The bacterial RNA polymerase-associated CarD protein couples promoter activity to DNA supercoiling

Domain: neuroscience
Published: 2026-02-03
Editorial outcome: major_revision

Abstract
RNA polymerase (RNAP) must unwind duplex DNA prior to transcription initiation. In bacteria, unwinding starts at a DNA motif called the promoter -10 element. Specifically, non-template strand bases interact with RNAP to trigger the process. A protein called CarD can support -10 element opening in many microbes. Whilst this most often activates transcription, CarD may repress if the DNA open complex is too stable. For Rhodobacter sphaeroides, a purple photoheterotrophic alpha-proteobacterium, CarD is particularly important as many -10 elements have a key DNA sequence defect. Here, we use genomic tools to map transcription initiation, binding of RNAP, CarD interactions, and DNA topology, globally in R. sphaeroides. We show an association between CarD-controlled transcription, and negative DNA supercoiling, which alters global gene expression if perturbed. Using biochemical tools, we show that promoter co-regulation by CarD and supercoiling results from the -10 element defect inherent to R. sphaeroides promoters. If this flaw is corrected, regulation by CarD and supercoiling is disrupted. As supercoiling dissipates during stress, we suggest CarD couples housekeeping transcription to the environment via DNA topology.

Introduction
Throughout life, transcription is catalysed by a highly conserved enzyme called RNA polymerase (RNAP). In bacteria, the core enzyme has 5 subunits: an α dimer, β, β‘ and ω1. Whilst this core is catalytically active, accurate recognition of promoter DNA sequences requires a dissociable σ factor. Frequently, bacteria encode multiple σ factors, with different promoter specificities. Most transcription is directed by the primary housekeeping σ factor, designated σ70 in Escherichia coli, which recognises two hexameric DNA motifs: the -35 and -10 elements. These sequences, named according to their position upstream of the transcription start site (TSS, +1) have a consensus sequence of 5’-TTGACA-3’ and 5’-TATAAT-3’, respectively. Proteins in the σ70 family share 4 structural domains: σ1, σ2, σ3, and σ42. Domain σ4 recognises the -35 element, whilst σ2 contacts the -10 sequence3–5. Upon binding the -10 motif, σ2 facilitates DNA melting and an open complex is formed. Specifically, bases -11A and -7T flip out of the DNA base stack and occupy pockets within σ24,6,7. Hence, positions -11A and -7T are highly conserved. The general principles of promoter recognition and DNA unwinding are common to many bacteria.

Rhodobacter sphaeroides is a purple photoheterotrophic alpha-proteobacterium and has a housekeeping σ factor called σ938. Surprisingly, over 50% of R. sphaeroides -10 elements have a sequence defect and lack the -7T base9. Despite the implications for DNA opening, such promoters drive high-level expression of essential genes, including rRNA operons. This is permitted by CarD, a transcription factor discovered in Mycobacterium tuberculosis10. Found in diverse bacterial phyla, CarD stabilises DNA opening and so compensates for the missing -7T base11–13. To do this, CarD binds the β subunit and uses a conserved tryptophan as a wedge to help separate the DNA strands12–14. Whilst this typically activates transcription, over-stabilisation of DNA opening can be inhibitory11. For example, CarD represses its own transcription in this way15.

Separation of DNA strands can also be influenced by nucleic acid topology16. For instance, over- or under-twisting DNA generates positive or negative supercoils respectively17. Strand unwinding is easier in the latter case. Inside cells, biological processes can generate DNA supercoils. Thus, during transcription, RNAP creates negative and positive supercoils, behind and ahead of the transcription elongation complex respectively18,19. This is primarily due to a phenomenon known as the twin supercoiling domain model19. Briefly, the crowded intracellular environment prevents rotation of the bulky RNAP molecule around the double helix during enzyme translocation. To compensate, the DNA twists and supercoils arise. Globally, correct DNA organisation is maintained by topoisomerases. For example, DNA gyrase introduces negative DNA supercoils at sites across the chromosome. Even so, global supercoiling levels can change dramatically in response to environmental conditions20–24.

In this work, we have mapped global patterns of supercoiling and transcription in R. sphaeroides. We identify exact sites of transcription initiation, the presence of σ93, and the binding of CarD. The data reveal frequent synergy between CarD-dependent gene control and DNA supercoiling. For example, if DNA gyrase is inhibited, CarD is released from many promoters, rendering them less active. This can be recapitulated in vitro, with purified CarD and RNAP. In this way, we show that co-dependence of promoters, on CarD and negative DNA supercoiling, results from the -10 element defects (i.e., the absent -7T base) outlined above. Hence, correcting the motif circumvents the simultaneous need for both CarD and under-twisted DNA. Similarly, our data show promoters, co-repressed by CarD and negative supercoiling, also have defective -10 elements. If this flaw is remedied and the DNA relaxed, CarD can revert to its more frequent role of transcription activation. We show that genes activated by CarD and negative supercoiling are frequently linked to housekeeping functions, including protein synthesis. We suggest CarD links the expression of these genes to environmental conditions, via negative supercoiling, which dissipates during slow growth.

Results
Global analysis of transcription initiation in Rhodobacter sphaeroides
Global patterns of transcription in R. sphaeroides are poorly defined, and current genome-scale approaches have hardly been used. To address this, we mapped global CarD and σ93 binding using ChIP-seq. Simultaneously, we measured total transcription with RNA-seq and identified transcription start sites with cappable-seq. The data, collected during aerobic growth, are integrated as genome-wide maps in Fig. 1a. The results are further summarised in Supplementary Data 1 and 2. We identified 8,150 TSSs, including 1248 of the 2252 sites previously identified by Myers et al. (Supplementary Fig. 1)25. The greater number of TSSs identified here likely represents the improved sensitivity of cappable-seq compared to older methods26. Overall, the distribution of TSSs in R. sphaeroides resembles other proteobacteria, including E. coli and Salmonella Typhimurium27–29. Thus, 61% of TSSs are located within coding DNA (Fig. 1b). Of intergenic TSSs, 89% are correctly orientated to drive mRNA transcription. The remaining intergenic TSSs were incorrectly orientated or between convergent genes (Fig. 1c, left). We note that recent work has suggested transcription may sometimes initiate independently of a canonical promoter in vitro30,31. However, available evidence indicates this is rare in vivo, as multiple processes remove the post-termination RNAP complex from the DNA31,32.

Bidirectional promoters occur frequently in Rhodobacter sphaeroides
A subset of TSSs were closely spaced and on opposite DNA strands (supplementary data 3). These correspond to bidirectional promoters, with inherent -10 element symmetry, from which transcription can proceed in either possible direction. Such promoters are found throughout prokaryotes, but have an unusual configuration in R. sphaeroides27. Briefly, for bidirectional promoters in most bacteria, -10 element positions -11A and -7T co-locate to the same base pair, on opposite DNA strands. As such, TSSs are separated by 18bp. In R. sphaeroides, such divergent TSSs are most often 23bp apart. In this configuration, promoter positions -12T and -11A reciprocally co-locate (Supplementary Fig. 2a, b). The likely reason is the frequent absence of base -7T in R. sphaeroides. Overall, 8.8% of R. sphaeroides promoters are bidirectional. Similar to other bacteria, bidirectional promoters are much more likely to be intergenic and between divergent genes than their directional counterparts (Fig. 1c, right)27,28.

Promoters associated with CarD have sub-optimal -10 elements
To understand σ factor and CarD association with each TSS, we examined our ChIP-seq data. In total, we identified 1167 and 588 peaks for σ93 and CarD, respectively. Of the 8150 TSS found by cappable-seq, 39% were bound by σ93, and 48% of these sites were also associated with CarD (Fig. 1d, e). Only 2% of CarD-bound TSSs were not also recognised by σ93. It is notable that intergenic TSSs are much more likely to be associated with σ93 or CarD (Fig. 1b).

To better understand the properties of promoters bound by CarD, we made DNA sequence logos (Fig. 1f)33. Note that, prior to alignment, promoter regions were split into discrete sections. This avoids sequences being out of register, due to variation in spacer and discriminator length, during alignment. In subsequent logos, the height of each letter indicates sequence preference and information content. Compared to all other promoters, those associated with CarD have defective -10 elements due to poor conservation of base -7T. Conversely, the -12T and -11A bases, also critical for -10 element function, were conserved regardless of CarD binding. Given the role of base -7T in DNA unwinding, it seems likely that promoters associated with CarD have inherent defects for strand separation. This is consistent with prior reports that CarD controls transcription by stabilising the open complex12–14.

