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Plasmid supercoiling decreases during the dark phase in cyanobacteria: a clarification of the interpretation of chloroquine-agarose gels

In cyanobacteria DNA supercoiling varies over the diurnal light/dark cycle and is integrated with the circadian transcription program and (Woelfle et al. [2007], Vijayan et al. [2009], PNAS). Specifically, Woelfle et al. have reported that DNA supercoiling of an endogenous plasmid became progressively higher during prolonged dark phases in Synechococcus elongatus PCC 7942. This is counterintuitive, since higher levels of negative DNA supercoiling are commonly associated with exponential growth and high metabolic flux. Vijayan et al. then have reverted the interpretation of plasmid mobility on agarose gels supplemented with chloroquine diphosphate (CQ), but not further discussed the differences. Here, we set out to clarify this open issue in cyanobacterial DNA supercoiling dynamics. We first re-capitulate Keller’s band counting method (1975, PNAS) using CQ instead of ethidium bromide as the intercalating agent. A 500x–1000x higher CQ concentration is required in the DNA relaxation reaction (topoisomerase I) than in the agarose gel buffer to quench all negative supercoiling of pUC19 extracted from Escherichia coli. This is likely due to the dependence of both, the DNA binding affinity of CQ and the induced DNA unwinding angle, on the ionic strength of the buffer. Lower levels of CQ were required to fully relax in vivo pUC19 supercoiling than were used by Woelfle et al. Next, we analyzed the in vivo supercoiling of endogenous plasmids of Synechocystis sp. PCC 6803, at two different CQ concentrations. These experiments indicate that negative supercoiling of plasmids does not increase but decreases in the dark phase, and progressively decreases further in prolonged darkness.


Introduction 1
In vivo, the DNA double helix usually exists in a torsionally strained state, often just denoted as negative 2 DNA supercoiling. The energy stored in underwound DNA can be channeled locally at gene promoters 3 to open and "read" DNA in transcription and replication [Dorman, 2019]. In Bacteria, the level of DNA 4 supercoiling is homeostatically controlled by enzymes but also depends on the transcription and replication, 5 and in turn influences the transcription rates of many genes. In most analyzed bacteria and conditions, 6 increased negative supercoiling is observed during exponential growth and decreased during stationary 7 phase or upon exposure to stress. The DNA supercoiling level of the circular chloroplast genome of the 8 single-celled algae Chlamydomonas reinhardtii showed fluctuations with the diurnal light/dark cycle [Salvador 9 et al., 1998]. Mori and Johnson [2001] first suggested that supercoiling could underlie the regulation of 10 hundreds of genes during the light/dark (diurnal) cycle in cyanobacteria, the bacterial ancestors of chloroplasts. 11 Indeed, genome compaction varied over the diurnal cycle in Synechococcus elongatus PCC 7942 [Smith 12 and Williams, 2006]. Woelfle et al. [2007] then used an old method, separation of plasmid topoisomers by 13 chloroquine-supplemented agarose gel electrophoresis, to show that the supercoiling level of its endogenous 14 plasmid varies over the light/dark cycle, and variation continued in constant light after entrainment to light/dark 15 cycles. The latter observation is considered a hallmark of the presence of a circadian clock. Specifically, they 16 noted a drop in negative supercoiling of the plasmid already 20 min after the transition to light, as well as 17 continuous increase in DNA supercoiling in prolonged 72 h darkness. However, a comparison with a later 18 1/10 paper using the same method [Vijayan et al., 2009] shows discrepancies in the interpretation of the agarose 19 gels, specifically the relative mobilities of more and less supercoiled plasmid samples. We first review the 20 principles of agarose gel electrophoresis of circular DNA in the presence of DNA intercalators. To establish the 21 effect of chloroquine (CQ) on plasmids, we recapitulated Keller's band counting method but using CQ instead 22 of ethidium bromide (EtBr) as the intercalating agent, and the pUC19 plasmid isolated from Escherichia coli 23 (E. coli) culture in exponential growth. Next, we extracted endogenous plasmids from the cyanobacterium 24 Synechocystis sp. PCC 6803 (Synechocystis) grown under light/dark conditions (12 h/12 h). Using different 25 CQ concentrations, we show that, opposite to the interpration by Woelfle et al. [2007], negative supercoiling 26 increased (lower ∆Lk) quickly upon the onset of the light phase and progressivly decreased (higher ∆Lk) 27 during a prolonged 48 h dark phase.

