1
Asymmetric chromosome segregation and cell division in DNA damage-1
induced bacterial filaments 2
3
Suchitha Raghunathan1,2, Afroze Chimthanawala1,3, Sandeep Krishna1,4, Anthony G. 4
Vecchiarelli5 and Anjana Badrinarayanan1* 5
6 1National Centre for Biological Sciences (Tata Institute of Fundamental Research), 7
Bangalore, India, 2Transdisciplinary University (TDU), Bangalore, India, 3SASTRA 8
University, Tanjore, India, 4Simons Centre for the Study of Living Machines, 1National 9
Centre for Biological Sciences (Tata Institute of Fundamental Research), Bangalore, India, 10 5Molecular, Cellular, and Developmental Biology Department, Biological Sciences 11
Building, University of Michigan, Ann Arbor, Michigan, USA 12
Correspondence to: *[email protected] 13
14
Keywords 15
Escherichia coli, DNA damage, filamentation, cell size maintenance, chromosome 16
segregation, cell division, time-lapse imaging, SOS response, bacteria. 17
18
Running title: Division dynamics and cell size maintenance in DNA damage-induced 19
bacterial filaments 20
21
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2
Abstract 22
Faithful propagation of life requires coordination of DNA replication and 23
segregation with cell growth and division. In bacteria, this results in cell size homeostasis 24
and periodicity in replication and division. The situation is perturbed under stress such as 25
DNA damage, which induces filamentation as cell cycle progression is blocked to allow for 26
repair. Mechanisms that release this morphological state for re-entry into wild type 27
growth are unclear. Here we show that damage recovery is mediated via asymmetric 28
division of Escherichia coli filaments, producing short daughter cells with wild type size 29
and growth dynamics. Division restoration at this polar site is governed by coordinated 30
action of divisome positioning by the Min system and modulation of division licensing by 31
the terminus region of the chromosome, with MatP playing a central role in this process. 32
Collectively, our study highlights a key role for concurrency between chromosome (and 33
specifically terminus) segregation and cell division in daughter cell size maintenance 34
during filamentous divisions and suggests a central function for asymmetric division in 35
mediating cellular recovery from a stressed state. 36
37
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3
Introduction 38
For successful cell division to occur, accurate DNA duplication and segregation 39
must be completed. In bacteria, chromosome replication initiates bi-directionally from an 40
‘origin’ and finishes opposite to this position, at the ‘terminus’ (Reyes-Lamothe and 41
Sherratt, 2019; Kleckner et al., 2014, 2018). Several factors ensure that cells divide only 42
upon completion of this process by regulating the multiprotein division machinery called 43
the ‘divisome’ (Galli and Gerdes, 2012; Dewachter et al., 2018; Kleckner et al., 2018; 44
Männik et al., 2016). For example, E. coli encodes negative regulators of the tubulin 45
homolog FtsZ, that is required to initiate the assembly of the divisome at the division 46
plane. Nucleoid occlusion by SlmA prevents the formation of the FtsZ-ring at locations 47
where chromosomal DNA is present and MinCDE oscillations direct the position of the Z-48
ring near mid-cell (Tonthat et al., 2011; Bernhardt and de Boer, 2005; Tsang and 49
Bernhardt, 2015). Recent studies have also suggested coordination of division with the 50
terminus via proteins such as MatP and ZapAB that act as a bridge between the DNA as 51
well as the divisome (Espéli et al., 2012; Mercier et al., 2008; Männik et al., 2016). 52
Together, in unperturbed laboratory conditions, this results in daughter cells that 53
replicate and divide in a periodic manner and that do not show much deviation in birth 54
and division cell sizes (Donachie, 1968; Taheri-Araghi et al., 2015; Wallden et al., 2016; 55
Micali et al., 2018; Campos et al., 2014; Si et al., 2019; Harris and Theriot, 2016). Such size 56
maintenance has been described in other bacteria as well as eukaryotic systems 57
(Chandler-Brown et al., 2017; Lambert et al., 2018; Soifer et al., 2016). 58
Recent investigations in bacteria have proposed that cells follow an ‘adder’-based 59
principle that governs size homeostasis either via regulation at the stage of birth 60
(replication initiation) or division or both (Taheri-Araghi et al., 2015; Wallden et al., 2016; 61
Campos et al., 2014; Si et al., 2019). In addition, some studies have also proposed a role 62
for cell shape or concurrency between processes of replication and division in size control 63
(Micali et al., 2018; Harris and Theriot, 2018). This homeostatic state is perturbed under 64
conditions of stress, a situation that can be often faced by bacterial cells in their 65
environment (Horvath et al., 2011; Justice et al., 2008; Yang et al., 2016; Heinrich et al., 66
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2019). For example, under DNA damage, a cell cycle checkpoint blocks cell division until 67
damage has been repaired. The bacterial SOS response is activated upon the binding of 68
RecA to single-stranded DNA that is exposed as a consequence of DNA damage 69
(Mukherjee et al., 1998; Jonas, 2014; Kreuzer, 2013). As part of this response, a cell 70
division inhibitor (such as SulA in E. coli) blocks polymerization of FtsZ, resulting in cellular 71
elongation or filamentation (Kreuzer, 2013; Mukherjee et al., 1998). Cell division 72
inhibition during damage is a conserved process, even though the effectors may vary 73
across bacteria. SOS-induced division inhibition is carried out by SidA in Caulobacter and 74
YneA in Bacillus (Mukherjee et al., 1998; Jonas, 2014; Modell et al., 2011; Mo and 75
Burkholder, 2010). SOS-independent DNA damage-induced division inhibitors have also 76
been identified, suggesting that this is an important step in DNA repair (Modell et al., 77
2014). Along with blocking division, chromosome cohesion or de-condensation is also 78
initiated in these filaments. It is thought that this can aid recombination-based repair 79
(Vickridge et al., 2017; Odsbu and Skarstad, 2014). Indeed, cells have also been shown to 80
change shape and size under other forms of stress including host environments, heat 81
