Transcriptional Response of Streptomyces coelicolor to Rapid Chromosome Relaxation or Long-Term Supercoiling Imbalance

Negative DNA supercoiling allows chromosome condensation and facilitates DNA unwinding, which is required for the occurrence of DNA transaction processes, i.e., DNA replication, transcription and recombination. In bacteria, changes in chromosome supercoiling impact global gene expression; however, the limited studies on the global transcriptional response have focused mostly on pathogenic species and have reported various fractions of affected genes. Furthermore, the transcriptional response to long-term supercoiling imbalance is still poorly understood. Here, we address the transcriptional response to both novobiocin-induced rapid chromosome relaxation or long-term topological imbalance, both increased and decreased supercoiling, in environmental antibiotic-producing bacteria belonging to the Streptomyces genus. During the Streptomyces complex developmental cycle, multiple copies of GC-rich linear chromosomes present in hyphal cells undergo profound topological changes, from being loosely condensed in vegetative hyphae, to being highly compacted in spores. Moreover, changes in chromosomal supercoiling have been suggested to be associated with the control of antibiotic production and environmental stress response. Remarkably, in S. coelicolor, a model Streptomyces species, topoisomerase I (TopA) is solely responsible for the removal of negative DNA supercoils. Using a S. coelicolor strain in which topA transcription is under the control of an inducible promoter, we identified genes involved in the transcriptional response to long-term supercoiling imbalance. The affected genes are preferentially organized in several clusters, and a supercoiling-hypersensitive cluster (SHC) was found to be located in the core of the S. coelicolor chromosome. The transcripts affected by long-term topological imbalance encompassed genes encoding nucleoid-associated proteins, DNA repair proteins and transcriptional regulators, including multiple developmental regulators. Moreover, using a gyrase inhibitor, we identified those genes that were directly affected by novobiocin, and found this was correlated with increased AT content in their promoter regions. In contrast to the genes affected by long-term supercoiling changes, among the novobiocin-sensitive genes, a significant fraction encoded for proteins associated with membrane transport or secondary metabolite synthesis. Collectively, our results show that long-term supercoiling imbalance globally regulates gene transcription and has the potential to impact development, secondary metabolism and DNA repair, amongst others.

INTRODUCTION 40 experimental conditions. Subsequently, the log2 value of the fold change was calculated. 169 Thetranscripts with a log2 value between -1.5 and 1.5 were eliminated from subsequent analyses, 170 with the exception of NAPs and topoisomerases encoding genes, for which the threshold was based 171 only on the p-value significance. The gene distribution analysis was performed by mapping the 172 positions of transcription start points of identified genes or the first gene in a regulated operon 173 determined on the basis of the RNA-seq data, to the S. coelicolor chromosome. To identify gene 174 clusters of interest, we determined the percent of affected genes within a 250 kbp fragment of the S. 175 coelicolor chromosome, subsequently moving the calculation window by 125 kbp in every step. 176 Those regions in which the percentage of affected genes was higher than 5% were classified as 177 supercoiling-sensitive clusters. 178

Reverse-transcription and quantitative PCR (RT-qPCR) 179