Promoters associated with CarD exhibit increased negative DNA supercoiling
We reasoned that negative DNA supercoiling might impact CarD-dependent control of transcription. To understand this, we used psora-seq34,35. Briefly, psoralen is a molecule that preferentially intercalates with underwound DNA. Hence, in combination with next-generation sequencing, psoralen was used to map global patterns of DNA supercoiling (Supplementary Data 4). Figure 2a shows maps of psoralen binding for each R. sphaeroides chromosome. The maps are presented at both high resolution (track 2, signal averaged across 5kb bins) and low resolution (track 3, 100kb bins). At low resolution, chromosome 1 partitions into origin and terminal halves, with the latter being more negatively supercoiled. The smaller chromosome 2 has no large-scale supercoiled domains. Rather, the entirety of chromosome 2 is more negatively supercoiled than chromosome 1. For both chromosomes, high-resolution maps reveal local patterns of supercoiling. In part, these can be explained by transcription. For example, R. sphaeroides has 3 rRNA operons; rrnA is located near the origin of chromosome 1, whilst rrnB and rrnC occupy a similar position on chromosome 2. The highest psoralen binding values locate to these highly transcribed regions (labelled in Fig. 2a). Negative supercoiling was also associated with transcribed DNA more generally (compare psora-seq and RNA-seq data in Fig. 2a). To understand the relationship between CarD binding and supercoiling, we compared the psora-seq and ChIP-seq profiles (Fig. 2b). CarD-bound promoters are significantly enriched for psoralen binding and so negative supercoiling, compared to unbound and random genomic loci. Some examples are shown in Fig. 2c. In each case, the CarD-associated TSS is within a negatively supercoiled DNA region. To confirm this association was specific to CarD, and not a property of all promoters, we also determined psoralen binding values for σ93 peaks, with or without an overlapping signal for CarD. Promoters bound by both CarD and σ93 were significantly more negatively supercoiled than those bound by σ93 alone (Supplementary Fig. 3).

Decreased DNA supercoiling impacts CarD occupancy at transcription initiation sites
Our data show that CarD-associated promoters are enriched for negative DNA supercoiling (Fig. 2b). To investigate the relevance of this, we repeated our ChIP-seq, RNA-seq, and psora-seq assays, with subinhibitory concentrations of novobiocin, a competitive inhibitor of DNA gyrase. We expected novobiocin to reduce negative supercoiling and so the psora-seq signal20,36. We also expected changes in DNA supercoiling to impact patterns of CarD binding and transcription. A summary of the psora-seq data, for different groups of chromosomal loci, is shown in Fig. 3a. As expected, treatment with novobiocin caused significant DNA relaxation across all subsets of chromosomal regions: the average psora-seq signal for CarD-bound, CarD-unbound, and random loci significantly decreased by 26%, 50% and 38%, respectively (Fig. 3a). To understand the impact on CarD binding, we compared ChIP-seq signals for each CarD-bound promoter, with and without novobiocin. The results are shown as a volcano plot in Fig. 3b, where each data point represents a single ChIP-seq peak. There is a clear trend for novobiocin to reduce CarD binding. Most likely, this is because promoter open complexes are destabilised, leading to the release of CarD and RNAP.

Reduced DNA supercoiling predominantly inhibits transcription at CarD-bound promoters
Since both CarD and negative supercoiling enhance strand separation, we expected novobiocin to alter σ93 binding at, and transcription from, many CarD-bound promoters. For example, in situations where RNAP cannot open the duplex efficiently, and requires assistance from both CarD and negative supercoiling, novobiocin should inhibit transcription. Conversely, if CarD and negative supercoiling over-stabilise DNA unwinding, transcription could increase. To test these predictions, we quantified novobiocin induced changes in σ93 binding (Fig. 3c) and transcription (Fig. 3d) at CarD targeted genes. Significant reductions in σ93 binding, and transcription, were detected at 138 and 260 CarD gene targets, respectively. Less frequently, σ93 binding (24 peaks) and transcription (108 genes) increased. Hence, negative supercoiling more often activates, rather than represses, transcription from CarD-bound promoters. Two example regions are shown in Fig. 3e. In both cases, novobiocin induces DNA relaxation, loss of both CarD and σ93 binding, and lower gene expression.

To understand the relationship between altered CarD binding and transcription, the data in Fig. 3b were reannotated. This was done according to gene expression changes adjacent to each CarD peak. The reannotated data are shown in Supplementary Fig. 4. Data point position and colour indicate changes to CarD binding, and adjacent gene expression, respectively. There is a clear tendency for reduced CarD binding and transcription inhibition to coincide (Supplementary Fig. 4a, blue data points). Conversely, increased transcription is associated with either no change, or a modest increase, in CarD binding (red data points). We conclude that there are two major classes of CarD-associated promoter. The first and largest corresponds to the 260 genes where transcription decreases upon DNA relaxation. We refer to these as CarD-bound relaxation-repressed promoters. The second class controls the 108 genes where transcription increases upon DNA relaxation. We refer to these as CarD-bound relaxation-activated promoters.

Different classes of CarD-bound promoter have similar sequence properties
To understand the properties of the two promoter classes, we generated DNA sequence logos (Fig. 3f)33. Visual comparison revealed no major difference between the motifs. Thus, we used a simple position weight matrix (Supplementary Fig. 4b) to score core promoter elements and determine the significance of any subtle variation. We also quantified GC-content of the discriminator region, located between the -10 element and TSS, that also influences DNA unwinding37. Overall, the two promoter classes were remarkably similar (Fig. 3g). Of the small differences detected, statistical significance was only apparent for the -10 element score. We conclude that relaxation-repressed and relaxation-activated promoters, bound by CarD, cannot be easily identified by gross sequence properties. Rather, CarD-bound promoters, as a whole, are notable for their shared -10 element defect; absence of base -7T (Fig. 1f). Hence, we speculate that DNA supercoiling, rather than DNA sequence alone, could also be a key consideration. Indeed, we note that relaxation-repressed promoters bound by CarD tend to be in regions of lower negative supercoiling compared to their relaxation-activated counterparts (Supplementary Fig. 4c).

Activation of transcription by CarD in vitro requires negative DNA supercoiling
To more precisely understand the role of DNA supercoiling, we first focused on the most common class of CarD-bound promoter; those repressed by DNA relaxation. The rrnBP1 promoter is an excellent example (Supplementary data 5) having previously been identified as CarD activated9,10,38. To understand the role of supercoiling, we used in vitro transcription assays. The template for transcription was plasmid pRLG770, having the R. sphaeroides rrnBP1 promoter upstream of the E. coli rrnB T1 and T2 terminators9. Before use, plasmids were treated with topoisomerase I, to remove all DNA supercoiling. These DNA molecules have low mobility during electrophoresis (Fig. 4a, lane 1). We then repeated topoisomerase treatment in the presence of ethidium bromide. In this scenario, negative supercoils are introduced in a manner dependent on the concentration of the DNA intercalator (Supplementary Fig. 5a)39. Hence, by using different amounts of ethidium bromide, we generated a set of plasmids with increasing amounts of negative supercoiling (Fig. 4a, lanes 2-6). For comparison, raw negatively supercoiled plasmid DNA, isolated directly from cells, is shown in lane 7. Note that standard gel electrophoresis cannot separate plasmid topoisomers with high, but different, levels of supercoiling (lanes 4-6). However, the expected differences are revealed in the presence of chloroquine (Supplementary Fig. 5b). These differences are indicated schematically in Fig. 4a. Results of in vitro transcription assays, with the different templates, are shown in Fig. 4b. The RNAI transcript, generated from the plasmid replication origin, serves as an internal control. In the absence of CarD, both rrnBP1 and RNAI transcription increased in response to greater negative supercoiling (Fig. 4b, lanes 1–7). However, this increase was modest for rrnBP1 (compare rrnB and RNAI transcript levels in lanes 1–7). Addition of CarD had no impact on RNAI transcription but specifically increased rrnB promoter activity (compare lanes 1–7 with 8–14). Furthermore, CarD was only able to act on DNA that was negatively supercoiled to a sufficient level. Thus, CarD had little or no impact on transcription from the relaxed (lane 8) or modestly supercoiled (lanes 9 and 10) DNA templates. Conversely, CarD activated rrnB transcription by >8-fold when negative supercoiling was sufficiently high (lanes 11-13). Critically, the level of negative supercoiling in raw plasmid DNA, isolated directly from cells, was sufficient for near maximal activation by CarD (lane 14). A quantification of rrnB transcript levels, in the presence and absence of CarD, is shown in Fig. 4c. In related experiments, similar results were obtained with the rpsL promoter, PrpsL, that drives expression of a small ribosomal protein subunit (Supplementary Fig. 6, lanes 9–16). Thus, CarD could activate PrpsL in the presence (lanes 13–16) but not absence (lanes 9–12) of negative supercoiling. Consistent with our genome-scale data, we conclude that activation by CarD frequently requires sufficient negative supercoiling. The most likely explanation is that this DNA topology favours DNA unwinding and allows CarD to overcome the energetic barriers imposed when base -7T is absent.