shock and osmotic fluctuations (Justice et al., 2008; Heinrich et al., 2019; Wehrens et al., 82
2018; Kysela et al., 2016; Caccamo and Brun, 2018; Bos et al., 2015; Yang et al., 2016). 83
Together, this highlights the plasticity with which bacteria such as E. coli sample a range 84
of cell sizes including filamentous and non-filamentous cell lengths. While the process by 85
which cell division is regulated to result in elongation under DNA damage has been well-86
characterized (Mukherjee et al., 1998; Suzuki et al., 1967; Jonas, 2014; Modell et al., 2014, 87
2011; Mo and Burkholder, 2010; Kantor and Deering, 1966; Adler and Hardigree, 1965), 88
how such a state is exited after repair to reinitiate wild type, periodic replication and 89
division remains unclear. 90
In this study, we probe the mechanism by which filamentous E. coli reinitiate 91
chromosome segregation and cell division after DNA repair. We use single-cell, time-92
resolved fluorescence microscopy to follow the kinetics of division restoration after cells 93
face a pulse of DNA damage and observe that size is maintained in daughter cells 94
generated from dividing filaments. Size homeostasis in daughters is not governed by the 95
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growth laws (in the filament) and is instead dictated by cell division. We further find that 96
division restoration is controlled by two steps: determining where and when to divide. 97
This stepwise process, regulated by a combination of MinCDE oscillations, FtsZ levels and 98
terminus segregation, is accompanied by asymmetric partitioning of repaired 99
chromosomes, resulting in the production of daughter cells of the right size and devoid of 100
DNA damage, thus facilitating recovery from a stressed state. 101
102
Results 103
DNA-damage induced filaments maintain daughter cell size during division 104
To understand how damage-induced filamentous E. coli (Supplementary video 1) 105
re-initiate cell division and wild-type growth after DNA damage, we followed division 106
restoration in cells after treatment with a sub-inhibitory dose of the DNA damaging agent, 107
Mitomycin-C via time-lapse imaging (1μg/ ml; (Dapa et al., 2017)) (Fig. 1A). While 108
unperturbed wild type cells divided at ~mid-cell (Fig. 1B), we found that a significant 109
proportion of cells deviated from this division pattern as they increased in length (Fig. 1C-110
D). Damage-treated cells close to wild type length (5-10 μm) tended to divide in the 111
middle resulting in production of two daughter cells of similar sizes. In contrast, 112
filamentous cells divided polarly (or asymmetrically) to produce a short ‘daughter’ cell 113
(SD) of size similar to wild-type and a long cell that continued to filament (LD) (Fig. 1C and 114
1E; Supplementary video 2). The probability of a cell to undergo asymmetric division 115
increased with increasing cell length with 85% cells dividing asymmetrically at lengths 116
>12μm. Varying durations of damage exposure (30, 60 or 90 min) resulted in different 117
degrees of filamentation. However, in each case, a filamentous cell at division resulted in 118
production of an SD that was close to wild type size (Fig. 1F-G and Fig. S1A-B). This 119
asymmetry in division was observed in damage-induced filaments independent of growth 120
media (Fig. S1C). To further test whether the switch from mid-cell to asymmetric division 121
was a consequence of filamentation, we treated E. coli with the cell division inhibitor, 122
cephalexin, where genome integrity is maintained and chromosome replication and 123
segregation takes place as wild type. However, cells are unable to divide, resulting in 124
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elongation in length (Rolinson, 1980; Chung et al., 2009). Cephalexin-treated filamentous 125
cells also generated daughter cells (SD) of fixed length via asymmetric division upon 126
removal of the inhibitor (Fig. S1D; Supplementary video 3). Together with previous 127
observations (Wehrens et al., 2018; Taschner et al., 1988; Begg and Doanachie, 1977), our 128
data suggest that filamentous cells, irrespective of types or durations of stress treatment, 129
engage in this form of division that produces daughter cells of wild type size. 130
To characterize the recovery process further in DNA damage-induced filaments, 131
we followed the fate of the filament (LD) and the SD over time. We observed that only 132
16±2% of filaments returned to wild type cell size during recovery in the three treatment 133
regimens (Fig. 1H). However, filamentous cells underwent multiple divisions in a 1 hr time 134
period, generating daughter cells (SD) of wild type size at each division (Fig. 1I). In the 135
same time, wild type cells undergo three divisions on average, suggesting that more 136
daughter cells are produced from a filament than a cell of same size as wild type during 137
recovery. In contrast to the filaments, daughter cells of wild type size (SD) displayed 138
growth and division dynamics similar to non-damage conditions (Fig. 1H and 1J). As an 139
example, in Fig. 1E, we followed the fate of an SD (4.6 μm) generated from a filament and 140
found that the daughter cell reinitiated wild type growth dynamics soon after division. 141
Time taken between divisions for SD was close to the distribution seen for wild type, while 142
LD continued to grow and divide as damage-treated cells (Fig. 1J). Consistently, the DNA 143
damage marker, RecA, formed multiple foci in LD at the time of division, while SD had RecA 144
localization as seen for wild type cells (Lesterlin et al., 2014; Rajendram et al., 2015; 145
Vickridge et al., 2017) or elongated cells with no DNA damage (cephalexin treated) (Fig. 146
S1E). In line with these observations, we found that viable cell count as well as cell length 147
distribution was restored to close to that of wild type in the three treatment regimes (Fig. 148
S1F-G). Thus, even though the filament may or may not recover from damage exposure, 149
recovery of the population may be mediated via several rounds of asymmetric divisions 150
from a single filament, resulting in daughter cells of wild type size and growth dynamics. 151
Daughter cell size is governed by cell division dynamics of filaments 152
