some indications might suggest that there are exceptions (26). However, E. coli strains with a second copy of oriC have been constructed successfully and clearly been shown to be functional (2, 3). The main difference between the two studies is that Liang et al. did use a core oriC sequence only, while Wang et al. cloned an oriC with the flanking genes (2, 6). These flanking genes might actually be able to render an oriC copy functional since for example transcription from the upstream mioC gene was found to be critical for initiation of DNA replication (27, 28). We have found recently that the oriC-neighboring genes show an overrepresentation of the GATC motif and suggested that this might contribute to proper origin function (19, 29). It will be of great importance to further investigate the underlying mechanisms in times of synthetic replicons with increasing size (4, 30). To clarify the role of oriC size for ectopic origin function we transferred the 5.1 kbp ectopic oriC used in the Wang et al. study into strain #2 of Liang et a. (Fig. 3). Respective CGH experiments of logarithmic growing cells in deed showed functionality of this ectopic replication origin with a similar copy number as the native oriC (Fig. 3 D). A smaller oriC region of about 1.2 kbps inserted at the ectopic position of strain #2 did show the same activity (Fig. 3 C). We noticed that the oriC region in the genome sequence annotation (accession number NC_000913.3) is actually lacking one of the DnaA boxes (Fig. 3 A). Notably, Liang et al. used this annotated region as ectopic oriC copy. We hypothesized that this missing DnaA box might render ectopic oriCs non-functional and that the intergenic region between mnmG and mioC including this DnaA box is sufficient for replication initiation. Indeed, a respective oriC insertion of 449 bps was functional in initiating replication at the ectopic site (Fig 3 B). V. cholerae-oriII insertions are functional replication origins in the E. coli chromosome context A reasonable approach to engineer a bacterium with two instead of a single chromosome is to learn from the few examples where naturally occurring bacteria have two chromosomes. The best studied example is Vibrio cholerae, which has two chromosomes with very different replication origins (8, 9, 31). The replication origin of chromosome II (oriII) has been shown to be functional in E. coli and was used as basis for a synthetic secondary chromosome in a bottom-up approach (5, 10). We have seen above that the ability of an origin to drive replication of a plasmid-like replicon does not necessarily mean that it is also capable of replicating chromosome-size molecules. To test if oriII from V. cholerae is a functional replication origin in a chromosome context of the heterologous host E. coli, we analyzed the replication of four E. coli strains which encode an ectopic oriII in addition to the native oriC (6). Flow cytometry measurements of the DNA content in exponentially growing cells showed a wider distribution with an increased average DNA content for strains #17, #19, #20 and, to a lesser extent, #15 (Fig. 4 A,E,I,M compare to Fig. 1 A). Respective run-out experiments revealed subpopulations with other chromosome numbers as the two, four and eight observed in wildtype cells (Fig. 4 B, F, J, N compare to Fig. 1 B). It is important to note that direct conclusions on replication timing of oriII and oriC are not possible based on rifampicin/cephalexin experiments because the two origins have different sensitivities towards rifampicin (13). The uneven numbers of chromosomes after rifampicin run-out could indicate perturbed regulation of DNA replication or a segregation 4 ACS Paragon Plus Environment