An improved rrnBP1 sequence responds differently to CarD and DNA supercoiling
We reasoned that rrnBP1 co-dependence on CarD and negative supercoiling might be removed by introducing the preferred -7T base. For example, CarD may activate such an optimised promoter when the DNA is relaxed. For comparison, we also examined the impact of a deleterious -12A mutation. Usually thymine, this base is transiently unpaired during DNA unwinding. Contact with the template strand is then restored prior to a stable open complex being established6,40. The various rrnBP1 derivatives tested are shown in the top half of Fig. 5a, and their differently supercoiled derivatives are in Supplementary Fig. 5c. Results of in vitro transcription assays are in Fig. 5b and data from three replicate experiments are quantified in Fig. 5c, which shows fold changes in transcription with and without CarD. As expected, wild-type rrnBP1 was only active in the presence of CarD and negative supercoiling (Fig. 5b, lanes 1–4). The -12A mutation rendered the promoter inactive in all conditions (lanes 5–8). Strikingly, the -7T mutation allowed full activation by CarD with a relaxed DNA template (compare lanes 4 and 10, also see Fig. 5c). Overall promoter activity increased substantially in the presence of negative supercoiling (lane 11) and CarD was unable to stimulate substantial further transcription (lane 12, and Fig. 5c). The impact of the -7T mutation was abolished when combined with the -12A base change (lanes 13–16, and Fig. 5c). We conclude that the requirement of CarD for negative supercoiling can be overcome by the -7T mutation. Similarly, CarD can override the absence of -7T if DNA has the correct topology. However, promoters lacking -12T cannot be activated by CarD.

Repression of transcription by CarD in vitro requires negative DNA supercoiling
We next turned our attention to the less common class of CarD-bound promoter; those activated by DNA relaxation. Our genome-scale data predict that CarD and negative DNA supercoiling should synergistically reduce transcription at such promoters (Fig. 3 and Supplementary Fig. 4). To test this, we again used in vitro transcription assays. The template for transcription was plasmid pRLG770, having the R. sphaeroides carD promoter, PcarD, upstream of the E. coli rrnB T1 and T2 terminators9. Note that PcarD remains bound by CarD, upon novobiocin treatment, and transcription increases (Supplementary Data 5). Conversely, CarD was previously shown to repress PcarD in the context of a negatively supercoiled DNA template in vitro15. Taken together, and consistent with our earlier speculation, these data suggest CarD may switch roles, from repressor to activator of PcarD, upon DNA relaxation. The results of in vitro transcription assays are shown in Fig. 5b lanes 17-20. Consistent with our prediction and our genome-scale data, PcarD was activated by CarD when the DNA template was first relaxed using topoisomerase I (lanes 17 and 18). Conversely, CarD repressed transcription, from the same promoter, in the context of a negatively supercoiled template (lanes 19 and 20). The likely explanation is that negative supercoiling and CarD, together over-stabilise PcarD opening whilst, in the absence of supercoiling, sub-optimal opening creates CarD dependence. If correct, promoter mutations, that enhance DNA opening, may negate PcarD activation in the context of a relaxed DNA template.

An improved PcarD sequence responds differently to CarD and DNA supercoiling
To test our prediction, we repeated our in vitro transcription analysis using PcarD having the -7T mutation. For comparison, we again tested the -12A mutation. The PcarD derivatives tested are shown in Fig. 5a (lower half). The results are shown in Fig. 5b (lanes 21–32), and quantification is in Fig. 5d. The -12A mutation abolished promoter activity in all conditions (Fig. 5b, lanes 21-24). The -7T mutation, expected to stabilise DNA opening, rendered PcarD moderately active in the absence of CarD and negative DNA supercoiling (lane 25). As predicted, this transcription was not further activated by CarD (lane 26 and Fig. 5d). In the presence of negative supercoiling, overall promoter activity increased substantially (lane 27) but remained subject to CarD-mediated repression (lane 28 and Fig. 5d). Thus, PcarD co-repression, by CarD and negative supercoiling, is not negated by the -7T mutation in the same way as rrnBP1 activation is disrupted. We speculate PcarD lacks base -7T to permit nuanced transcriptional changes in response to diverse DNA topologies.

Chimeric promoters derived from rrnBP1 and PcarD are CarD activated
The ability of CarD and negative supercoiling to co-activate transcription at some promoters, whilst co-repressing others, is curious. There is no marked sequence difference between the promoter classes (Fig. 3f, e). Furthermore, our experiments with rrnBP1 and PcarD indicate DNA topology, rather than sequence, may determine the regulatory impact of CarD. To test this further, we made a series of chimeric promoters. Hence, rrnBP1 sequence elements were replaced with equivalent PcarD motifs. We also examined the impact of the -7T mutation and DNA topology in this context. The chimeric promoter sequences are shown in Fig. 6a and results of in vitro transcription assays, using these DNA templates, are in Fig. 6b. Quantification of data from replicate experiments, showing fold changes in transcription, with or without CarD, are in Fig. 6c. Consistent with prior experiments, activation of rrnBP1 required both CarD and negative supercoiling (lanes 1–4) and this requirement was removed by the -7T mutation (lanes 5–8). Introduction of the PcarD 5’-TGn-3’ motif, which facilitates RNAP binding but not DNA opening, rendered rrnBP1 more active. However, activation by CarD still required DNA supercoiling (lanes 9-12 and Fig. 6c). The PcarD discriminator, which is more AT-rich than the equivalent rrnBP1 sequence, should facilitate DNA opening37. Consistent with this, CarD was able to significantly activate the promoter in the absence of negative supercoiling (lanes 13–14 and Fig. 6c). However, both supercoiling and CarD were required for full activation (lanes 15-16). Together, the PcarD extended -10 hexamer, and more AT-rich discriminator, allowed substantial rrnBP1 activation by CarD in the absence of negative supercoiling (lanes 17-18 and Fig. 6c). Indeed, CarD could only further activate transcription modestly when negative supercoils were present (lanes 19-20 and Fig. 6c). When further combined with the -7T mutation, we detected high basal rrnBP1 activity (lane 21). This was further activated by CarD (lane 22) but only in the absence of negative supercoiling (lanes 22–24). It is notable that no changes render rrnBP1 subject to repression by CarD. We conclude that DNA topology may impact the regulatory role of CarD more than promoter sequence alone.

CarD-bound promoters control key housekeeping functions
Genes encoding factors for information storage and protein synthesis are often regulated in response to growth rate41,42. Further, in starved cells, levels of negative DNA supercoiling are lower22,43,44. Hence, CarD might link the expression of such genes to growth rate via supercoiling changes. To test this, we quantified gene functions linked to reduced CarD binding at promoters upon DNA relaxation (blue points in Fig. 3b). As a control, we also identified gene functions associated with σ93-bound promoters lacking CarD. The gene functions are categorised in Fig. 7. Genes expressed from promoters, where CarD binding is reduced by DNA relaxation, are enriched for functions related to information storage and processing. Specifically, functions related to the synthesis of ribosomes and tRNAs (categories a and d, Fig. 7) are overrepresented. Note that, whilst other gene categories are linked to CarD-bound promoters and similarly impacted by DNA supercoiling, their functional categories are not specifically enriched (compare Fig. 7a, b).