29
A Short History of Topoisomer Separation 30 The method to measure plasmid supercoiling by agarose gels is the subject of this contribution and requires 31 some background. Textbook plasmid agarose gels show three distinct bands: the "linear" form which 32 underwent a double strand break, likely during extraction; and the oc (open circular) band, which has only 33 one or more single strand breaks and thus is still circular. The latter travels slowest on the gel. And thirdly, 34 intact plasmids travel fastest as a smeared band, widely known as the ccc band, for the covalently closed 35 circular form of the plasmid. The band is smeared since it actually contains several distinct species, denoted 36 topoisomers, that travel at slightly different speeds in the gel. Topoisomers are identical plasmid molecules 37 (isomers) that differ only by their level of negative DNA supercoiling, quantified as the plasmid's linking number 38 (Lk) [Crick, 1976]. The terminology is from the mathematical field of topology, and Lk is the number of 39 crossings of the two helix strands around each other. In relaxed (non-supercoiled) DNA the double helix 40 adopts its minimal free energy structure with 10.4 -10.5 bp per helix turn, e.g. a circular DNA with 1000 bp 41 has Lk 0 = 95 -96 such crossings. Levels of DNA superocoiling are usually provided as the difference to 42 this relaxed state linking number, ∆Lk 0 = Lk − Lk 0 and as the supercoiling density σ = ∆Lk0 Lk0 . Negatively 43 supercoiled DNA has ∆Lk 0 < 0 and positively supercoiled DNA has ∆Lk 0 > 0. Plasmids isolated from E. 44 coli typically have σ = −0.08 -−0.06, depending on the growth phase [Liu et al., 2018]. Relative topoisomer 45 abundances follow a Boltzmann distribution around a mean level of σ [Pulleyblank et al., 1975, Depew and 46 Wang, 1975, Keller, 1975. 47 Supercoiled plasmids (ccc) form a more compact molecule than relaxed plasmids. This is achieved by a 48 higher level structure where two double helices wind around each other to compensate for the torsional strain 49 on the double helix. This structure, a so-called plectoneme, is the origin of the term supercoiling, since it is a 50 doubly coiled structure: the DNA double helix forms a higher order "double helix". The more compact plasmids 51 migrate quicker through the polymeric meshwork of an agarose gel. If plasmids are artificially relaxed with a 52 nicking-closing enzyme (NC), such as bacterial topoisomerase I (TopoI), they are still ccc but now migrate at 53 a speed similar to the oc form. NC enzymes introduce a single strand break (nick). The torsional strain in the 54 supercoiled helix leads to rotation of the single stranded ends. The enzyme then ligates (closes) the ends 55 again and releases a plasmid with less supercoiling. These reactions will result in a Boltzmann distribution of 56 plasmids around ∆Lk 0 = 0, with some positively and some negatively supercoiled [Depew and Wang, 1975]. 57 These latter species are already more compact then the relaxed form and they form distinct bands "below" the 58 oc band. 59 To separate the in vivo levels of supercoiling on agarose gels an intercalating substance can be added 60 to the agarose gel and the gel buffer. Intercalating substances reduce the helix rotation angle between the 61 two bases they bind to, i.e., they locally unwind the helix [Lerman, 1961, Pritchard et al., 1966. This absorbs 62 some of the torsional strain of the underwound helix of negatively supercoiled DNA, and thus reduces the 63 apparent level of supercoiling. When adding the right amount of the intercalator, supercoiling is reduced to an 64 extent which can be separated on the agarose gel. A similar principle can be used to generate a series of 65 plasmid topoisomers, starting from the in vivo level of supercoiling and all the way to the fully relaxed form. 66 The intercalator is simply added to the reaction mix, supercoiled plasmids are partially relaxed and the NC  [Wang, 1971, Kirkegaard andWang, 1985]. In contrast, on agarose gels the DNA now 76 changes its migration speed and positively supercoiled plasmids travel again faster than the relaxed form. 77 Shure et al. [1977] suggested that with intercalators with lower affinity the topoisomer separation in agarose 78 gels 'less sensitive to variations in experimental conditions' and first introduced the use of chloroquine (CQ, all 79 amounts refer to chloroquine diphosphate) instead of EtBr. have tested the relative mobility changes with different CQ levels, and assume that they are in the range were 83 the plasmids are still negatively supercoiled. That is, plasmids that migrated faster on the gel were interpreted 84 as originally (in