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defect. Both phenomena could be caused by a second active replication origin, thus indicating that the ectopic oriII is functional. To verify this assumption we performed CGH to determine the DNA copy number profile for the four engineered E. coli strains. In all four strains we found a copy number peak at the position of the native oriC as expected (Fig. 4 C, G, K, O). In addition, all four copy number patterns showed one additional peak. Notably, this additional peak appeared in the region were the respective oriII copy was inserted into the genome (Fig. 4 D, H, L, P; tab. 1). The results provide further proof that the ectopic oriIIs are functioning as replication origins in the context of the E. coli chromosome. To test if the ectopic replication origins are spontaneously functional we collected samples of strains #17 and #20 in the stationary growth phase and performed CGH (supplementary figure Fig. S5). The genomic copy numbers in the origin regions were not higher than the whole genome average, indicating both replication origins to be silent in this growth phase and not spontaneously functional (Fig. 5 C+D). Simulation of genomic copy number patterns of multi-origin chromosomes Bacterial cells use the copy number gradient from ori to ter for the regulation of gene expression (32, 33). For example, highly transcribed genes are generally located near the replication origin to boost their expression. It was established that small-scale copy number variation can lead to multiple orders of magnitude changes in gene expression within regulatory circuits and could even switch the deterministic control (34). The use of targeted gene copy number variation through additional replication origins for synthetic circuit design is largely unexplored. A prerequisite is, however, deep knowledge on the timing of origin firing. In E. coli all copies of oriC are initiated in synchrony independent of the actual number of origins (35). In contrast, replication at oriII in V. cholerae starts only after about 2/3 of the primary chromosome have been replicated (13, 14). We have found evidence previously that also a small oriII-driven synthetic secondary chromosome in E. coli initiates later than oriC (5). This finding could indicate that the replication timing is dependent on the replication origin and the associated genes only and is functional in a heterologous host. If replication occurs independent at two origins, the respective initiation timing within a cell can be deduced from the copy numbers of the replication origins. A lower copy number corresponds to later initiation and a higher copy number to earlier initiation. Consequently, copy numbers of the origins in a two-oriC strain were found to be the same according to their synchronous initiation (3). A second parameter in this context is the meeting point of the replication forks coming from both origins, corresponding to the minimal copy number between the two replication origins. If the two origins initiate simultaneously the minimum should consequently be right in the middle assuming a constant replication rate (3). We have simulated the correlation of copy number difference and minimum position for the four strains with ectopic oriII copies described above (Fig. S6 A, C, E, G). Experimental values for the origin copy numbers and the minima positions were calculated using a curve fitting algorithm (see Methods section for details). Comparison of the experimental data with the simulation shows a clear discrepancy in many cases (Fig. S6). Notably, the simulation above was based on the restriction of “independent replication of the two origins”. The missing correlation of origin copy numbers and minima positions in the experimental data suggest that the two origins do actually not replicate independent from one another. We have simulated genomic copy numbers in response to initiation timing at two replication origins more systematically incorporating a scenario where oriII initiates after replication forks coming from oriC have passed and copied oriII (Fig. 5 A, B). This would be a situation where the two replication origins are not independent. While a correlation between the ori copy numbers and the minima between them was seen for the scenario where the two origins initiate independently, this correlation was 5 ACS Paragon Plus Environment


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lost when oriII was initiated after it was replicated from orI (Fig. 5 B, compare solid to dashed black line). Taken together, the comparison of the genomic copy number plots with simulated values suggest that initiation of DNA replication at oriII and oriC does not occur simultaneously and is not independent. The exact replication mode appears to be dependent on the position of the ectopic oriII, because respective copy numbers vary greatly from being higher (#20) to being equal (#17) or lower (#15 & #19) compared to oriC (Fig. 4 O, G, C, K). We simulated copy number distributions with constant replication timing but variation of the oriII position (Supplementary fig. S7). Similar to what was found with varied replication delays the copy number of oriII increased dramatically at the position where it is replicated first and then initiated. It is important to note that our simulation can show the origins to be interdependent but the in vivo replication pattern is expected to be much more irregular compared to the simulation because many factors will influence what happens in individual cells. Replication timing of two linear chromosomes in E. coli might be similar to replication of the two chromosomes in Vibrio cholerae As outlined above, the two origins on the engineered E. coli chromosomes influence their replication one another. To examine the replication timing of both origins in the chromosomal background independently, we analyzed a strain in which the E. coli chromosome is split in two linear replicons (Fig. 6) (6). Flow cytometry measurements of the DNA content in an exponential growing culture showed a wider distribution as compared to the wildtype strain (Fig. 6 A compare to Fig. 1 A). Interestingly, the run-out experiment resulted in cells with a DNA content corresponding to either 4+4 or 8+8 linear chromosomes (Fig. 6 B). This could implicate that the copy number of the two replicons is regulated to be equal, which is also true for the two chromosomes occurring naturally in Vibrio cholerae (13, 14). However, this is not necessarily a dedicated regulation mechanism but might simply be caused by dying of cells that did not lose one of the sub-chromosomes. Notably, the oriIIbased replicon synVicII got lost from the E. coli population without selective pressure (5). Similar results were observed for an oriII-based non-essential replicon in V. cholerae (23). The relatively broad distribution of DNA contents in cells might indicate some heterogeneity (Fig. 6). Analysis of the DNA copy numbers by CGH show that the oriC-driven replicon has a higher copy number compared to oriII (Fig 6 C+D). The copy number ratio of oriII to oriC (wt) was 0,75 +/- 0,01. This ratios fits very well with the copy number ratios of the oriII-driven synthetic, secondary chromosome synVicII in E. coli suggesting that copy number regulation is not dependent on replicon size (5). Again, it has to be considered that the copy number might be changed because of chromosome loss and the numbers are therefore not actually related to a timing mechanism. It might also be some peculiarity of this single example of genome architecture analyzed here. Interestingly, there was a special site on the primary chromosome of V. cholerae found to control chromosome II replication (15). Notably, this special site was not included in the engineered E. coli strains analyzed here. A very recent study confirmed the important role of this so called crtS site in timing of chromosome II replication in V. cholerae (36). It would be highly interesting in the future to study the effect of this site on the strains analyzed here. In summary, our data show that oriII of V. cholerae can be used as orthogonal replication origin for large replicons in heterologous hosts with conservation of its ability to initiate replication.