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How is daughter cell size maintained in dividing filaments? Recent investigations 153
have extensively characterized various mechanisms for chromosome segregation 154
checkpoints and adder-based cell size maintenance principles in wild type cells (Taheri-155
Araghi et al., 2015; Campos et al., 2014; Si et al., 2019; Harris and Theriot, 2016; Arjes et 156
al., 2014; Kleckner et al., 2014; Hill et al., 2012; Campos et al., 2018). To address this 157
question in the current context, we analysed the growth and division dynamics of 158
filaments. In damage-induced filaments, we found that length added between divisions 159
was not fixed (Fig. 2A) and did not correlate with length of the cell at birth, with several 160
instances of consecutive divisions without any elongation in between each division. To 161
test whether there is periodicity in division timing, we measured time between 162
consecutive division events. While wild type cells divided every ~20min, filamentous cells 163
displayed a large distribution of times between consecutive division events (Fig. S2A). 164
Although a lack of periodicity in division timing was observed, we found that 67±4% of 165
divisions took place at the opposite pole of the previous division, suggesting that there 166
may be no preference for old or new pole during division licensing (See Fig. S2D-G and 167
materials and methods for details on how divisions are computationally analysed). 168
After damage filament length increased exponentially with a characteristic rate, 169
as seen in wild type conditions (Fig. S2B-C), with some fluctuation which probably arises 170
because of stochasticity in damage and/ or repair. When length added or removed was 171
plotted as a function of birth length in filaments, no clear trend was observed (Fig. 2A). 172
However, when length added/ removed was plotted as a function of division times, we 173
observed that length removed (SD cell length) neither increased nor decreased with inter-174
division time, while length added increased as time between divisions increased (Fig. 2B). 175
This is in contrast to wild type cells where length added/ removed are constant (Fig. 2D-176
E). Overall, our observations suggest that the adder mechanism seen in steady state 177
conditions breaks down in filamentous cells undergoing asymmetric division (see 178
methods section for analysis). This could occur if growth rate changes, or if the 179
relationship between time to division and birth length deviates from what is expected 180
(eq. 3 in supplementary results for a model of this relationship), or both. In case of 181
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elongated cells, we find that cells continue to grow exponentially at a characteristic rate 182
regardless of length, while the relation between time to division and birth length becomes 183
much more noisy. Indeed, when we plotted division time as a function of birth length, a 184
decreasing trend was most clearly observed only for wild type and less so (with more 185
noise) in the case of damage-treated cells (Fig. 2C and 2F). However, two properties of 186
filament division remain constant: a. length removed (SD cell size) is invariant and b. 187
division takes place poleward, asymmetrically. Taken together, this supports the idea that 188
daughter cell size is governed by regulation of cell division rather than filament growth 189
dynamics. 190
Cell division positioning and licensing have distinct regulatory mechanisms 191
Role of Min system in divisome positioning 192
As stated above, we noticed that cells transitioned from mid-cell to asymmetric 193
divisions as they grew longer in length (Fig. 1C). To test if the Min oscillation system 194
(Wehrens et al., 2018; de Boer et al., 1989; Raskin and de Boer, 1999; Tsang and 195
Bernhardt, 2015; Bisicchia et al., 2013; Dewachter et al., 2018) could determine division 196
licensing events in damage-induced filaments, we first asked whether Min oscillations are 197
maintained in these long cells. We imaged MinD-GFP and, consistent with Wehrens et al., 198
2018, found that it localized at several positions along the length of the cell and in an 199
equidistant manner, with a distance of 7 µm on average between each Min localization in 200
MMC or cephalexin-treated cells (Fig. S3A). As expected, we observed that cells had 1-2 201
Min localizations at lengths between 5-10µm and the number of Min localizations 202
increased with increasing cell length (Fig. S3B). Multiple Min localizations would suggest 203
that the number of divisome localizations would also proportionally increase. However, 204
when we imaged the localization of FtsZ (Supplementary video 4) in filaments, we found 205
that the number of FtsZ rings did not scale with increasing cell length and the position of 206
FtsZ shifted from away from mid-cell as cell length increased (Fig. S3C). Importantly, 207
deletion of the min operon resulted in loss of cell size maintenance with divisions that 208
could occur at mid-cell as well (Fig. 3A and S3E). Thus, as in wild type conditions, the Min 209
system may dictate daughter cell size maintenance in damage-induced filaments as well. 210
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Even though daughter cell size maintenance was inaccurate in a min deletion, we 211
found that in both wild type and Dmin cells division occurred only one-site-at-a-time (Fig. 212
3D, S2D-G and S3E). Since we did not observe multiple FtsZ rings or multiple constrictions 213
in recovering filaments, we wondered whether nucleoid occlusion or negative regulation 214
of FtsZ polymerization (Kreuzer, 2013; Mukherjee et al., 1998; Tonthat et al., 2011; 215
Bernhardt and de Boer, 2005) may play a role in restricting the numbers and hence timing 216
of division, resulting in division occurring one-site-at-a-time. However, we found that 217
daughter cell length is maintained in sulA or slmA deletions and the number of 218
constrictions did not increase (Fig. 3B-C and S3D). We then wondered whether FtsZ levels 219
itself could be limiting in these cells (Bi and Lutkenhaus, 1990), thus permitting only one 220
division event at a time. To test this, we over-expressed FtsZ from an arabinose-inducible 221
promoter during damage or cephalexin recovery. In the case of damage-induced 222