vivo) more supercoiled (more negative supercoiling, lower Lk). This is reflected in their gel 85 band annotation and the equation they used to calculate an average relative mobility (RM ) of each sample: where "Rel" denotes the migration distance of the upper (slowest migrating) band, actually the still present further reverted the interpretation of migration speed to: where the annotated gels indicate that they inpret the upper, slowest migrating band as the oc form. They 96 also observe a fast migrating band in all samples which for unexplained reasons they denote as a "rel" for 97 relaxed. They calculated a "mean" migration distance of the supercoiled topoisomers. The equation likely 98 contains an error, since oc − rel would be a negative value. Independent of these unclarities the subtract the 99 RM of Woelfle et al. from 1. This implies that they interpret faster migrating topoisomers as originally less 100 negatively supercoiled. At high intercalator concentration these should be more positively supercoiled and 101 travel faster. Choosing more neutral band identifiers makes these differences clearer: where "upper" and "lower" refers to their position on the original gel images, and "target" is the mean or 103 median of the topoisomer distribution. Despite the opposite meaning of RM , both publications interpret a 104 higher RM as a higher level of negative DNA supercoiling. 105

3/10
This different interpretation of the gels in Woelfle et al. [2007] would imply that negative supercoiling of 106 plasmids rapidly increases upon onset of the light phase and would progressively decrease in prolonged 107 darkness. Vijayan et al. [2009] measured supercoiling only in constant light conditions, and did not mention 108 or discuss these discrepancies. Thus, the DNA supercoiling dynamics within the natural light/dark cycle of 109 cyanobacteria remains unclear.
110 Figure 1. pUC19 on 1.2 % agarose gel supplemented CQ. Samples of the pUC19 plasmid, isolated from E. coli, were partially relaxed with C relax = 0 -3000 µg mL −1 CQ and topoI as indicated (lanes). The sample "C" is untreated pUC19 as control. Size marker "S" is the GeneRuler 1 kb DNA Ladder. The samples analyzed on 6 agarose gels, each supplemented with a CQ concentration (C gel = 0 -8 µg mL −1 ) as indicated (lower right). The top band in each gel is the open circular form. The band between 2.5 kb and 3 kb is the linear form of the plasmid. [Keller, 1975] analyzed the linking number of the circular genome of the simian virus 40 (SV40, 5.2 kb), 112 propagated in and isolated from African green monkey cells (CV-1). For this, the DNA was subjected to in vitro 113 relaxation with an NC enzyme purified from human tissue culture cells (KB-3). Full relaxation of the SV40 114 DNA in the gel buffer was achieved at C gel = 0.06 µg mL −1 , or with 394.3 g mol −1 , C gel = 0.15 µM. In the topo 115 I reaction C relax ≈ 4.5 µM (lane 9 in Figures 1 and 3 of [Keller, 1975]) were required to fully relax the DNA. 116 The in vivo supercoiling level of the SV40 DNA was ∆Lk 0 ≈ 24 and σ ≈ 0.05. 117 Here, we propagated the pUC19 vector plasmid (2686 bp) in in E. coli DH5α and harvested plasmids in 118 exponential phase on LB medium. A plasmid relaxation series was performed with the bacterial NC enzyme 119 TopoI obtained from New England Biolabs. To find the appropriate CQ concentrations to reproduce Keller's 120 band counting method, we first analyzed the EtBr concentrations used in the original publication, considered 121 the ca. 1000x lower binding affinity of CQ, and adjusted concentrations from there. Figure 1 shows that the 122 lowest CQ concentration in the gel (C gel = 1 µg mL −1 ) was sufficient to detect a clear change in the migration 123 of all pUC19 topoisomers. Fully relaxed plasmids (C relax = 0 -200 µg mL −1 ) had already shifted to positive 124 supercoiling and migrated faster than without CQ. Topoisomers treated with C relax = 800 -1000 µg mL −1 were 125 all close to full relaxation at C gel = 1 µg mL −1 . The untreated in vivo sample ("C") appeared fully relaxed 126 at C gel = 2 µg mL −1 , and shifted to positive supercoiling at C gel > 4 µg mL −1 . The topoisomers relaxed 127 at the highest concentration (C relax = 3000 µg mL −1 ) appeared with band patterns almost identical to the 128 untreated control in all gels. With a molecular weight of 515.86 g mol −1 (chloroquine diphosphate), fully 129 relaxing concentrations of CQ were thus C gel ∼ 3.9 µM and C relax ∼ 5.8 mM. Counting topoisomers across 130 gels shows that the untreated control has a ∆Lk 0 = −15 -−16. With 2686 bp length, Lk 0 ≈ 257 and thus 131 σ ≈ 0.06, as expected for a plasmmid isolated from E. coli.