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Methods Bacterial strains, media, plasmids and oligonucleotides All bacterial strains, plasmids and oligonucleotides used in this study are listed in table S1-2. Cells were grown in LB medium [with Ohly® yeast extract; LBO] or AB medium (37) supplemented with 10 µg/ml thiamin, 100 µg/ml uridine and either 0.2 % glucose and 0.5 % casamino acids (AB-Glu-CAA) or 0,4% sodium acetate (AB-So) at 37 °C. Antibiotic selection was used at the following concentrations: chloramphenicol 35 µg/ml; kanamycin 100 µg/ml. Deleted and ectopic origins of replication were verified via PCR. Strain construction Origins oriC-449 and oriC-1.2 (compare to fig. 3A) were integrated at genomic position 344.066 bp (NC_000913.3) which is the site that was used by Liang et al. for ectopic oriC insertion in strain #2. Using overlap extension PCR a fragment was prepared containing oriC-449 and a cat-cassette flanked by FRT sites with primers 1279+1280 and 1337+1338+1282, respectively (Table S2). OriC-1.2-cat was directly amplified with primers 1343 and 1344 from strain WX322 (2). Integration was performed via lambda red recombination using the E. coli strain AB330 (38). Ectopic oriCs were then transferred into strain #2 via P1 transduction(6). OriZ (=oriC-5.1) was directly transduced from strain WX338 (2) into strain #2. Its insertion site is at position 345.246 (NC_000913.3). Colony PCR with primers 766 and 1074 were performed for verification of integrations. Flow cytometry Cells fixed in ethanol were washed and diluted in 0.1 M phosphate buffer, pH 9.0 (PB buffer). Then total protein content was stained overnight with 3 µg/ml FITC solution (PB buffer) at 4°C. Afterwards cells were stained with Hoechst 33258 (5) For run-out experiments cells were treated with 150 µg/ml rifampicin and 10 µg/ml cephalexin for more than three generations (2-3 h) allowing to finish ongoing rounds of replication (39, 40). Samples were analyzed on LSR II flow cytometer (BD Biosciences) and flow cytometry measurements were carried out as described (14). Data was processed with the software FlowJo (Treestar, Ashland, USA). The average DNA content/cell ratio was determined as average Hoechst fluorescence per cell. The average cell mass was determined as the average of FITC fluorescence per cell. The average DNA/mass was found by dividing average DNA content by average cell mass. E. coli MG1655 exponentially growing cells in AB-So were prepared and stained with Hoechst 33258 as described. These cells function as internal standard for calibration of the flow cytometer measurements and were added to every sample. Comparative genomic hybridization Exponentially growing cells in LBO (OD600 =0.15) were mixed in a 1:1 ratio with cold killing buffer. Samples were kept on ice during the whole procedure. Strain #1 (wildtype) grown in AB Glu-CAA until stationary phase was used as a reference and also treated with killing buffer. All samples were centrifuged at 4 °C and cells were resuspended in 300 µl immunoprecipitation Buffer and transferred into a new reaction tube. Cells were sonicated via Bioruptor® Plus (Diagenode Diagnosics) (48 cycles á 30 s with 30 s cooling) to receive DNA fragments of around 500 bp. Cellular debris was centrifuged and supernatant was transferred to a new reaction tube. 300 µl TE buffer and 2 µl RNase A (10mg/ml) were added and samples were incubated at 65 °C for 90 min. DNA was isolated with phenol/chloroform. 400 ng DNA (20 ng/ml in 20 µl) were labelled with Cy3-dCTP (sample) or Cy57 ACS Paragon Plus Environment