Physiologically relevant changes in DNA supercoiling modulate gene control by CarD
To test our model further, we first examined CarD binding to, and transcription from, 3 relaxation-repressed promoters, identified in our genomic assays, in living cells. Two of the associated genes belong to the enriched functional categories: RSP_4322 and rmpI, which are implicated in tRNA biogenesis and ribosome function, respectively. Gene RSP_1952 encodes a cold shock protein, is transcribed from a CarD-bound promoter, and is also repressed upon DNA relaxation. Results comparing signals from ChIP-qPCR (to measure CarD binding) and RT-qPCR (to infer mRNA levels) assays, for growing and starved cells, are shown in Supplementary Fig. 7a, b. As expected, levels of CarD binding and transcription are dramatically reduced in starved cells when supercoiling decreases. However, these data must be interpreted with caution; levels of CarD and σ93 are both lower in starved cells, and this must also contribute (Supplementary Fig. 7c, d). Hence, to further validate our model, we took an additional approach. Briefly, R. sphaeroides is capable of aerobic or photosynthetic growth45. The latter case, cell division is much slower, and this likely impacts supercoiling. To test this, we used plasmid pBBRBB to transform R. sphaeroides. Transformants were cultured, either aerobically or photosynthetically, before plasmid DNA was extracted. Levels of supercoiling were then determined using chloroquine gel electrophoresis. As expected, greater negative supercoiling was apparent during faster aerobic growth (Supplementary Fig. 7e). To understand if these physiologically relevant changes in supercoiling could impact transcription activated by CarD, we used the extracted plasmids as templates for in vitro transcription. Our logic was that pBBRBB contains numerous housekeeping promoters, which are frequently bound by CarD and repressed by DNA relaxation. Furthermore, this avoids the confounding effects of changing CarD and σ93 levels in vivo. The results are shown in Supplementary Fig. 7f. Whilst CarD was able to stimulate transcription from all DNA templates, the impact of CarD was greater when supercoiling levels were higher.

Methods
Strain and plasmids
Strains and plasmids used for this study are provided in Supplementary Data 6. Unless stated otherwise, R. sphaeroides strain 2.4.1 was grown in M22+ media at 30°C62. For starter cultures, a single colony was used to inoculate 10 ml of M22+ media in a 30 ml universal tube. After incubation for 2–3 days, at 30°C, starter cultures were used to inoculate 100 ml of M22+ media, in a 2-litre baffled conical flask, at an initial OD of 0.05. These cultures were incubated at 30°C and aerated with rapid shaking at 250rpm. Anaerobic photosynthetic growth was achieved by completely filling a 50 ml vessel with freshly inoculated culture. These were incubated with stirring at room temperature, next to a lamp fitted with a 20W 2700K bulb. When required, novobiocin was added at a final concentration of 2 μg ml-1, followed by 20 min of further incubation.

All molecular cloning and DNA manipulations were done with standard procedures. HiFi Gibson assembly (New England Biolabs) was used for cloning promoter fragments in pRLG770. Site directed mutagenesis was performed with the Q5 SDM kit (New England Biolabs). Oligonucleotides used are included in Supplementary Data 6.

Mapping of global transcription using cappable-seq and RNA-seq
Total RNA was isolated from exponentially growing R. sphaeroides as previously done for Bacillus subtilis29. Cells were lysed using an MP Biomedical FastRNA Pro Blue Kit. Genomic DNA was removed, and RNA isolated, using a Norgen Total RNA purification plus kit. Cappable-seq libraries were prepared by Vertis Biotechnologie AG26,27 and sequenced using an Illumina NextSeq 500 system (75 bp read length). Standard RNA-seq library preparation was done by Azenta Life Sciences, and 150bp paired-end sequencing done using an Illumina HiSeq. Biological duplicates were analysed in all cases. Raw sequencing data are accessible from ArrayExpress using accession code E-MTAB-15087. Cappable-seq data were processed as described previously to identify TSSs27,29. Briefly, FASTQ files were aligned to the R. sphaeroides genome (GenBank accension numbers CP000143.2 and CP000144.2) with Bowtie 2 (Galaxy Version 2.4.2) using default settings63. Coverage at each genomic position was calculated with the genomecov function of Bedtools (version 2.26)64. Chromosomal positions with a minimum read depth of 4, and at least three times higher coverage than the previous position, in both biological replicates, were called as TSSs. Read depth at each genome position was normalised to the total reads aligning to chromosome 1 and 2. The normalised read depth for each TSS for both replicates was averaged and used as a measure of activity. Using this value, all TSSs were categorised into deciles reflecting their activity. TSSs were assigned to the nearest gene less than 300bp downstream on the same strand.

For differential gene expression analysis of RNA-seq data, read counts for each replicate were calculated from Binary Alignment Map (BAM) files using featureCounts (version 2.6)65 and parsed with the exact function of EdgeR (Galaxy version 3.34.0)66. Bidirectional TSS were identified as in prior work27,67. Data were visualised with Integrative Genome Visualiser (IGV)68. Circular plots were generated using shinyCircos-V2.069.

Promoter sequence analysis
To generate promoter consensus sequences, optimal, and appropriately positioned, DNA elements were identified upstream of each TSS. The 5bp region upstream of the TSS was taken as the discriminator. For the -10 element, the closest match to the sequence 5’-TATAAT-3’, separated from the TSS by between 4 and 8 bp, was used. The 17bp upstream of the selected -10 element was taken as the spacer sequence. A -35 sequence, best matching the consensus 5’-TTGACA-3’, was then selected between 16 and 18 bp upstream of the designated -10 motif. Individual consensus motifs were generated, by aligning discrete promoter regions, to account for variation in element spacing. The exception was the generation of bidirectional promoter motifs. Sequence logos were generated with Weblogo370. The position weight matrix used to score different CarD-bound promoter classes is in Supplementary Fig. 4b.

Mapping of global protein-DNA interactions using ChIP-seq
Exponentially growing R. sphaeroides, with or without 2 μg ml-1 novobiocin, were crosslinked with 1% (v/v) formaldehyde for 20 minutes. The reaction was quenched with 0.5 M glycine, and cells were resuspended in lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 0.1% (v/v) sodium deoxycholate and 1 protease inhibitor cocktail tablet per 50ml) with 2 mg ml-1 lysozyme. After sonication, 20 μl of chromatin was kept as an input control. Approximately 150 μg of chromatin was immunoprecipitated by the addition of anti-CarD antibodies (a gift from Wilma Ross, University of Wisconsin-Madison) or anti-σ antibodies (Biolegend, clone 2G10). Protein-DNA complexes were immunoprecipitated with a 1:1 mix of Protein A/G Dynabeads (Thermo Fisher). Before eluting complexes, beads were washed with lysis buffer, high salt (500 mM NaCl) lysis buffer, wash buffer (20 mM Tris pH 8, 250 mM LiCl, 0.5% (v/v) NP-40, 0.5% (v/v) sodium deoxycholate and 1 mM EDTA) and TE. Elution, protein degradation (1 hour incubation with proteinase K at 42°C), and reverse crosslinking (4 hours at 65°C) was done in elution buffer (50 mM Tris pH 7.5, 10 mM EDTA and 1% (v/v) SDS). DNA was purified from the eluate using a Qiaquick PCR cleanup kit (Qiagen). Sequencing libraries were then prepared using the NEB next Ultra II kit (New England Biolabs). Library concentrations were determined by qPCR using a NEB next library quantification kit (New England Biolabs) and pooled before being sequenced using an Illumina NovaSeq instrument. Data is available using ArrayExpress accession code E-MTAB-15087. Reads were aligned, and coverage determined, as described above. Data were normalised so that the average read depth per base was the same for each experiment. Peaks with a minimum width of 200bp, and normalised read depth of 50, were called as binding sites and this also defined the peak boundaries. To determine changes in protein binding, read counts for each binding site were extracted from BAM files, using the featureCounts function of Rsubread (version 2.6.4)71. Read count changes were identified using EdgeR (version 3.33.7)66. Sites where CarD and σ93 binding sites overlapped, with each other or TSSs, were identified using the subsetByOverlaps function of the Genomic Ranges package (version 1.44)72. Peaks were defined as overlapping if any part of one peak fell within the boundaries of the other. Peaks for CarD or σ93 were associated to genes using the findOverlap function of the Genomic Ranges package with additional conditions72. Briefly, a CarD peak was assigned to a gene if the upstream peak boundary was upstream of the gene start codon and if the downstream boundary overlapped the coding sequence. For σ93 peaks, the same rules were used. Additionally, in cases where the entirety of a short gene (e.g., for a tRNA) fell within the peak boundaries, the peak was assigned to that gene. All σ93 peaks beginning inside a gene coding region were designated intragenic, if not already assigned to an overlapping gene.