For RT-qPCR analyses, RNA from S. coelicolor mycelia cultivated in liquid 79 medium for 24 180 hours was isolated using the GeneJET RNA isolation kit (Thermo Scientific, USA) according to the 181 manufacturer's procedure (except that the concentration of the lysozyme in the suspension buffer 182 was increased to 10 mg/ml). The isolated RNA was digested with TURBO DNase I (Invitrogen) to 183 remove traces of chromosomal DNA and then purified and concentrated using the GeneJET RNA 184 Cleanup kit (Thermo Scientific). A total of 500 ng of RNA was used for cDNA synthesis using a Maxima 185 In previous work, we found that exposing S. coelicolor to novobiocin, initially led to the rapid 206 relaxation of DNA, followed by gradual restoration of topological homeostasis (Szafran et al., 2016), 207 as had been seen in other bacteria (Ferrandiz et al., 2010). The return to topological balance 208 appeared to stem from changes in the expression of topoisomerase encoding genes, which in 209 S. coelicolor corresponded to a strong induction of gyrAB but only slight inhibition of topA (Szafran et 210 al., 2016); this is different from the simultaneous induction of gyrAB and inhibition of topA 211 transcription observed in other bacteria (Ahmed et al., 2015;Unniraman and Nagaraja, 1999). Here, 212 we sought to identify other genes in S. coelicolor whose transcription was also directly affected by 213 the rapid chromosome relaxation. To this end, we treated S. coelicolor liquid cultures with 10 g/ml 214 novobiocin (the concentration equal to MIC 90 , Fig. S1). We analyzed the changes in global transcript 215 strain. Based on previous work (Gmuender et al., 2001;Szafran et al., 2016), 10 minutes of 217 novobiocin exposure was presumed to be sufficient to affect promoters directly sensitive to gyrase 218 inhibition, triggering the primary transcriptional response, but was deemed insufficient time to 219 induce a secondary transcriptional response (which depends on any primary-induced transcriptional 220 regulators). 221 In analyzing our RNA-Seq data, we found that the rapid chromosome relaxation caused by 222 novobiocin (Fig. 1A and S3A-B) led to a 2.71-fold induction of the gyrBA operon transcription (Fig.  223 1B), confirming that the primary transcriptional response could be induced by 10 minutes of gyrase 224 inhibition. We next set out to identify genes exhibiting distinct changes in transcription in 225 comparison to the untreated control (at least 2.83-fold), and found 121 genes that were sensitive to 226 the rapid loss of chromosome supercoiling. These genes constitute 1.5% of the S. coelicolor open 227 reading frames (ORFs) (see supplementary file 'novobiocin' for the complete list of the identified 228 genes). Mapping of the transcription start sites (TSSs) of the identified genes to the S. coelicolor 229 chromosome revealed that the novobiocin-sensitive genes were unevenly distributed along the S. 230 coelicolor chromosome ( Fig. 1C and S3C). Surprisingly, in contrast to E. coli, in which 106 genes were 231 induced whereas 200 genes were repressed by gyrase inhibition (Peter et al., 2004), in S. coelicolor 232 most of the novobiocin-sensitive genes (117 of 121) were upregulated by chromosome relaxation, 233 with only 4 genes (sco0498, sco1377, sco3726 and sco3918) being strongly downregulated (log2-234 fold>1.5). 235 We speculate that the gene upregulation after novobiocin exposure rather than their 236 transcriptional repression may be due to the high GC content (72%) of the S. coelicolor genome 237 (Bentley et al., 2002). In E. coli, promoter regions of relaxation-induced genes are notably enriched in 238 AT base pairs, whereas relaxation-repressed promoters have increased GC content (Peter et al., 239 2004). In S. coelicolor, the increase in GC content in the promoter regions to levels above the average 240 would be expected to result in increased DNA stability, which in turn may inhibit promoter unwinding. Conversely, more AT-rich promoter regions would be expected to favor promoter 242 upregulation. To test this hypothesis, we compared the AT content of 66 novobiocin-induced 243 promoters with the AT content of randomly chosen promoter regions. As predicted, we found that 244 novobiocin-induced genes showed approximately 2-3% higher AT content compared with the 245 randomly selected promoters and 8-9% higher in comparison to the average of the S. coelicolor 246 genome (28%). Notably, the AT-rich region was significantly expanded in the novobiocin-sensitive 247 genes relative to the random promoters (Fig. 1D). Unexpectedly, AT-content analysis revealed a GC-248 rich region located approximately 100-200 bp upstream of the novobiocin-induced genes (Fig. 1D (Zhang et al., 2007). Interestingly, a high fraction of the novobiocin-sensitive genes (~30%) encode 263 proteins involved in either antibiotic production (10 genes, including the