132
In summary, the intercalator concentrations required to fully quench the original level of supercoiling of 133 SV40 and pUC19 were ≈30x and ≈1000x higher in the reaction buffers than in the gels, respectively. The CQ 134 concentrations were 100 -1000x higher than the EtBr concentrations in Keller [1975]. EtBr binds to DNA with 135 relatively high affinity, e.g. K a = 1.5 × 10 5 M −1 in 0.2 M Na + [Gaugain et al., 1978], and each intercalated 136 molecule unwinds the helix by 26 -28 • [Keller, 1975]. CQ has a lower affinity to DNA, depending on ionic 137 strength of the buffer, e.g.

146
To clarify the unresolved issue whether DNA supercoiling is higher during the light or dark phases in cyanobac-147 teria, we measured supercoiling levels of endogenous plasmids of Synechocystis sp. PCC6803 ("Moscow 148 strain" PCC-M) during diurnal light/dark (12 h/12 h) conditions and a prolonged dark phase (12 h light, followed 149 by h24 h darkness). A large sample volume (25 mL) was required to obtain enough plasmids, and direct 150 mixing of the sample with an equal volume of pre-cooled (−20 • C) pure undenatured ethanol was key to 151 successful plasmid extraction. However, the yield was still very low (0.5 -1 µg, but incl. genomic DNA), 152 limiting the amount of gels that can be run from one sample. Multiple sets of putative topoisomer bands were 153 detected at all sampled time points by agarose gel electrophoresis supplemented with chloroquine at either 154 1 µg mL −1 or 20 µg mL −1 (Fig. 2A, B). Synechocystis contains three short plasmids, two at 2.4 kb 155 McFadden, 1993, 1994] and one at 5.2 kb [Xu and McFadden, 1997]. Restriction analysis and Southern blots 156 confirmed that the most pronounced topoisomer bands were obtained for plasmid pCA2.4_M. The migration 157 distances on the gels were different between samples and the relative differences reversed between the 158 two CQ concentrations. This change of relative migration speed indicates that plasmids are still negatively 159 supercoiled at the low, but positively supercoiled at the high concentration. Plasmids which were originally 160 more relaxed migrate slower and faster at the low and high concentrations, respectively. Next, we quantified 161 the agarose gels by generating electropherograms of each lane in ImageJ. Baseline correction, peak detection 162 and peak area quantification were performed in R (Fig. 2C). For each sample an average linking number 163 deficit ∆Lk was calculated and plotted as time series over the light/dark cycles (Fig. 2D). Plasmids were 164 more negatively supercoiled (lower ∆Lk) during light phases, and reached a maximal level only 30 min after 165 transition to the light phase. This level was maintained or only slightly increased throughout the 12 h light 166 phase. During the dark phase the transition was slower, and plasmids became progressively more relaxed 167 during the 12 h, and relaxation continued at equal pace during an additional 12 h dark phase. The maximal 168 linking number differences between light and dark phase were only ∆∆Lk ≈ 1, and ∆∆Lk ≈ 2 after the 169 prolonged dark phase. 170 In summary, negative supercoiling of pCA is higher during the day, with a quick increase directly after the 171 onset of light, and lower during the night; extended night relaxes plasmid even more. These results are the 172 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 NcoI light/dark/light light/dark/dark CQ=1 μg/mL  Plasmid topoisomer analysis by intercalator-supplemented relaxation series and agarose gel electrophoresis 178 is an old and elegant method. However, the gels are difficult to interpret, and the effect of intercalator concen-179 trations must be checked for each plasmid and conditions (gel and reaction buffers). Ideally, Keller's band 180 counting method should be applied to each newly analysed plasmid. The low yields when extracting endoge-181 nous plasmids from cyanobacteria, the time-intensive calibration routine, and non-standard electropherogram 182 analysis make it somewhat challenging to properly analyse in vivo supercoiling in a given species. 