filaments, we did not observe more than one constriction on average in both wild type 223
and FtsZ overexpression conditions, irrespective of the length of the cell (Fig. 3E and S3F). 224
Given that chromosome segregation is perturbed in these conditions, we reasoned that 225
the effect of FtsZ overexpression would be better observed in cephalexin-treated cells, 226
where 30% cells had more than one constriction site in wild type background (Fig. S3D). 227
We found that overexpression of FtsZ in cephalexin recovery resulted in the formation of 228
multiple constriction sites in long cells (Fig. S2G, S3D and S3F). However, division timing 229
did not become faster here or in the damage-recovery case (Fig. 3F) and division still 230
occurred one-site-at-a-time (Fig. S2D-G and S3F). It is possible that another divisome 231
component, such as FtsN (Coltharp et al., 2016), may be limiting in filaments, thus 232
resulting in single division events. While we cannot rule out regulation of the divisome at 233
the level of protein activity, transcriptional profiling of cells during recovery from MMC 234
treatment did not show down-regulation of key division components (Fig. 3G). Thus, 235
although the Min system governs division site locations, some other factor(s) determine 236
division site licensing/ timing. 237
Regulation of division licensing: impact of chromosome and terminus segregation 238
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Given that chromosomes in DNA damage-induced filaments are no longer 239
segregated, it is possible chromosome dynamics in filaments could contribute to control 240
of division licensing. To ascertain the role of chromosome segregation in regulation of 241
division timing, we followed chromosomes after DNA damage via imaging the nucleoid-242
associated protein, HupA (Youngren et al., 2014; Marceau et al., 2011), tagged with 243
mCherry after MMC or cephalexin treatment (Fig. 4A; Supplementary video 5). Since 244
filamentous cells carry several copies of their chromosomes, yet division licensing occurs 245
towards a cell pole, we wondered whether there could be differences in chromosome 246
segregation dynamics across the length of the filament. Estimation of location of least 247
intensity of HupA fluorescence (as a proxy for nucleoid-free regions and thus 248
chromosome segregation) revealed that such regions were not near mid-cell in a majority 249
of damage or cephalexin-treated elongated cells (Fig. 4B). In contrast to wild type, where 250
median relative position of nucleoid-free gap from a cell pole was 0.46 (~mid-cell), the 251
location of this gap was broadly distributed between the cell pole and mid-cell with 252
median position of 0.33 and 0.34 from a cell pole for MMC and cephalexin-treated cells 253
respectively. Indeed, the location of the FtsZ-ring also coincided with where the 254
chromosomes were most segregated at the time of division in our damage-recovery 255
conditions (Fig. S4A-B). 256
Interestingly, we observed that early divisions in long filaments resulted in 15±6% 257
of daughter cells that were anucleated. These cells were distinct from mini-cells (Adler et 258
al., 1967) in that their cell size was close to that of wild type (3.5µm) (Fig. 4C). We found 259
that 38% of anucleate divisions occurred at the first division event during recovery. This 260
percentage reduced to <7% by the third division. Thus, we hypothesized that if 261
chromosome segregation has no effect on division dynamics then the time from divisome 262
assembly to cell division should be the same between anucleated and nucleated divisions. 263
However, if segregation had an effect, then the dynamics would vary between the two 264
types of divisions. In the case of divisions that resulted in production of anucleated cells, 265
we found that the average period between FtsZ assembly at the division site to cell 266
division was 7 min. In contrast, in case of nucleated divisions, we noticed that FtsZ 267
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localized in the nucleoid-free regions for significantly longer, with an average of 12 min 268
prior to division (Fig. 4D). 269
We wondered what could be the rate limiting step in allowing division to occur, 270
thus resulting in persistence of FtsZ in nucleoid-free regions in the case of nucleated cell 271
divisions. Given that the terminus region of the chromosome is the last to be segregated 272
prior to division in wild type cells, we followed the dynamics of the terminus during 273
division in recovering filaments using the parS-ParB locus labelling system (Espéli et al., 274
2012; Nielsen et al., 2006) (Fig. S4C-D). While the bulk of the chromosome segregated 275
well-before division, and divisome components downstream of FtsZ, (ZapA and FtsN; 276
Supplementary video 6-7), localized 8 min and 6 min prior to division respectively, a single 277
terminus focus could be observed to persist in the chromosome-free region, where 278
constriction had begun to occur. The constriction completed to division just prior to or 279
concomitant with the termini splitting into two foci on either side of the division plane, 280
within 2 min on average (Fig. 4E, S4C-D). If terminus segregation is indeed key to division 281
licensing to generate nucleated, viable daughter cells, then perturbing this function 282
should affect division dynamics during recovery. A key player in modulating terminus 283
segregation specifically is MatP, which has been implicated in a. structuring the ter 284
macrodomain (via prevention of MukBEF interaction with the terminus) and b. connecting 285
the ter region with the divisome (Espéli et al., 2012; Lioy et al., 2018; Mercier et al., 2008; 286
Nolivos et al., 2016). We found that a matP deletion was compromised in damage 287
recovery (Fig. S4E). These cells had a significant increase in anucleate cell production, with 288
50±3% of divisions being anucleated. This is in contrast to wild type or deletions of slmA 289
or sulA that do not affect terminus segregation, but instead regulate divisome assembly 290
(Fig. 4F). In addition, nucleated daughter cell size maintenance was inaccurate in matP 291
deleted cells (Fig. 4G). These data are consistent with the idea that chromosome and 292
more specifically, terminus segregation acts as the licensor of nucleated divisions in 293