actinorhodin activator 264 sco5085/actII-orf4 and genes within the coelibactin biosynthetic cluster sco7681-sco7688) or 265 putative membrane transporters (at least 25 genes) ( Table 2). This suggests that rapid chromosome 266 relaxation functions as the stress signal that triggers the transcription of genes whose products are 267 involved in stress adaptation (i.e., those encoding regulatory proteins) and/or interspecies 268 competition. Considering that decrease of gyrase activity and rapid chromosome relaxation may 269 result from exposure to different cell membrane destabilizing agents that affect transmembrane 270 potential and ATP synthesis, or other biologically active molecules produced by competing bacterial 271 species (Prakash et al., 2009), it is conceivable that the intrinsic response to such agents should 272 encompass the induction of transport proteins that may restore homeostasis to the intracellular 273 environment and/or function to eliminate toxic compounds. 274 In summary, the significant fraction of novobiocin-sensitive genes encompassed those 278 encoding membrane transporters, antibiotic synthesis and regulatory proteins. The novobiocin-279 sensitive genes were nonuniformly distributed along the chromosome, and most of them were 280 upregulated. The upregulation was reflected by the increased AT-content of their promoter regions 281 and the presence of a upstream GC-rich DNA stretch. 282

Long-term exposure to topological imbalance globally affects gene transcription 283
Having established that approximately 120 genes were directly impacted by gyrase inhibition, 284 we were next interested in assessing the response of S. coelicolor to the long-term effects of altered 285 chromosome supercoiling. To date, there is little understood about the long term effects of altered 286 supercoiling, since most of studied bacteria were able to quickly restore topological homeostasis. First, we compared the number of S. coelicolor genes that were strongly affected by 306 topA-down-or upregulation (cultivation in the absence or presence of 1 g/ml thiostrepton). We 307 identified 552 genes whose transcription was changed by topA-downregulation and 214 genes 308 altered by the induction of topA transcription (at least 2.83-fold change; log2-fold >1.5), which 309 corresponded to 7.0% and 2.7% of the S. coelicolor predicted coding sequences, respectively ( were sensitive to both changes in supercoiling conditions (Fig. 2C). This may imply that long-term 316 topological imbalances of all types may trigger shared transcriptional response pathway(s). 317 Nevertheless, a substantial fraction of genes responded specifically to increase of negative 318 supercoiling resulting from TopA depletion (408 of 552, more than 70%), whereas topA-upregulation 319 regulated specifically only 32% (70 of 214) of identified genes (Fig. 2C). The observed difference in 320 the number of the supercoiling sensitive genes (SSGs), as well as their specificity for particular 321 supercoiling conditions, may be presumably explained by the scale of topological imbalance, which is 322 more pronounced under the former than under the latter conditions ( Fig. 2A and S3). However, our 323 results also show that, even though the topological balance was only slightly distorted at topA 324 induction, the transcription of a relatively high number of genes was still affected, suggesting the 325 presence of mechanism(s) that maintains a modified level of gene transcription. Markedly, similar 326 observations were made recently for S. pneumoniae transcriptional activity of genes under TopA 327 inhibition conditions (Ferrandiz et al., 2016), where the expression of many genes was specifically 328 altered. Collectively, these findings may suggest that transient supercoiling imbalances could lead to 329 long-term rearrangement of bacterial chromosome topology and long-standing changes in gene 330 transcription even after the global supercoiling level is restored. 331 Overall, we observed that a persistent supercoiling imbalance induced by constitutive change 332 of TopA expression affected the transcription of approximately 3-7% of S. coelicolor genes. 333 Interestingly, the inhibition of TopA in S. pneumoniae (which was followed by restoring of 334 supercoiling imbalance) affected only 2% of the genome while long term TopA depletion in S. 335 coelicolor had more pronounced effect on the gene transcription. This suggests that the response to 336 topological imbalance depends on possibilities to modify transcriptional program, being the most 337 pronounced when the topoisomerase genes expression cannot not be readjusted. 338