183 6/10 Can these methods be updated to the requirements of current day laboratory practice? Mitchenall et al. 184 [2018] reported separation of topoisomers of pBR322, pUC19, and a 339 bp DNA minicircle on a commercial 185 capillary gel electrophoresis platform (Qiagen QIAxcel Advance System), where EtBr is a component of 186 the gel and used by the detection system for quantification. The system is quite sensitive and samples of 187 100 ng DNA sufficed. Moreover, the range of separated topoisomers was broader than on agarose gels. 188 This would in principle allow to use fewer runs to achieve full separation of in vivo levels of supercoiling. 189 However, the intercalator type and concentration can not be manipulated in this gel cartridge-based system. 190 We recently succeeded to separate pUC19 topoisomers by adding comparatively large concentrations of CQ 191 (C gel = 1 -3 mg L −1 ) to the commercial (and secret) gel delivered for the AATI Fragment Analyzer (now owned 192 by Agilent). But the results were not consistent between runs. Likely, the undisclosed fluorescent nucleic acid 193 marker has effects on DNA supercoiling. More work and alternatives to the existing commercial systems are 194 required to establish topoisomer separation by capillary gel electrophoresis as a standard procedure. This 195 could potentially greatly reduce the amount of sample required per run. 196 The analysis of electropherograms is also a limiting factor for many laboratories. No standards or 197 established tools exist on how to calculate average linking number differences between samples. Commerical 198 gel image analysis software is not geared towards this application. To this end Ziraldo et al.
[2019] provide a 199 plug-in for the ImageJ image analysis software that could streamline the analysis of agarose gels. Here, we 200 also used ImageJ to extract electropherograms from gel images, and peak detection routines from a mass-201 spectrometry R package, msProcess (see Methods) which is not maintained anymore, to calculate average 202 linking numbers in R. A novel R package, bioanalyzeR 10x CutSmart reaction buffer (NEB: B7204, 500 mM potassium acetate, 200 mM Tris-acetate, 100 mM 227 7/10 magnesium acetate, 1000 µg mL −1 BSA, pH 7.9), filled up to 24 µL with nuclease-free water, and incu-228 bated for 15 min at 37 • C to allow for equilibration of the CQ intercalation. Then 1 µL (5 U) topoI (NEB: 229 M0301) was added and the reaction mix incubated for another 15 min at 37 • C. Then, the reaction was 230 stopped by incubation at 65 • C for 20 min and samples were purified with the NucleoSpin Gel and PCR 231 Clean-up kit (Machery-Nagel) to remove the enzyme and the intercalator. This protocol is published at 232 https://dx.doi.org/10.17504/protocols.io.rbcd2iw. Synechocystis Plasmid Restriction Analyses. A pooled sample of plasmid extracts from Synechocystis 258 was subjected to rectriction by NcoI (Thermo Scientific: FD0573), which has a single cut site in pCA2.4_M but 259 not in the similarly sized pCB2.4_M. 1.5 µL of FastDigest buffer and 1 µL of restriction enzyme were added to 260 12.5 µL of plasmid samples. Reactions were incubated for 30 min at 37 • C and stopped by addition of 3 µL of 261 6x DNA loading dye with 1 % SDS and incubation at 80 • C for 15 min. The resulting 18 µL were loaded directly 262 onto the agaorse gels 263 Chloroquine Agarose Gel Electrophoresis. Agarose gels with different concentrations of chloroquine 264 diphosphate salt (CQ, Sigma: C6628-50G, CAS: 50-63-5) were used to determine the relative migration 265 speed of supercoiled topoisomers. 