filaments. Taken together, this suggests that coordination between Min-mediated 294
divisome localization and MatP-mediated terminus positioning/ segregation regulates 295
daughter cell size maintenance, thus facilitating recovery from DNA damage (Fig. S5). 296
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Discussion 297
In laboratory conditions, wild type E. coli maintains a distinct periodicity of cell 298
growth and division that appears to be coupled with chromosome replication and 299
segregation (Donachie, 1968; Taheri-Araghi et al., 2015; Wallden et al., 2016; Micali et al., 300
2018; Campos et al., 2014; Arjes et al., 2014; Kleckner et al., 2014; Hill et al., 2012). 301
However, it is becoming increasingly evident that bacteria can exist in diverse 302
morphological states, in part dictated by their environmental conditions (Yang et al., 303
2016; Justice et al., 2008; Heinrich et al., 2019; Jonas, 2014; Muraleedharan et al., 2018; 304
Kysela et al., 2016; Caccamo and Brun, 2018; MacCready and Vecchiarelli, 2018). Even E. 305
coli can become highly filamentous under conditions of stress such as during infection 306
(Horvath et al., 2011; Justice et al., 2004). Transitions into filamentous morphologies are 307
thought to confer several advantages such as avoiding phagocytosis via the host immune 308
response or providing a means to dilute the effects of any inhibitors present in the 309
surroundings (Yang et al., 2016; Justice et al., 2008; Horvath et al., 2011; Justice et al., 310
2004). Recent reports have also suggested that filamentation (at least in the case of E. coli 311
treated with ciprofloxacin) may be the first step towards the emergence of antibiotic 312
resistance as daughter cells carry mutations making them ciprofloxacin resistant (Bos et 313
al., 2015). Thus, it becomes important to understand how bacterial cells enter and exit 314
these filamentous states to ensure survival under stress. Indeed, several studies have 315
characterized mechanisms by which filamentation is induced under DNA damage 316
(Kreuzer, 2013; Mukherjee et al., 1998; Suzuki et al., 1967; Jonas, 2014; Modell et al., 317
2014, 2011; Adler and Hardigree, 1965; Uphoff, 2018). However, once DNA repair has 318
been completed, how cells restore wild type growth is unclear. 319
Here we show that cell cycle restoration involves asymmetric chromosome 320
segregation and cell division in filaments, resulting in the production of daughter cells of 321
wild type cell size and with undamaged DNA. While the filament itself may or may not 322
recover, several short daughters generated from a single filament go on to replicate and 323
divide as wild type cells. The concept of asymmetric partitioning of cellular components 324
during stress seems to have been co-opted by several bacterial systems (Schramm et al. 325
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2020) including M. tuberculosis, where one daughter cell inherits the growing pole, while 326
the other has to assemble a growth pole de novo. This results in difference in susceptibility 327
to antibiotic treatment between the two cell types; the daughter cell with the growing 328
pole is more sensitive to cell wall synthesis inhibitors when compared to cells that 329
inherited the non-growing pole (Aakre and Laub, 2012; Aldridge et al., 2012). The 330
swarmer cells of P. mirabilis and V. parahaemolyticus (Muraleedharan et al., 2018; 331
MacCready and Vecchiarelli, 2018) use the Min-system to regulate asymmetric cell 332
division to allow for such division while still preserving the population of filamentous cells. 333
Dim-light stress induces filamentation in the photosynthetic cyanobacterium S. 334
elongatus, which then divides asymmetrically via positioning by the Min system (Liao and 335
Rust, 2018). Even in E. coli filaments generated in non-DNA damage conditions, studies 336
have reported poleward division events (Adler and Hardigree, 1965; Wehrens et al., 2018; 337
Taschner et al., 1988; Begg and Doanachie, 1977; Mileykovskaya et al., 1998). Taken 338
together, this suggests that switching from mid-cell to filamentation-based division may 339
be a universal method for cells under stress to ensure viable cell divisions. 340
Consistent with previous reports (Wehrens et al., 2018), we find that damage-341
treated cells switch from mid-cell to polar division in a size-dependent manner. Our 342
results suggest that the growth dynamics of filaments does not regulate size homeostasis 343
at division. Instead, size of the daughter cell is determined by a combination of Min 344
oscillations and chromosome segregation (Fig. S5). Cell growth may or may not occur to 345
accommodate both these events, consistent with the observation that consecutive 346
divisions can take place independent of addition of length in between, likely due to the 347
accumulation of already duplicated termini that undergo segregation. Previous studies in 348
wild type conditions have proposed that division timing could be modulated by factors 349
such as rates of constriction or concurrency between processes of chromosome 350
segregation and division (Micali et al., 2018; Lambert et al., 2018). In damage-induced 351
filaments, we find that cell division is composed of two distinct steps of division site 352
location and licensing. This results in a one-site-at-a-time cell division, which may serve 353
as a checkpoint to ensure accurate and complete chromosome segregation. As seen for 354
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E. coli filaments under other stresses (Wehrens et al., 2018), the MinCDE system 355
contributes to determining the location of divisome assembly. As cell length changes, the 356
location of the Min oscillation nodes closest to the poles are likely to remain at a constant 357
relative position, whereas the relative position of oscillation nodes away from the poles 358
are likely to keep changing as the cell length changes. This would also explain why cells 359
switch from mid-cell to poleward division as they increase in length. Subsequent to this, 360
divisome assembly may be stabilized due to chromosome segregation away from mid-361
cell. Indeed, a recent study has shown that physical forces (such as molecular crowding) 362