Mapping of global DNA supercoiling using Psora-seq
Experiments were done as described by Visser et al. with the following changes35. Genomic DNA from exponentially growing cells was extracted with CTAB resuspended in 225 μl of water. Genomic DNA was sheared to a size of 400–500 bp using a Biorupter (Diaganode). 50 μl of sheared genomic DNA was kept as an input sample, and the remainder was immobilised on streptavidin T1 dynabeads (Thermo-Fisher). After washing and reverse crosslinking as per Visser et al, eluted DNA was precipitated and resuspended in 20 μl water. Library preparation and sequencing was done by Azenta Life Sciences. Sequencing was done using an Illumina NovaSeq in 150 bp paired-end configuration. Data is available using the ArrayExpress accession code E-MTAB-15087. FASTQ files were trimmed using trimmomatic to remove Illumina adaptors (TruSeq3 setting) and hard trimmed to keep the first 65 bp. Trimmed reads were aligned to the genome as described above. Read counts for 500bp genomic windows were determined using the multiBamSummary package of deepTools2 (Galaxy Version 3.5.4)73. Windowed read counts were normalised using a scaling factor (of 1) divided by the average window read count. Each window’s psoralen binding value was calculated by taking the Log2 of the normalised read count divided by the normalised input read count. Psoralen binding values derived from Psora-seq windows that overlapped CarD binding sites were determined using the subsetByOverlaps function of the Genomic Ranges package (version 1.44)72. Circular plots were generated using shinyCircos-V2.069.

Relaxation of plasmid DNA using topoisomerase I
Negatively supercoiled plasmid DNA (5 μg) was treated with 10 units of calf thymus topoisomerase I (Takara Biosciences) in 50 μl of the provided reaction buffer for 90 minutes at 37°C. If required, ethidium bromide was added at concentrations indicated. Reactions were stopped by the addition of phenol:chloroform:isoamyl alcohol (25:24:1), and DNA was recovered from the aqueous phase by ethanol precipitation. Plasmid isoforms (200 ng) were separated using 1% (v/v) agarose gels, containing ±5 μg ml-1 chloroquine. DNA bands were visualised by post-staining with ethidium bromide (~1 μg ml–1).

ChIP-qPCR
Chromatin was immunoprecipitated as above from 40 ml R. sphaeroides grown aerobically to the exponential or stationary phase in M22+ media. Quantitative PCR was performed using Luna Universal qPCR Master Mix (New England Biolabs) on an Agilent AriaMX system, with primer pairs shown in Supplementary Data 6. Each sample had 3 technical replicates and was one of 2 biological replicates. Enrichment was calculated as % input, with Ct values of input control being scaled to 100%.

RT-qPCR
Total RNA was purified from exponential and stationary phase R. sphaeroides cultures as above. Cell mass approximately equivalent to 10 ml at OD 0.5 was used. Total RNA was treated with DNase I (Zymogen) before clean-up with a Qiagen RNeasy kit. RT-qPCR was performed using the Luna Universal Probe One-Step RT-qPCR kit (New England Biolabs) on an Agilent AriaMX system. Primers are included in Supplementary Data 6. Fold enrichment was calculated using the 2-ΔCT method, normalised to the constitutively expressed PutR control.

Purification of R. sphaeroides RNA polymerase
R. sphaeroides σ93 RNAP was purified from strain 2.4.1 encoding a C-terminally His tagged β′ subunit (a gift from Wilma Ross and Rick Gourse, University of Wisconsin–Madison)9. Cultures were grown in LB medium and cells harvested in exponential phase (OD ~0.5). Pellets were resuspended in 20 ml of BugBuster (Millipore) protein extraction reagent diluted to the working concentration in Ni2+ buffer (20 mM Tris pH 7.9, 600 mM NaCl and 5% (v/v) glycerol). Resuspended cells were supplemented with 1 cOmplete EDTA-free protease inhibitor tablet (Roche), 4 mg of lysozyme, ~150 units of DNase I (Roche), and 35 units of RNase A (Qiagen) before room temperature incubation with gentle agitation for 1 hour. The suspension was sonicated for 2 min (30s on/off, 25 % amplitude) before clarification by centrifugation (45,000 xg for 20 min at 4°C) and filtration (0.2 μM PES filter). Lysate was loaded onto a His-trap HP column (Cytiva) pre-equilibrated with Ni2+ buffer. Unbound and weakly bound proteins were removed by washing with 8 column volumes of Ni2+ buffer and then Ni2+ buffer + 25 mM imidazole. Bound proteins were eluted with stepwise washes with 50, 100, 200 and 300 mM imidazole in Ni2+ buffer. RNAP was eluted in 200 mM and 300 mM imidazole fractions, which were pooled and concentrated with a 50 KDa Vivaspin concentrator (Merck). Concentrated protein was dialysed overnight into 2x storage buffer (40 mM Tris pH 7.9, 400 mM KCl, 2 mM DTT, 0.2 mM EDTA) before further concentration. An equal volume of 100% glycerol was added before storing the protein at -20°C.

Purification of R. sphaeroides CarD protein
The pETSUMO plasmid for expression of an N terminally His -SUMO tagged CarD was gifted by Dr Wilma Ross and Prof Rick Gourse (University of Wisconsin–Madison)9. E. coli T7 Express cells (New England Biolabs) carrying the plasmid were grown in LB at 37°C until the exponential phase, at which point cells were shifted to 18°C, induced with 1 mM IPTG and incubated overnight. Cell lysis and Ni2+ chromatography was performed as above. 200 mM and 300 mM imidazole fractions containing CarD were pooled and concentrated. To remove His -SUMO tag, 40 μg Ulp1 protease was added to ~12 mg CarD and dialysed overnight at 4°C into Ulp1 buffer (20 mM Tris pH 7.9, 150 mM NaCl and 1 mM β-mercaptoethanol). Precipitate was removed through filtration (0.45 μM PES filter) and protein was passed over His-trap HP column as before, except tag-free CarD was present in flow through. CarD was concentrated with a Vivaspin concentrator before the addition of an equal volume of 100% glycerol for storage at -20°C.

Western blotting
R. sphaeroides cells, equivalent to a suspension having an OD of 0.5, were pelleted and resuspended in 200 μl lysis buffer (1x BugBuster (Merck), 20 mM Tris HCl pH 7.9, 600 mM NaCl and 5% (v/v) glycerol) and incubated with 1 μl of Lysonase (Merck) for 20 minutes at room temperature before 10 cycles of sonication on a Bioruptor (Diagenode). For each sample, 5 μg of lysate was resolved by SDS-PAGE before transfer to nitrocellulose. After blocking, anti-CarD (a gift from Wilma Ross, University of Wisconsin-Madison) or anti-σ factor primary antibodies were used at 1:5000 and 1:1000 dilutions, respectively. Secondary antibodies, anti-rabbit HRP (Thermo-fisher Scientific. cat no 31464) for CarD and anti-mouse HRP (Sigma-Aldrich. cat no 12-349) for σ factor, were used at 1:10000 dilutions. Western blots were visualised with the SuperSignal™ West Pico PLUS ECL substrate kit (Thermofisher).

In vitro transcription assays
In vitro transcription reactions contained 0.01 μM DNA template, 0.05 μM σ93 RNAP and, where applicable, 2 μM of CarD in transcription buffer (20 mM Tris pH 7.9, 170 mM NaCl, 10 mM MgCl2, 1 mM DTT and 0.1 mg ml-1 BSA). Reactions were initiated by the addition of an NTP mix (200 μM ATP/GTP/CTP, 10 μM UTP and 2 μCi [α-32P] UTP) and incubated for 20 minutes at 30°C. Reactions were stopped by the addition of 30 μl acidic phenol (pH 4.3). After centrifugation, the aqueous phase was added to an equal volume of formamide containing stop solution, before separating transcripts using an 8% (w/v) denaturing polyacrylamide gel. Gels were exposed overnight using Bio-Rad phosphor screens, and signals visualised with a Bio-Rad Personal Molecular Imager, using Quantity One software (version 4.6.9). Plasmid template DNA was purified from NEB 5-α E. coli cells (New England Biolabs) using ZymoPURE II Plasmid Midiprep Kit (Zymogen). For quantification, band densities were measured using Bio-Rad Image Lab (version 6.1). The value for reactions with CarD was divided by the same value for the corresponding reaction without CarD to give the fold activation by CarD.