1.2% agarose gels in 0.5x TBE (Roth: 3061.2; 0.5x: 5.4 g/L Tris base, 266 2.75 g/L boric acid, 4 mL/L of 0.5 M EDTA (pH 8.0), pH 8.3) were prepared by heating to boiling. After 267 cooling (hand-warm) CQ was added to the indicated final concentration from a stock solution (10 mg mL −1 ) 268 and the mixture poured into the gel chamber. The running buffer was 0.5x TBE buffer with the same CQ 269 concentration as the gel. For each sample, 250 ng (E. coli/pUC19 after treatment with T5 exonuclease) or 270 1 -2 µg (Synechocystis, PlasmidSafe reaction mix was directly loaded) of plasmid DNA were mixed with 271 loading dye and filled up to 30 µL with water. Gels were run for 16 -24 h at 40 V (1.8 V cm −1 ) in a Peqlab 272 gel chamber, covered in aluminum foil to protect from light. Gels were then washed two times for 30 min in 273 250 mL 0.5x TBE buffer to remove the CQ, and stained with 25 µL Sybr Gold in 225 mL 0.5x TBE buffer for 274 8/10 3 -24 h and imaged on a BioRad Imaging System (ChemiDoc MP). Washing and staining were also performed 275 light-protected. The protocol is published at https://dx.doi.org/10.17504/protocols.io.nbtdann.

276
Southern Blots of Agarose Gels. Probes for Southern blots of the pCA2.4_M plasmid were generated 277 by colony PCR, using forward primer: ACAGGGGTAAATGAGTGCCG, reverse: GCAAGCAGTCCTCCACAAGA, and the 278 PCR programm: 3 min, 95 • C; 35x(95 • C 30s, 50 • C 30s, 72 • C 1 min); 72 • C 5 min. The products were isolated 279 by cutting the band from an agarose gel and clean-up with the NucleoSpin Extract II kit (Macherey-Nagel). 280 Labelled DNA was generated using the same PCR program with the DIG Easy Hyb (Roche, 1603558) mix, 281 according to manufacturer's instruction. Southern blots were generated using the CDP-Star kit with slight 282 modifications as follows. Gels were blotted onto a nitrocellulose membrane using a vacuum blotter (90 min 283 with 5 -7 mmHg, in 10x SSC buffer). The membrane was pre-hybridized for 1 h at 50 • C. The probes were 284 denatured (95 • C, 15 min) and cooled on ice, then transfered to the blot and hybridized over night at 50 • C. 285 The membran was washed 2x 5 min mit 2x SSC, 0.1 % SDS at 50 • C, then with 2x 15 min mit 0.1x SSC und 286 0.1 % SDS at 65 • C, and cross-linked with UV light for 10 min. All further steps were performed according to 287 the CDP-Star manual and blots were imaged on a BioRad Imaging System (ChemiDoc MP).

288
Analysis of Gel Electropherograms & Calculation of ∆Lk. Electropherograms were extracted in ImageJ 289 for each lane and analyzed in R, using LOESS smoothing and peak detection functions from the msProcess 290 R package (version 1.0.7) (https://cran.r-project.org/web/packages/msProcess/). A baseline was 291 determined in two steps using the msSmoothLoess function (Fig. 2C, top panel). The first step used the full 292 signal and served to determine the coarse positions of peaks. The final baseline was then calculated from the 293 signal after removal of peak values. This baseline was subtracted from the total signal to detect peaks (bands) 294 with the msPeakSimple function from msProcess and calculate peak areas. For topoisomer analysis peaks 295 were detected with the msPeakSimple function and the assigned consecutive integral linking number values 296 ∆Lk i with an estimated offset of a reference peak from the relaxed form with Lk = 0. The areas under the 297 peaks A ∆Lk were calculated and the average linking number deficit of a sample was then determined as the 298 center of mass of all N identified topoisomer bands. 300 Note, that the true ∆Lk of bands from the relaxed plasmid was not determined, eg. by Keller's band 301 counting method [Keller, 1975]. The calculated ∆Lk values refer to an assignment of a reference band that 302 was present in all lanes to ∆Lk = −8, and this can be considered a minimal estimate of the true |∆Lk|, 303 based on the distance of topoisomer and relaxed DNA bands in a topoI relaxation experiment (not shown). 304