squeeze or confine DNA towards mid-cell in filaments (Wu et al., 2019), which could 363
facilitate better or faster separation of chromosomes nearer the poles during segregation.364
Our data are consistent with the idea that once the division machinery has 365
assembled at a potential site of division, licensing occurs only when no DNA is 366
encountered by the divisome; thus even in the absence of chromosome segregation 367
anucleate cells can be produced (Mulder and Woldringh, 1989). However, upon initiation 368
of chromosome resegregation, it is likely that a division checkpoint is triggered by the 369
terminus region of the chromosome, via negatively regulating the ability of division to be 370
completed. Consistently, we find that chromosome segregation can impose a delay in 371
division, with the terminus being the last region of the chromosome to be segregated. 372
Specifically affecting terminus segregation via deletion of matP results in perturbation to 373
damage recovery, with a significant increase in anucleate cell production. These cells also 374
have a broad distribution of nucleated daughter cell sizes. Hence concurrency between 375
terminus segregation and cell division contributes to daughter size maintenance during 376
asymmetric cell division. Given that MatP has two independent functions of ter region 377
organization and ter anchoring with the divisome (Männik et al., 2016; Bailey et al., 2014; 378
Nolivos et al., 2016), it would be important to understand which of these activities is 379
specifically necessary in the recovery process. The system characterized in this study now 380
provides the ability to assess these rate-limiting steps of division licensing and further 381
probe the mechanisms of asymmetric chromosome segregation that preferentially results 382
in daughter cells devoid of damage. 383
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In sum, our study highlights the requirement for coordination between two 384
independent mechanisms of divisome regulation for division accuracy in filaments. The 385
Min system facilitates localization of FtsZ in nucleoid-free regions. In parallel, modulation 386
of terminus region dynamics by MatP regulates the licensing of cell division. Concurrency 387
between terminus positioning and segregation with cell division ensures daughter size 388
maintenance in dividing filaments. Together, this results in the production of viable 389
daughter cells with wild type growth and division dynamics and contributes to the 390
restoration of cell size distribution, even when the filament itself may not recover. The 391
conservation of asymmetric division in a range of stress-induced bacterial filaments 392
underscores the importance of this mechanism in facilitating robust exit of cells from the 393
DNA damage checkpoint via the generation of fit, damage-free daughter cells. 394
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Author contributions 395
SR: conception of project, experimental design, execution of experiments, data analysis 396
and writing of manuscript. AC: experimental design and execution of experiments and 397
analysis related to RecA. SK: data analysis and interpretation, writing of manuscript. AV: 398
reagents, tools and interpretation. AB: conception of project, experimental design, 399
writing of manuscript and funding. 400
401
Declaration of interests 402
The authors declare no competing interests. 403
404
Acknowledgements 405
The authors are grateful to Thomas Bernhardt, Steven Sandler, Suckjoon Jun, Bill 406
Soederstroem, Fred Boccard, Olivier Espeli, Christian Lesterlin, Ramanujam Srinivasan and 407
Manjula Reddy for generous sharing of strains and plasmids. The authors acknowledge 408
assistance from Ismath Sadhir in RNA extraction, Nitish Malhotra in RNA-seq analysis, 409
Aditya Jalin, Alex Sam Thomas and Aalok Varma in writing Matlab scripts as well as the 410
Central Imaging and Flow Facility (CIFF) and Next-generation genomics facility (NGGF). 411
The authors thank Fagwei Si, Piet de Boer, Raj Ladher, Michael Laub, Tung Le and 412
members of the AB lab for helpful discussions/ feedback on the manuscript. This work is 413
supported by funding from NCBS-TIFR (AB and SK), by an HFSP Career Development award 414
(AB) and funding from the Simons Foundation (SK). 415
416
Materials and Methods 417
Bacterial strains and growth conditions 418
Strains and plasmids used in the study are listed in Table 1. Transductions were conducted 419
with P1 phage following the protocol in (Thomason et al., 2007). For Escherichia coli, cells 420
were grown at 37°C in either rich media (LB: for 1 L dissolved 10 g tryptone, 5 g yeast 421
extract and 10 g NaCl in doubled distilled water) or minimal media (M9-Cas: for 1 L 422
dissolved 5 g glucose, 1 g casamino acids, 1 ml of 0.5% thiamine, 1 ml of 1M MgSO4 and 423
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200 ml 5x M9 salts in double distilled water). DNA damage was induced with 1 μg/ml of 424
mitomycin C (MMC) for 30, 60 or 90 min (LB) or 90 min (M9-Cas) (unless otherwise 425
indicated). Cell division was inhibited using 5 μg/ml of cephalexin for 60 min (LB) or 90 426
min (M9-Cas). 427
Fluorescence Microscopy 428
Imaging was performed on a widefield, epifluorescence microscope (Eclipse Ti-2E, Nikon 429
with motorized xy-stage, Z-drift correction), 63X plan apochromat objective (NA1.41), 430
pE4000 light source (CoolLED), OkoLab incubation chamber and Hamamatsu Orca Flash 431
4.0 camera. Images were acquired using the NIS-elements software (version 5.1). 432
Microfluidics imaging was performed using the CellASIC-ONIX2 Microfluidic System, 433
Temperature Controlled CellASIC-ONIX2 Manifold XT and CellASIC ONIX Plate for Bacteria 434
Cells (B4A) (Merck). Details of the imaging and acquisition setting are described here 435
(Raghunathan and Badrinarayanan, 2019). 436
Time-course and Time-lapse imaging 437
For the recovery time-course, cells were grown overnight in LB or M9-Cas, back diluted 438
to ~OD600 0.01 and allowed to grow to OD600 ~0.1. Culture was then treated with MMC or 439
cephalexin for 60 min (LB) or 90 min (M9-Cas). Cells were pelleted, washed with fresh 440
media, resuspended to OD600 ~0.1 and then allowed to recover from damage treatment. 441
Cultures were maintained in log phase (OD600 ~0.1 - 0.4) throughout the experiment. For 442