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dCTP (reference), mixed and hybridized with whole genome microarrays from Agilent (8x15k) as described in Johnsen et al. 2011 (41). Arrays were scanned on an Agilent SureScan High Resolution Scanner. Spot intensities were extracted via AgilentScan Control software Ratios of dye intensities were calculated and normalized to the array wide average via R. Biological replicates of the CGH experiments showed good reproducibility (Fig. S8). Replication initiation sites (local maxima of copy number) and DNA-polymerase meeting points (local minima of copy number) were determined in several steps. First a loess fitting was applied to the microarray data to get a locally weighted average (shown as green line in CGH plots). For this average line, we detected maxima and minima. The maxima and minimal positions were used to dissect the data set in subsets delimited by the extrema. For these subsets a linear regression line was determined and the coordinates of the intersection points were taken as final maxima and minima. Simulation of genomic copy number dependent on two-origin replication DNA replication was simulated using an arbitrary but constant replication rate. A replicon consists of 1000 segments. After initiation at the origin position, the segments are copied one by one in both directions and stored in fragments. Due to the bidirectional replication, each fragment is elongated by one segment each time step on both ends. Replication speed is assumed to be the same for all replication forks. A replication fork collision happens when the next segment in the replication process of a fragment is already part of another fragment. In this case, the fragments are merged and replication continues at the remaining ends if no collision occurred on these ends (replication complete). If all fragments are merged and the whole replicon is amplified, the fragment is considered a replicon. Another round of replication can occur on either a replicon or still on a fragment to model overlapping replication rounds and perturbed initiation of origins. In analogy to natural replication, replicons accumulate by the replication process. The simulation was stopped after the creation of 200,000 replicons + fragments and the frequency of segment replication in all replicons and fragments was counted. Two origins were placed on the initial replicon at position 300 and 700 and replication initiation frequency of oriI was set to 900 + x time steps. Hence, the next initiation at oriI took place long after the replicon was fully amplified (replicon replication time = 500 time steps or less), simulating non-overlapping chromosome replication. The x is a random number that further delays the initiation of individual replicons to reflect the natural noise of the initiation timing and to desynchronize daughter replicon initiation similar to the situation in bacterial cultures. OriII initiated exactly d time steps after oriI in this simulation. For the analysis we simulated all values of d between zero (synchronous initiation) and 900 and counted the segment frequency in 200,000 replicons + fragments (Fig. 5). Acknowledgements We thank all members of the Waldminghaus lab, especially Nadine Schallopp for excellent technical assistance. We are grateful to David Sherratt, Xindan Wang, Xiquan Liang, Federico Katzen and Alexander Böhm for providing strains and we would like to thank the Flow Cytometry and Genomics Core Facility (ZTI, Marburg) for providing respective devices. Christoph Weigel is acknowledged for helpful comments. This work was supported within the LOEWE program of the State of Hesse.


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Supporting Information - Tables S1 and S2 with strains and oligonucleotides used in this study - Fig. S1: Growth curves of strains containing an additional, ectopic oriC or oriII - Fig. S2: PCR-based verification of native and ectopic oriC copies. - Fig S3+4: Sequencing results of ectopic oriC - Fig. S5: CGH analysis of stationary phase cells of strains with additional oriII - Fig. S6: Position of the minima in CGH plots in strains with an ectopic oriV - Fig. S7: Simulation of marker frequency analysis with different oriII positions.