Long-term supercoiling imbalance impacts the expression of discrete gene clusters 339
Remarkably, supercoiling sensitive genes whose transcription was affected by the long-term 340 topological imbalance were nonuniformly distributed along the S. coelicolor chromosome, similar to 341 genes induced in the novobiocin-treated cells. SSGs induced by topA-downregulation or topA-342 upregulation showed characteristic grouping, suggesting they were organized into supercoiling-343 sensitive clusters, which were in turn separated by supercoiling insensitive regions (Fig. 3A). To 344 quantify these observations, we calculated the percentage of genes affected by different supercoiling 345 conditions within 250 kbp chromosome regions along the whole chromosome. We found that gene 346 clustering was strongly detectable under topA-downregulation conditions, with 6-7 chromosomal regions containing more than 5% of genes affected negatively or positively in each cluster (Fig. 3B). 348 Notably, those clusters containing the positively and negatively regulated genes partially overlapped, 349 further supporting the existence of DNA domains that are particularly sensitive to changes in DNA 350 supercoiling. Interestingly, gene clustering was also detectable, although less pronounced, under 351 topA-upregulation conditions, where the distribution of the supercoiling-sensitive regions along the 352 chromosome was comparable to that observed in the TopA-depleted strain ( Fig. 3B and S4A). 353 Analogous clustering of SSGs has been described previously for E. coli and S. pneumoniae (Ferrandiz 354 et al., 2016, Peter et al., 2004. Collectively, these observations suggest that the organization of SSGs 355 into topologically separated domains is a feature conserved among bacteria, presumably ensuring 356 the coordinated expression of the associated genes. Remarkably, the loci of the genes affected by 357 topological imbalance showed increased density in the central part of the chromosome, however the 358 initiation of the chromosomal replication region (oriC) was found to be outside the identified SSG 359 clusters, and the oriC proximal genes, which predominantly encode proteins involved in DNA 360 replication, were insensitive to the changes in chromosome supercoiling (Fig. 3). 361 Interestingly, among several identified gene clusters impacted by supercoiling imbalance, 362 one cluster was particularly enriched in SSGs, encompassing ~20% of all genes positioned within the 363 250 kbp region. Closer examination of this region revealed that 26 out of 34 genes within 30 kbp 364 (between sco4667 and sco4700) were supercoiling sensitive. We therefore termed this region the 365 supercoiling-hypersensitive cluster (SHC). Most of the SHC genes encoded products of unassigned 366 function and these genes themselves were poorly transcribed under standard culture conditions. 367 However, they were strongly upregulated under both topA-downregulation and topA-upregulation 368 conditions (Table 3). We confirmed these observations and excluded the effect of growth medium or 369 genetic modifications in the PS04 strain, by measuring the relative transcript level in PS04 and 370 control strain cultures grown in liquid 79 medium using RT-qPCR and oligonucleotides specific to the 371 first (sco4667) and one before last (sco4699) genes of the SHC region. The transcription of both 372 genes appeared to be highly dependent on the level of TopA: their transcription significantly 373 increased when TopA was depleted, decreased when TopA was at the wild type level, and increased 374 slightly again at topA induction (Fig. S4B). These observations further supported the proposal that 375 SHC upregulation was correlated with relative degree of topological imbalance. Since we did not 376 observe increased expression of genes in the SHC region following a 10 minute exposure to 377 novobiocin, we speculate that its activation requires not only a supercoiling imbalance, but also the 378 activity of other factors. 