time-course, samples for microscopy were collected at time points indicated. 1 ml of 443
culture was pelleted, resuspended in 100 μl and then spotted on 1% agarose pad and 444
imaged (Chimthanawala and Badrinarayanan, 2019). 445
For time-lapse experiments, damaged-induced cells were pelleted and washed with fresh 446
media and were either loaded into the microfluidics device or spotted on a 1.5% agarose 447
pad (made with appropriate growth media) and imaged. All time-lapse images were taken 448
every 30 sec or 2 min for ~3 hrs. For the FtsZ overexpression or FtsN imaging experiments, 449
cells were induced with either 0.3% arabinose or 0.1% rhamnose respectively for 1 hr 450
prior to imaging. For MinD-GFP imaging, cells were induced with 0.2mM IPTG for 90 min 451
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prior to imaging. Inducers were maintained in the agarose pads or microfluidic plates. 452
Experiments were performed in LB unless otherwise indicated. 453
RNA sequencing 454
Protocol for RNA extraction described in (Badrinarayanan et al., 2017) was followed. 455
Briefly, cell pellets were collected during the recovery time-course at the indicated times. 456
RNA was extracted using the Direct-zol™ RNA MiniPrep (Zymo, Cat no. R2052A81) and 457
RNA Clean & Concentrator™-25 (Zymo, Cat no. R1018A82). RNA libraries were prepared 458
using the TruSeq Stranded mRNA Library Preparation kit at NCBS NGS facility. Libraries 459
were sequenced using Illumina MiSeq sequencing platform. Raw reads (single end; read 460
length = 50 bp) were obtained as .fastq files. The reference genome sequence (.fna) and 461
annotation (.gff) files for the same strain (accession number: NC_000913.3) were 462
downloaded from the ncbi ftp website ("ftp.ncbi.nlm.nih.gov"). The raw read quality was 463
checked using the FastQC software (version v0.11.5). BWA (version 0.7.12-r1039) 464
(Burroughs and Aravind, 2016; Li and Durbin, 2009) was used to index the reference 465
genome. Reads with raw read quality ≥20 were aligned using BWA aln -q option. 466
SAMTOOLS (version 0.1.19-96b5f2294a) (Li et al., 2009) was used to filter out the multiply 467
mapped reads. BEDTOOLS (version 2.25.0) (Quinlan and Hall, 2010) was used to calculate 468
the reads count per gene using the annotation file (.bed) in a strand specific manner. 469
Normalized counts per millions (cpmn) was obtained using TMM normalization of EdgeR 470
package. The cpmn across time was calculated relative to the no damage control. The log2 471
fold change in expression was plotted from this. 472
Image Analysis 473
Images acquired were visualized and processed using ImageJ (Schindelin et al., 2012, 474
2015). Segmentation was performed using Oufti (Paintdakhi et al., 2016). Foci tracking 475
was accomplished using the Spotfinder Z function of MicrobeTracker (Sliusarenko et al., 476
2011). For recovery time-course, custom Matlab scripts were used to extract cell length 477
and total fluorescence intensity information for each cell. Anucleate cells were identified 478
using a threshold of 0.02 a. u. for total intensity of HupA-GFP in the cells. For time-lapse 479
analysis, at each division, the longer cell was termed LD and the smaller cell given the 480
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identity of SD. A custom Matlab script was used to the extract the lengths of the LD and SD 481
at each division, the length added between divisions and time between divisions. 482
Daughter cells were classified as ‘recovers’ if at their first division, their length was <10µm 483
and they divided symmetrically. Cells that were longer at their first division and divided 484
asymmetrically were classified as ‘filaments’. RecA foci numbers were obtained using 485
Spotfinder Z and combined with cell length information from Oufti. Number of FtsZ rings 486
in a cell were obtained using Spotfinder Z and combined with cell length information from 487
Oufti. For analysis of constrictions and divisions, we used Oufti, which allows sub-pixel 488
segmentation of phase contrast images of cells in a time-lapse. The method is illustrated 489
in figure S2 in the supplementary methods section. In (a), phase profile for a wild type cell 490
before and after division is plotted. While the profile is, on average, a flat line for most of 491
the time imaged, one can identify a constriction prior to division (marked with *). Similarly 492
in (b), the phase profile for a filamentous cell (DNA damage-induced) undergoing division 493
is plotted. If the phase profile deviates by 20-25% of the cell width, it is called a 494
constriction. If the phase profile is discontinuous (with a gap), then it is marked as a 495
division by the segmentation algorithm automatically. For nucleoid tracking, fluorescence 496
intensity profiles of HupA-mCherry, obtained from Oufti was used. Data was first 497
smoothened using a Savitzgy-Golay filter (Luo et al., 2005). Following this the fluorescence 498
profile was inverted so that the regions of lowest fluorescence now had the highest 499
values. Peaks were determined using the peak prominence function after setting a 500
threshold of half the maximum intensity for each frame. Peaks correspond to lowest 501
fluorescence intensity in the cell. Regions right next to the cell poles were excluded. This 502
information was combined with FtsZ foci tracked using Spotfinder Z to obtain relative 503
positions of the lowest fluorescence intensity in the cell and FtsZ ring. Formation of FtsZ, 504
ZapA and FtsN foci, and segregation of the single terminus focus (ParB-GFP) to 2 foci at 505
the site of division was scored manually along with time to division. 506
507
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Main Figure legends 508
Figure 1: DNA-damage induced filaments maintain daughter cell size during division. a. 509
Representative time-lapse montage of filamentous cells during recovery. White asterisks 510
indicate divisions occurring towards a cell pole. Scale bar - 5 µm; time in min here and in 511
all other images. b. Cell length of two daughter cells generated from a single division in 512
wild type conditions. Each grey dot represents a single division event (n = 157). The red 513
line plots the expected values if all cells were dividing at their mid-point. c. Cell length of 514