376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417

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Fig. 1: DNA replication in E. coli strains with additional, ectopic oriC. Results are shown for E. coli MG1655 strain #1 with only the native oriC (A-D), strain #2 with an ectopic oriC copy about 1 mbps from oriC (E-H) and strain #6 with an ectopic oriC copy about 1.6 mbps from oriC (I-L). A, E, I 11 ACS Paragon Plus Environment


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Histograms of flow cytometry analyzed exponential growing cells. DNA content is plotted versus number of cells. Chromosome equivalents are indicated. B, F, J Flow cytometry analyses of DNA content in Rif/Ceph treated cells. C, G, K Profile of genome wide copy numbers based on comparative genomic hybridization (CGH). Grey dots represent values of single probes. Positions of oriC(wt) and additional oriC insertions in the respective strains are indicated and the genome position as distance from oriC(wt). Maxima are highlighted in yellow, minima in red. The black line represents the linear regression. The green line corresponds to the Loess regression (F = 0.05). See method section for calculation of regression lines, maxima and minima. Outer edges of the plots represent the respective other chromosome arm. Copy numbers above two were considered for analysis but are not shown in plots. D, H, L Overview of expected (outer circle) and experimental (inner circle) results of CGH analyses. Maxima are highlighted in yellow, minima in red.

Fig. 2: DNA replication in supposed oriC(wt) deletion strains. Results are shown for E. coli MG1655 strain #3 which should have an ectopic oriC copy about 1 mbps from oriC and a deletion of the native oriC (A-D) and strain #7 which should have an ectopic oriC copy about 1.6 mbps from oriC and a deletion of the native oriC (E-H). A, E, Histograms of flow cytometry analyzed exponential growing cells. DNA content is plotted versus number of cells. Chromosome equivalents are indicated. B, F 12 ACS Paragon Plus Environment

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544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561

562 563 564 565 566 567 568 569


Flow cytometry analyses of DNA content in Rif/Ceph treated cells. C, G Profile of genome wide copy numbers based on comparative genomic hybridization (CGH). Grey dots represent values of single probes. Positions of oriC(wt) and additional oriC insertions in the respective strains are indicated and the genome position as distance from oriC(wt). Maxima are highlighted in yellow, minima in red. The black line represents the linear regression. The green line corresponds to the Loess regression (F = 0.05). See method section for calculation of regression lines, maxima and minima. Outer edges of the plots represent the respective other chromosome arm. Copy numbers above two were considered for analysis but are not shown in plots. D, H Overview of expected (outer circle) and experimental (inner circle) results of CGH analyses. Maxima are highlighted in yellow, minima in red.

Fig. 3: DNA replication from ectopic oriC insertions of different sizes. A Overview of the oriC core region and flanking genes. DnaA boxes are highlighted in red. Different lengths of the oriC region, which were used in this and other studies, are indicated. Profiles of genome wide copy numbers based on comparative genomic hybridization (CGH) for E. coli strain SM143 with a 449 bp oriC fragment about 1.064 kb apart from native oriC (B), strain SM145 with a 1172 bp oriC fragment at the 13 ACS Paragon Plus Environment


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same position (C) and strain SM139, containing a 5160 bp oriC fragment about 1.065 kb apart from native oriC (D). Grey dots represent values of single probes. Positions of oriC(wt) and additional oriC insertions in the respective strains are indicated and the genome position as distance from oriC(wt). Maxima are highlighted in yellow, minima in red. The black line represents the linear regression. The green line corresponds to the Loess regression (F = 0.05). See method section for calculation of regression lines, maxima and minima. Outer edges of the plots represent the respective other chromosome arm. Copy numbers above two were considered for analysis but are not shown in plots.