379 The SHC genes include those encoding a putative two-component system (sco4667 and 382 sco4668) genes organized in a single operon and additional two putative transcriptional regulator-383 encoding genes positioned upstream (sco4671 and sco4673). The cluster also encompasses genes 384 whose products may contribute to DNA/RNA transactions, including: putative DNA helicase 385 to compensate for the changes in chromosome topology. According to previous reports (Bradshaw et 407 al., 2013) NAPs are present at high levels during wild type S. coelicolor vegetative growth. We 408 confirmed that under standard growth conditions, many NAP genes, including hupA, ssbA, lsr2, sihf 409 and hupS were highly transcribed (Fig. 4A), while other sporulation and/or stress responsive genes 410 like dpsB, dpsC and ssbB, were poorly transcribed. Next we assessed the impact of the chromosome 411 supercoiling changes on the expression of these genes. 412 Following TopA-depletion (conditions of increased negative supercoiling), we observed 413 transcript levels of DNA-organizing proteins to be statistically different (p < 0.05) (Fig. 4B). Of the 414 NAPs affected by changes in chromosome topology, hupA and hupS were amongst the most 415 impacted. Notably, however, the transcriptional effects were in opposite directions: hupA transcript 416 levels increased to 1.44 relative to wild type conditions, while hupS transcript levels decreased to 417 0.76 compared with wild type (Fig. 4B). To assess whether these effects were growth medium-418 specific, and to confirm the supercoiling dependence of these two HU-encoding genes, we grew 419 these strains in the rich 79 medium, and using RT-qPCR compared their expression under a range of 420 topA induction conditions. As before, we observed hupA upregulation and hupS downregulation 421 following TopA depletion if compared to the wild type level (Fig. 4C) Chromosome regulation stemming from gyrase inhibition with novobiocin had an immediate 474 impact on 1.5% of the genes in the S. coelicolor chromosome. Interestingly, among the genes affected by severe depletion or modest increase of TopA level, we identified genes whose 476 transcription was modified under both conditions. 477 Among the genes sensitive to any alteration of TopA level, 14 regulatory protein-encoding 481 genes were identified (Table 4), including the four regulators in the SHC region (sco4667 (sensor 482 kinase), sco4668 (regulatory protein), and putative transcriptional regulators (sco4671 and sco4673)). 483 Outside of this region was sco5803, which encodes the LexA repressor that controls the DNA damage 484 response (highly induced by topA-up-and downregulation). In E. coli, the LexA-regulon encompasses 485 at least 31 genes, including recA and lexA (the latter being negatively autoregulated), uvrA, ftsK, polB, 486 dinF, and dnaE2, alongside others involved in the DNA damage response (Fernandez De Henestrosa 487 et al., 2000). We found that the transcription of recA, recX, dnaE2, dinP or uvrA genes, which 488 presumably belong to the LexA-regulon in S. coelicolor, based on the predicted binding consensus 489 sequence (Novichkov et al., 2013), were similarly affected by changes in chromosome supercoiling, 490 indicating that the entire LexA regulon was impacted (Table 5 and 6 and Fig. S6). 491 The identification of putative LexA-dependent genes among the genes upregulated when 497 topA transcription was altered may suggest that these conditions trigger LexA-dependent DNA repair 498 pathway. However, the mechanism of LexA regulon induction by altered chromosome supercoiling 499 remains speculative. We assume that in Streptomyces, as in other bacteria, LexA activity is controlled 500 Streptomyces, induction of the DNA repair pathway was associated with the inhibition of cell division, 511 which could explain the inhibition of sporulation seen for the S. coelicolor TopA-depleted strain 512 (Ogino et al., 2008). 513 Since we did not identify LexA-regulon among novobiocin-sensitive genes, we speculate that 514 the DNA repair response may be the effect of long-term exposure to topological imbalance. Thus, 515 topological imbalance if cannot be compensated by response pathways could serve as a marker of 516 DNA damage.