Analysis of gene ontology
Predicted COG terms for all R. sphaeroides encoded proteins were obtained from EggNOG-mapper, which assigns terms based on orthology74. Assigned COG terms were manually curated, with unannotated proteins designated as S for function unknown. Non-coding RNA (tRNA, ncRNA, SRP RNA and rRNA) were added manually as separate terms.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Forest et al. employed a combination of genome-wide techniques (RNA-seq, ChIP-seq, Cappable-seq, and psoralen-seq), along with in vitro transcription assays, to investigate the interplay between CarD and DNA supercoiling, and how they together regulate promoter activity in Rhodobacter sphaeroides. The authors propose that CarD “couples housekeeping transcription to the environment via DNA topology.”\nI find the evidence convincing that CarD indeed links promoter activity to DNA supercoiling. However, the claim that CarD couples transcription to the environment via changes in DNA topology is more speculative. This would be more compelling if demonstrated under physiologically relevant conditions, such as stationary phase, rather than through novobiocin treatment alone. During stationary phase (or slower growth conditions/media), negative supercoiling is expected to decrease, which could provide a suitable context for testing whether CarD mediates environmentally responsive transcription. One caveat is that CarD level is reduced during stationary phase in R. sphaeroides (I assume the carD promoter itself might be supercoiling-sensitive?)\nA question remains incompletely resolved: how are CarD, the frequent absence of a -7T position, and negative supercoiling mechanistically linked? Is CarD-DNA binding intrinsically sensitive to DNA topology? The ChIP-seq data suggest so, but can this be tested in vitro? Given the \"tryptophan wedge\" mechanism proposed for CarD in Mycobacterium tuberculosis, can the authors speculate on how Rhodobacter CarD, RNA polymerase, and supercoiling might function together mechanistically.\nExpanding the Discussion, together with a “model” figure (Figure 7?), would greatly help readers.\nWhat were the controls used in the ChIP-seq experiments? Polyclonal antibodies against CarD and σ93 were used for immunoprecipitation, but polyclonal antibodies are notoriously prone to nonspecific binding. Since carD and rpoD are essential genes, deletion controls are not feasible. However, R. sphaeroides is genetically tractable, so epitope-tagging could be an alternative, though I acknowledge it might require substantial effort. Including some control experiments to address antibody specificity would help reassure readers.\nAdditionally, can a consensus DNA-binding motif for CarD be derived from the ChIP-seq data?\nRegarding psoralen-seq, each bin spans 5 kb and could contain multiple genes or promoters. How reliable is it to assign a CarD-dependent promoter to such broad bins?\nThe presentation of data throughout the manuscript could be improved. As it stands, the manuscript is mainly for aficionados in bacterial transcription field i.e. not accessible for a wider readership. For example, in Figure 1f, the x-axis should be labeled using the conventional +1, -10, and -35 nomenclature, rather than sequential positions from 1 to 60. It would be helpful to annotate, for example the -7 position as well. I recommend adopting the labeling style used in Henry et al. (PNAS, 2020; reference 6).\nIn Figure 3d (left panel), some CarD-associated promoters are upregulated following novobiocin treatment, rather than downregulated as one might expect. These cases are intriguing: what are these promoters, and what are their distinguishing features? A deeper analysis could yield additional insights into the relationship between CarD and supercoiling, yet this was not discussed/investigated in the manuscript.\nFinally, in Figure 3c, the manuscript refers to changes in ChIP-seq peak height as “upregulated” or “downregulated.” I assume these terms are reserved for transcriptomic data?\nAlso, eliminate spaces between numbers and percent signs (e.g., use “50%” instead of “50 %”).", "reviewer_2": "This study offers compelling observations on the roles of CarD and DNA supercoiling in transcriptional regulation in R. sphaeroides. However, several aspects of data interpretation require closer alignment with existing mechanistic and structural work. In particular, a more thorough analysis of promoter architecture—including the -35 and extended -10 motifs—alongside consideration of non-canonical initiation mechanisms such as post-termination complexes (PTCs), is essential. The integration of ChIP-seq, Psoralen-seq, RNA-seq, and cappable-seq represents a significant strength, enabling a genome-wide view of regulatory dynamics. Together, these approaches provide complementary insights into promoter activity, DNA topology, and protein-DNA interactions. With the revisions and clarifications outlined above, this work could make a substantial contribution to our understanding of transcriptional regulation in bacteria. This work presents exciting observations but the interpretations need to be grounded more firmly in the mechanistic literature. A more rigorous analysis of promoter architecture (especially the -35 and extended -10 motifs), consideration of non-canonical initiation via PTCs, and improved data presentation will significantly strengthen the manuscript.\nMajor Points for Revision:\n1. -35 element and promoter assignment (fig. 1):\nThe lack of -35 element analysis is a puzzling. Why is this not included in the assignment of bona fide promoters? R. sphaeroides appears to have canonical -35 elements and lacks RbpA (PMID: 33168725), so their inclusion in promoter analysis is essential.\n2. clarification of classification in cappable-seq:\nIt is unclear whether the cappable-seq approach filters out transcript reads without recognizable promoter elements. How are genic and intergenic reads defined by the authors?\n3. Interpretation of intergenic/nongenic transcription in context of post-termination complexes (PTCs):\nMuch of the cappable-seq signal appears intergenic. There is a growing body of literature showing that RNAP can initiate transcription without sigma via post-termination complexes (PTCs) (PMIDs: 39779919, 37406096, 37459525). These initiate at -10-like elements without -35 motifs and may recruit sigma and CarD (PMID: 31974358). The authors must consider:\n-Whether some of the observed TSSs originate from PTCs rather than bona fide promoters.\n-The presence of bidirectional symmetry and absence of -35 motifs as features of PTC-derived initiation.\n-That PTCs are highly sensitive to supercoiling (PMID: 39779919).\n-That R. sphaeroides appears to encode a RapA-like factor (WP_119001171.1), which clears PTCs.\nThat CarD and sigma association with intergenic TSSs may reflect stabilization of PTC bubbles rather than standard promoter interactions.\n4. Missing -35 and extended -10 motif analysis:\nExplicit analysis of these motifs would help determine which TSSs are likely sigma-dependent versus PTC-derived. Without it, many TSSs may be misclassified.\n5. Mechanistic interpretation of CarD and supercoiling (including Fig. 4C):\nRecent findings in M. tuberculosis suggest that CarD’s effects are inhibited on negatively supercoiled DNA (PMID: 40060575). The authors should:\n-Discuss this preprint in more detail and reconcile the discrepancy with their findings in a more mechanistic way.\n-Clarify that novobiocin reduces negative supercoiling, likely destabilizing RPo indirectly, thus complicating their conclusions.\n-Consider the potential impact of novobiocin on PTC activity.\n-Quantify transcription in all lanes in Fig. 4C to compare CarD+ vs. CarD– at increasing negative supercoiling levels.\n-Discuss why RNAI is used as a control and provide its promoter sequence—CarD has no apparent effect here, which needs explanation.\n-More clearly quantify results from Figs. 4 and 5 to support conclusions about the effects of -10 element mutations.\n6. Analysis and rigor (Figs. 4 and 5):\n-Indicate the number of replicates for each experiment.\n-Quantify transcription in all lanes in Fig. 4C to compare CarD+ vs. CarD– at increasing negative supercoiling levels. The current quantification in 4c is just CarD. Also where is the error bar in the red bar?\n-Provide P-values and statistical significance.\n-In the supplement, label each gel lane and replicate clearly (e.g., replicate 1, 2, 3).\n7. Mechanisms (Fig. 3D and Line 143):\n-The statement that CarD depletion causes \"open complex collapse\" should also consider that it might promote faster escape into elongation.\n-Novobiocin treatment results in both upregulation and downregulation of transcription—even at CarD-associated promoters—this duality needs discussion. It is currently ignored.\n- Different promoters may respond to CarD in different ways depending on their kinetic properties (PMID: 37075846); this should be acknowledged.\n8. Discussion:\nThe discussion should incorporate:\n-The broader literature on CarD function, supercoiling effects, initiation kinetics, and promoter escape.\n-The likely contribution of topoisomerases and DNA topology to transcription across bacteria.\n-A critical evaluation of whether all observed TSSs represent bona fide promoters.