Fig. 4: DNA replication in E. coli strains with an ectopic oriII from V. cholerae. Results are shown for strains #15 (A-D), #17 (E-H), #19 (I-L) and #20 (M-P). All four strains have an ectopic insertion of the V. cholerae replication origin oriII at positions indicated in the middle and right panel. A, E, I, M Histograms of flow cytometry analyzed exponential growing cells. DNA content is plotted versus number of cells. Chromosome equivalents are indicated. B, F, J, N Flow cytometry analyses of DNA content in Rif/Ceph treated cells. C, G, K, O Profile of genome wide copy numbers based on comparative genomic hybridization (CGH). Grey dots represent values of single probes. Positions of oriC(wt) and additional oriC insertions in the respective strains are indicated and the genome position as distance from oriC(wt). Maxima are highlighted in yellow, minima in red. The black line 14 ACS Paragon Plus Environment

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597 598 599 600 601 602 603 604 605


represents the linear regression. The green line corresponds to the Loess regression (F = 0.005 for C and F = 0.05 for G, K, O). See method section for calculation of regression lines, maxima and minima. Outer edges of the plots represent the respective other chromosome arm. Copy numbers above two were considered for analyses but are not shown in plots. D, H, L, P Overview of expected (outer circle) and experimental (inner circle) results of CGH analysis. Maxima are highlighted in yellow, minima in red.

Fig. 5: Simulation of replication on a replicon with two origins and different initiation timing. A Schematics of two different scenarios dependent on the initiation timing of both origins. The second origin initiates before the replication fork from oriI passes through oriII (left) or after oriII is replicated (right). Initiation at oriII will correspondingly start on either one or two DNA strands. B Simulation of genomic copy number distributions depending on replication origin timing. The abscissa denotes the delay of oriII relative to oriI initiation. The ordinate denotes the chromosomal position. Replication speed for all forks is set to 1 chromosomal unit per time such that the indicated number of the abscissa also indicates the distance the replication forks have travelled from oriI since initiation. 15 ACS Paragon Plus Environment


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Origins are located at the 300 and 700 position as indicated. At delays above 400 the replication fork from oriI passes oriII before oriII initiates (compare to A). Copy numbers are indicated by a color gradient with a log scale as indicated. The solid black line represents the meeting point of the replication forks between both origins. The dashed line represents the theoretical meeting point based on the origin copy numbers assuming a constant replication rate. C and D Chromosomal copy numbers for two specific initiation delays in both replication scenarios.

Fig. 6: DNA replication of an E. coli strain with two linear chromosomes. Results are shown for strain #22 with a divided chromosome replicating based on oriII or oriC as indicated (A-D). A Histogram of flow cytometry analyzed exponential growing cells. DNA content is plotted versus number of cells. Chromosome equivalents are indicated. B Flow cytometry analyses of DNA content in Rif/Ceph treated cells. C Profile of genome wide copy numbers based on comparative genomic hybridization (CGH). Grey dots represent values of single probes. Positions of oriC(wt) and additional oriC insertions are indicated and the genome position as distance from oriC(wt). Maxima are highlighted in yellow, minima in red. The black line represents the linear regression. The green line corresponds to the Loess regression (F = 0.05). See method section for calculation of regression lines, maxima and minima. Outer edges of the plots represent the respective other chromosome arm. Copy numbers above two were considered for analysis but are not shown in plots. Chromosomal deletions are highlighted in blue with an additional asterisk. Linearization sites are indicated in orange. D Overview of expected (outer circle) and experimental (inner circle) results of CGH analyses. Maxima are highlighted in yellow, minima in red.

Table1: Chromosomal locations of oriC(wt) and ectopic replication origins (ori) in different E. coli strains. Strain #1 #2 #3 #6 #7 #15 #17

oriC(wt) position (kb) expected1 experimental2 0 -32 0 -38 0 -44 0 -43 0 -45 0 -18 0 -51

ectopic ori position (kb) expected experimental 1059 1059 1675 1675 500 372 1059 1118 16

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636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653


#19 #20 #22 SM139 SM143 SM145

1 2

0 0 0 0 0 0

-51 15 17 3.5 -13 -8.9

-1416 1675 -1416 1065 1064 1064

-1507 1764 -1418 1082 1099 1117

expected positions according to (2, 6) Positions obtained by comparative genomic hybridization analyses



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DNA Replication in Engineered Escherichia coli Genomes with Extra

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