Many transcriptional regulators are sensitive to specific supercoiling conditions 518
Although topA downregulation and topA-upregulation induced a common transcriptional 519 response, a significant fraction of the affected genes responded specifically to particular supercoiling 520 conditions (Fig. 2C). We identified 12 SSGs-encoding regulatory proteins that were affected 521 specifically by topA-upregulation (Table 7). These numbers are similar to those induced by 522 novobiocin treatment, but surprisingly, the sets of genes identified in both experiments did not 523 overlap. Amongst the many uncharacterized genes that were sensitive to topA-upregulation, 524 developmental regulators such as sigF (upregulated) and nsdA (downregulated) were identified; 525 however, these changes in expression were not associated with any obvious phenotype for the topA 526 up-regulated strain. 527 The increased chromosome supercoiling resulting from TopA depletion specifically influenced 530 a substantial number of genes encoding regulatory proteins. Among 35 of the TopA depletion-531 sensitive genes, we identified 18 putative transcriptional regulators, including seven genes encoding 532 TCSs (kinases and/or their putative phosphorylation targets), six genes encoding putative 533 acetyltransferases (GNATs family) and four genes encoding subunits of regulatory Clp proteases 534 (Table 8), alongside the sporulation-specific sigma factor encoding whiG. 535 Transcription of the gene encoding the sigma factor WhiG was one of the most significantly 538 upregulated regulatory genes under TopA depletion (over 21-fold induction; Table 8 and Fig. 5A). RT-539 qPCR confirmed that whiG transcription was stimulated by increased DNA supercoiling and 540 decreased to wild type levels upon induction of normal topA transcription (Fig. 5B). WhiG is typically 541 expressed at low levels during vegetative growth and is involved in the regulatory cascade that 542 governs Streptomyces sporulation (Elliot et al., 2001;Kelemen et al., 1996). WhiG directs the 543 expression of the whiI and whiH genes, but does not affect the transcription of other whi-family 544 regulators, such as whiA and whiB (Chater et al., 1989;Mendez and Chater, 1987;Ryding et al., 545 1998), we analyzed the transcriptional response of these genes to increased DNA supercoiling. As 546 expected, the transcription of both whiI and whiH was elevated following TopA depletion (although 547 they were not identified in the initial screening due to below-threshold q values, remarkably p <0.001 548 was still statistically relevant), suggesting the induction of a WhiG-dependent regulatory cascade by 549 changes in chromosome supercoiling (Fig. 5A). In S. coelicolor, in contrast to S. venezuelae, whiG 550 transcription was independent of any of six known whi genes (whiA, B, G, H, I and J) (Kelemen et al., 551 1996). However, whiG was shown to belong to the BldD-regulon, which also encompasses bldN, 552 bldM, and whiB (den Hengst et al., 2010;Elliot et al., 2001). In fact, in S. venezuelae, the whiG gene 553 was shown to be directly repressed by BldD, the master regulator that binds to DNA upon association 554 with c-di-GMP (Tschowri et al., 2014). This prompted us to test whether increased DNA supercoiling 555 influenced the transcription of other BldD-dependent genes. Indeed, upon TopA depletion, we 556 observed high upregulation of three BldD targets: bldM, bldN and whiG (Fig. 5A). The mechanism of 557 supercoiling-dependent upregulation of the BldD-regulon may, similar to that proposed for LexA, 558 result from decreased BldD DNA-binding affinity during higher DNA supercoiling. Thus, alleviating BldD binding during times of increased DNA supercoiling could lead to increased target gene 560 transcription. 561 Remarkably, even though elevated supercoiling induced key sporulation genes including 562 whiG, their induction did not lead to sporulation; in fact the TopA-depleted strain fails to form spores 563 (Szafran et al., 2013). This may indicate either a lack of additional regulators needed for 564 differentiation, or the activity of other signaling pathways that prevent sporulation (e.g. the DNA 565 damage response, as described for RecA/LexA above). The observed induction of sporulation 566 cascades does, however, suggest that DNA topology may function as a global regulator that triggers 567 sporulation cascades either in response to environmental stress or physiological conditions that 568 induce increased DNA supercoiling. . We showed here that in S. coelicolor supercoiling-sensitive genes are organized in discrete 574 clusters, a feature that seems to be a conserved strategy for topological regulation among many 575 bacteria. Supercoiling imbalance triggers the activation of genes involved in stress responses, 576 including DNA repair pathway, transmembrane transporters and chaperonins, as well as genes 577 involved in the production of secondary metabolites. Both increased and decreased DNA supercoiling 578 appear to have been detected as signals of DNA damage, inducing DNA repair genes and oxidative 579 stress response genes. The long-term response to supercoiling imbalance is based on the activation 580 of the set of primary and downstream regulatory proteins. Sporulation is triggered under stress 581 conditions in Streptomyces, and accordingly, we found that several differentiation-regulating genes 582 are affected by DNA supercoiling. In general, topology-governed regulons are expected to rely on the 583 supercoiling-dependent binding of various regulatory proteins to DNA.