\n-The roles of DksA seem less relevant and can be de-emphasized in favor of the points above.", "reviewer_3": "Forrest et al is a well-designed study on the effects of the transcription factor CarD in regulating Rhodobacter sphaeroides promoters. The authors combine various high-throughput data, including ChIP-seq, RNA-seq, transcript start site mapping (cappable-seq), and negative supercoiling mapping (with psoralen sequencing) to define the CarD regulon and supercoiling-sensitive genes of R. sphaeroides. These authors then use biochemical experiments with plasmids of different negative supercoiling to demonstrate that the rrnBP1 promoter requires both CarD and negative supercoiling for activation, with this requirement bypassed by a -7A -> T transversion in the -10 sequence. The authors thus conclude that “an optimal -10 element sequence, negative supercoiling, and CarD all enhance promoter activity, and that maximal R. sphaeroides transcription requires only two of these stimulatory factors.”\nI found the global study technically sound and the biochemical experiment especially compelling. Although a relationship between CarD, supercoiling, and open complex formation has been reported, the high-throughput studies are novel in R. sphaeroides and the biochemical experiment clearly demonstrates the relationship between CarD and negative supercoiling at the rrnBP1 promoter. However, these studies are not sufficiently complementary, with the conclusions are not justified by the experiments. I believe that the authors are conflating multiple distinct classes of CarD-dependent promoters in this study. I also have many questions about the proposed model and experimental approaches. Either more experiments or significantly more limited interpretations of the data is warranted.\nMajor concerns:\n1) As noted by the authors, CarD-dependent promoters can be repressive or stimulatory, but no effort is made to distinguish between these classes. Indeed, previous work in Mycobacteria suggests that CarD-mediated open complex formation could either promote promoter escape (activation) or inhibit it (repression) (Zhu et al, PMID: 31217290), suggesting that CarD/supercoiling could identically impact open complex formation at many promoters but cause vastly different transcriptional outcomes.\na. Only the rrnBP1 promoter is tested biochemically and it is not made clear the relationship between this promoter and the high-throughput experiments. To what extent is rrnBP1 an appropriate model for the relaxation-repressed, CarD-bound promoter class proposed in this study? Is the rrnBP1 promoter bound by CarD in ChIP-seq and significantly downregulated in response to chromosome relaxation? What is the sequence of the rrnBP1 promoter and how does it relate to the predicted CarD-dependent promoters (see further comments regarding sequence logo in 1b, below)? Biochemically validating additional CarD-associated novobiocin-repressed promoters is essential to strengthen the conclusions presented here.\nIt is also clear from this study and the literature that there is a class of CarD-repressed promoters, with the prime example the CarD promoter itself (Henry et al, PMID: 34152199). Henry et al showed that an extended -10 element and other spacer sequences mediated repression. In this work, the authors have a set of CarD-dependent relaxation-activated promoters (Figure 3) that are overlooked. Do these promoters have the same conserved sequences as those that are relaxation-repressed? Is the CarD promoter supercoiling-dependent and is it repressed or activated? Are the sequence features identified in Henry et al observed in any promoter classes? An ideal experiment would be to deplete CarD and reexamine transcription to distinguish between these promoter sets but some effort should be made to categorize and validate that the promoter features associated with different CarD/supercoiling regulation classes. The authors should also consider biochemically testing CarD-associated novobiocin-induced promoters. In these cases, is CarD functioning in synergy or opposition to supercoiling? Without these experiments, the authors should be extremely cautious in generalizing their biochemical result to encompass the full range of possible CarD/supercoiling interactions.\nb. The authors make an argument for -7T/-12T de-enrichment in CarD promoters. However, there are several issues with idea. In figure 1F, I cannot see the difference in the -7T/-12T as reported. The de-enrichment of -7T is well-supported by the literature (Henry et al, PMID: 33168725; 34152199) but T is still an enriched base in the sequence logo presented. The -12 T is clearly enriched as well. Is the interpretation based on pvalue? It does not make sense to quantitatively compare two p-values. I would suggest the bigger differences are that CarD-bound promoters lack -8T and a G at -33. Perhaps a quantitative analysis like in Henry et al (PMID: 33168725) would be more appropriate.\nMore worryingly, nothing is ever explored for -12T (or for any other promoter differences). Henry et al (PMID: 33168725) observed that -12T (without -7T) was conserved for 15 promoters activated by CarD in vitro. Further, the CarD promoter itself contains a -12T. What is the explanation for these discrepancies? What is the function, if any for these sequences? How do they relate to the different potential CarD/supercoiling promoter classes (1a, above)? Does -12T have any effect on CarD/negative supercoiling synergy?\nAt the end of the work, I am convinced that for rrnBP1 both CarD and negative supercoiling are required for activation. However, I have many remaining questions about the overall model.\n2) Why is rrnBP1 be regulated in this fashion? I agree with the authors that synergy is logical, but this operates more like an “AND” logic gate, meaning the effects are not tunable. Is it to quickly shutoff ribosomal synthesis during unfavorable conditions? I feel this idea would be better-supported if there are additional examples of this regulation. Perhaps this lack of synergy is unique to rrnBP1.\n3) How generalizable is this finding for all R. sphaeroides promoters? I also agree that “the combined impact of CarD and DNA topology must differ according to biological context”. Examining several more promoters and, ideally, in different regulatory classes (novo-activated, novo-inhibited, CarD-bound, CarD-free, etc) is essential for extending the experiments to infer CarD’s effects on individual promoters. This is especially true since CarD is itself is controlled by a negative feedback loop. Whether negative supercoiling at the CarD promoter increases or represses CarD expression will have consequences for the interpretation of the transcriptomic/ChIP data, especially if CarD is rapidly turned over.\n4) How does CarD/RNAP together sense negative supercoiling? Figures 2-3 show that CarD-bound sites are more negatively supercoiled than random sites. However, are these CarD-bound sites more negatively supercoiled than σ93-bound sites? One would imagine that any active TSS must be negatively supercoiled since negative supercoiling promotes promoter melting and RNAP escape. If CarD-free σ93-bound sites are more negatively supercoiled, CarD could perhaps compensate for the absence of negative supercoiling at promoters rather than require it for binding, which is the model presented here. This analysis should also be performed again in the novobiocin-treated dataset, as it would be interesting to know if σ93/RNAP remains more bound when CarD is present/absent, which would support the hypothesis that CarD stabilizes open complexes.\nRelatedly, for Figure 6B, because CarD is associated with σ93 (housekeeping) promoters, this set should be naturally enriched in metabolism and central dogma processes. I’d be curious to see the comparison to the remaining σ93-dependent non-CarD promoters, which are also likely enriched for these functions. Significant differences between these two sets would be more compelling than the comparison to random promoters, which are likely enriched for alternative sigma factor promoters that regulate stress response genes.\n5) Based on the Methods, it is not clear how the σ93 and CarD overlapping promoters were selected. Further definition of what “overlap” constitutes would be helpful. I also wonder where CarD binds at these promoters (e.g. around TSS, -10, or hundreds of bases upstream) and if this information could be used to refine the models for CarD/supercoiling behaviors.\n6) Is there a relationship between CarD and the bidirectional promoters noted in Figure 1? Is the hypothesis that CarD should activate these promoters and it should activate both? Is CarD bind to these bidirectional promoters?\n7) Line 147: Is the idea here that more CarD-bound genes are downregulated than CarD-independent? Can this be explained more clearly (e.g. more CarD-bound genes were downregulated)? There also appears to be more genes upregulated for CarD-independent promoters, is this correct?\n8) Line 151-154: I did not understand the data for “Novobiocin predominantly downregulates transcription and this is more pronounced for CarD bound genes”. Is the idea that more CarD-bound genes are downregulated upon treatment? To my eye, it appears that there are more genes downregulated for CarD-free promoters. I also suggest the statement “novobiocin caused significant DNA relaxation, loss of both CarD and σ93 binding, and lower gene expression” should be revised. Novobiocin-induced/DNA relaxation-induced genes have been observed in diverse organisms, and there are many genes (red) that are induced upon DNA relaxation in Figure 3D.