long daughter (LD) and short daughter (SD) generated from a DNA damage-induced 515
filament during recovery. Cells are treated with mitomycin C (MMC) for 60 min. Each grey 516
dot represents a single division event (n = 531). The red line plots the expected values if 517
all cells were dividing at their mid-point. d. Location of division is plotted as a function of 518
cell length in filamentous E. coli during recovery from DNA damage treatment (60 min; n 519
= 531). e. Cell length of long daughter (LD) and short daughter (SD) is tracked over time 520
during damage recovery. Decrease in cell length is indicative of division. f-g. As (b-c) for 521
cells treated with MMC for 30 min. h. Fate of SD and LD during recovery. Cell is classified 522
as recovered if it undergoes mid-cell division and filamentous if it continues to filament 523
after division (n ≥ 56) i. Number divisions per cell in 1 hr for all durations of damage 524
treatment. As a control, number of divisions wild type cells undergo is also shown (n≥100 525
division). j. Distribution of time between divisions for wild type (no damage control), LD 526
and SD during recovery from MMC (n ≥ 104). 527
Figure 2: Daughter cell size is governed by cell division dynamics of filaments. a. Length 528
added to a cell between divisions (black dots) and length removed at the latter division 529
(i.e., the length of the daughter cell) (blue dots) as a function of the birth length of the 530
cell for MMC-treated cells. b. Length added to a cell between divisions (black dots) and 531
length removed at the division (i.e., the length of the daughter cell) (blue dots) as a 532
function of the time between divisions for MMC-treated cells. c. Time between divisions 533
as a function of cell length at birth. Red line shows the relation that would be necessary 534
for the system to be an adder (equation (3) in supplementary results), given that cells are 535
growing exponentially with the rates given in Fig. S2B-C. d-f. (As a-c) for wild type cells. 536
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Figure 3: Cell division positioning and licensing have distinct regulatory mechanisms. 537
Determining where to divide: role of Min system. a-c. Cell length of long daughter (LD) 538
and short daughter (SD) generated from a DNA damage-induced filament during recovery 539
for ΔminCDE (n=186), ΔslmA (n=144) and ΔsulA (n=246) backgrounds respectively (grey 540
dots). As a reference, lengths for wild type during recovery are shown in orange. The red 541
line plots the expected values if all cells were dividing at their mid-point. d. Representative 542
time-lapse montage of division in wild type cells during damage recovery. e. Number of 543
constrictions per cell plotted as a function of cell length during DNA damage recovery for 544
wild type and cells over-expressing FtsZ (n ≥ 91). f. Time between divisions for no damage 545
(control) and for cells after treatment with MMC or cephalexin with (++FtsZ) or without 546
(wild type) FtsZ overexpression (n ≥ 93). g. Heat map of transcript levels (from RNA-seq) 547
of genes involved in cell division during a damage recovery time-course. As a control, 548
genes induced under the SOS response are also highlighted (bold). Log2-fold change 549
normalized to control without damage is plotted. 550
Figure 4: Cell division positioning and licensing have distinct regulatory mechanisms. 551
Regulation of division licensing: impact of chromosome and terminus segregation. a. 552
Representative time-lapse montage of division in cells during recovery. Grey – phase, red 553
– HupA-mCherry (chromosome); scale bar - 5 µm; time in min. b. Position of least intensity 554
of HupA fluorescence (gaps between chromosomes) plotted as a function of cell length 555
(from one pole to mid-cell) in recovering MMC or cephalexin-treated filaments. As 556
reference, these data are also shown for wild type cells with no damage treatment 557
(control) (n ≥ 150). c. Cell length distribution for wild type cells (no perturbation) is 558
plotted. Along with this, cell length distribution of daughter cells during DNA damage 559
recovery is plotted for nucleate cells and anucleate cells. To highlight the distinction 560
between these cell division events and minicell formation, cell length distribution of 561
minicells (from min deleted cells) is also shown (n ≥ 100) d. Time from FtsZ localization to 562
division completion is plotted for divisions that result in nucleated or anucleated SD cells. 563
(n ≥ 38) e. Distribution of time to division after FtsZ, ZapA and FtsN localization to division 564
site is plotted. Along with this, time to division after segregation of terminus (ter) during 565
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recovery after MMC treatment is also shown (n ≥ 93). f. Percentage of SD that are 566
anucleate, recover and filament is plotted for wild type and deletions of matP, sulA or 567
slmA during DNA damage recovery. (n ≥ 103) g. Daughter cell length distribution for 568
nucleated divisions in the case of matP deletion and wild type is plotted. As a reference 569
cell length distribution for cells without damage treatment is also plotted for each case (n 570
= 145). 571
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819
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Figure 1
a.
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.CC-BY 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprintthis version posted March 16, 2020. . https://doi.org/10.1101/2020.03.16.993485doi: bioRxiv preprint
Figure 2
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.CC-BY 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprintthis version posted March 16, 2020. . https://doi.org/10.1101/2020.03.16.993485doi: bioRxiv preprint
*
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Figure 3
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.CC-BY 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprintthis version posted March 16, 2020. . https://doi.org/10.1101/2020.03.16.993485doi: bioRxiv preprint
Figure 4
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.CC-BY 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprintthis version posted March 16, 2020. . https://doi.org/10.1101/2020.03.16.993485doi: bioRxiv preprint