\nMinor comments:\n1) The authors should carefully check over their referencing and reference the primary literature as often as possible:\na. Line 27-28: a review here would be more appropriate, as reference 2 (Zhang et al) were only able to visualize domains 2-4 and do not discuss the role of sigma domains in depth. Reference 3 also does not the primary source to show sigma and -10/-35 contacts.\nb. Line 30: Instead of references 2-4, which are not the references that show sigma domain 2 interacting with -7T/-11A, please cite Feklistov and Darst (PMID: 22136875), Zhang et al (PMID: 23086998), and Zuo and Steitz (PMID: 25866247).\nc. Line 108: The authors may also want to cite Lal et al (PMID: 27025941), the first report of psoralen use genome-wide in bacteria.\n2) Line 46-47: The authors are correct that lack of rotation of RNAP increases supercoiling. However, simple duplex DNA melting by RNAP also leads to torsional strain and consequent twisting of DNA.\n3) Line 75: Is there some way to distinguish between “active” and “infrequently used” promoters from cappable-seq? Perhaps the studies would provide better motif resolution if the highest-active promoters are selected.\n4) Please renumber the logo (indicating +1) in Figure 1F and mark the correct bases to examine to ease the burden on the reader.", "reviewer_author_response": "We thank the reviewers for their considerate assessment of our manuscript. In the vast majority of cases, we agree with the reviewers and have taken their suggestions on board when revising our paper. Thus, the updated paper includes substantial new experimental work and a deeper analysis of the existing genomic data.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nIn this manuscript, Forest et al. employed a combination of genome-wide techniques (RNA-seq, ChIP-seq, Cappable-seq, and psoralen-seq), along with in vitro transcription assays, to investigate the interplay between CarD and DNA supercoiling, and how they together regulate promoter activity in Rhodobacter sphaeroides. The authors propose that CarD “couples housekeeping transcription to the environment via DNA topology.”\nI find the evidence convincing that CarD indeed links promoter activity to DNA supercoiling.\nHowever, the claim that CarD couples transcription to the environment via changes in DNA topology is more speculative. This would be more compelling if demonstrated under physiologically relevant conditions, such as stationary phase, rather than through novobiocin treatment alone. During stationary phase (or slower growth conditions/media), negative supercoiling is expected to decrease, which could provide a suitable context for testing whether CarD mediates environmentally responsive transcription.\nWe have done additional stationary phase experiments that the reviewer suggests, testing binding of CarD using ChIP-qPCR and levels of transcription using RT-qPCR. Both binding of CarD, and transcription from the CarD activated promoters, is greatly reduced in stationary phase as expected. These data are presented in Figure S7a and S7b.\nOne caveat is that CarD level is reduced during stationary phase in R. sphaeroides (I assume the carD promoter itself might be supercoiling-sensitive?)\nThe reviewer is correct, CarD levels do decrease in stationary phase. Most likely, CarD is degraded. We also find that σ93 levels decrease substantially in starved cells (Figure S7c and S7d). As the reviewer notes, such changes make the above result difficult to interpret. Hence, we are cautious in our presentation of these data, noting that both supercoiling changes, and reduced levels of CarD/σ93, must contribute.\nWith this in mind, we have done an additional experiment that we think is more informative with respect to physiologically relevant changes in supercoiling. After comparing several different conditions, we found clear changes in plasmid supercoiling in R. sphaeroides cells grown aerobically compared to those dependent on photosynthesis. Rather than investigate gene expression effects in vivo, where many other regulatory factors at play, we used plasmids isolated from cells grown in each condition (and so having different levels of negative supercoiling) as templates for in vitro transcription. The data show that CarD readily actives transcription from the more negatively supercoiled plasmid obtained from aerobically growing cells. By comparison, CarD activated transcription poorly from plasmid templates obtained from photosynthetically growing cells. Hence, physiologically relevant changes in supercoiling do impact the ability of CarD to control transcription. The data are in Figures S7e and S7f.\nA question remains incompletely resolved: how are CarD, the frequent absence of a -7T position, and negative supercoiling mechanistically linked? Is CarD-DNA binding intrinsically sensitive to DNA topology? The ChIP-seq data suggest so, but can this be tested in vitro? Given the \"tryptophan wedge\" mechanism proposed for CarD in Mycobacterium tuberculosis, can the authors speculate on how Rhodobacter CarD, RNA polymerase, and supercoiling might function together mechanistically.\nExpanding the Discussion, together with a “model” figure (Figure 7?), would greatly help readers.\nWe apologise for the mechanistic link not being clear. Negative supercoiling (by virtue of the underwound double helix) CarD (via its tryptophan wedge) and -7T (due to base flipping and σ binding) all contribute to promoter open complex stability, and this is the mechanistic link.\nThus, at the rrnB promoter, which lacks -7T, both CarD and negative supercoiling are needed for transcription to occur. If the -7T base is introduced by mutation, it is possible to dispense with either CarD or negative supercoiling.\nWe don’t think CarD binding is intrinsically sensitive to DNA topology. Rather, if a stable open complex can’t be formed (e.g. because of a reduction in negative supercoiling) then the entire transcriptional apparatus (including CarD) is likely to vacate the promoter DNA prematurely.\nAs requested, we have added extra text. This appears at the end of Results sections “Decreased DNA supercoiling impacts CarD...” and “Reduced DNA supercoiling predominantly inhibits...”.\nWe also now mention this in the first paragraph of the Discussion. At this point we haven’t included a “model” figure. We tried several graphic representations but found it difficult to simultaneously show the different regulatory outcomes.\nWhat were the controls used in the ChIP-seq experiments? Polyclonal antibodies against CarD and σ93 were used for immunoprecipitation, but polyclonal antibodies are notoriously prone to nonspecific binding. Since carD and rpoD are essential genes, deletion controls are not feasible. However, R. sphaeroides is genetically tractable, so epitope-tagging could be an alternative, though I acknowledge it might require substantial effort. Including some control experiments to address antibody specificity would help reassure readers.\nWe always do mock immunoprecipitations (without antibody) for ChIP-seq experiments and that was also the case here. These controls yield little DNA, consistent with specific isolation of DNA binding proteins when antibodies are preset. Furthermore, the specific regions of enrichment for σ93 and CarD binding are not evident in the control experiment (see below). Note that the sequence reads from the control experiment are available on array express along with the ChIP-seq data for σ93 and CarD binding.\nanti-σ93\nanti-CarD\nno antibody\nThe reviewer is correct that the anti-CarD is polyclonal. To reassure the reviewer, we have done additional western blotting experiments. The anti-CarD is highly specific with no evidence of binding to other proteins. The new data are in Figure S7c.\nThe anti-σ93 is monoclonal and the epitope is precisely mapped. This is a commercially available antibody, that is highly specific and reacts with the primary sigma factors of numerous bacteria (the antibody was raised against E. coli σ70). Almost all published ChIP-seq studies of primary sigma factors have used this antibody. Figure S7d shows western blotting experiments using this antibody. The two more mobile bands detected are likely to be degradation products (σ factors are prone to proteolysis at the linker regions between domains) since their abundance precisely matches that of full length σ93 throughout the growth phases.\nAdditionally, can a consensus DNA-binding motif for CarD be derived from the ChIP-seq data?\nNo, and this is expected based on structural work showing that CarD makes very minimal contacts with the DNA. Rather, CarD predominantly interacts with the RNAP.\nRegarding psoralen-seq, each bin spans 5 kb and could contain multiple genes or promoters. How reliable is it to assign a CarD-dependent promoter to such broad bins?\nPromoters can reliably be assigned to the correct bin, but we agree resolution is a drawback of psoralen-seq. However, in the absence of other in vivo approaches, our experimental options are limited. This is why we have done extensive in vitro studies to back up the general trends that evident from our genome-scale experiments. Given that the in vivo and in vitro data are perfectly consistent, this seems a reasonable approach. We note that, in vivo, supercoiling is highly dynamic and must vary between cells at any given moment. As such, the genome-scale experiments are only intended to provide a population averaged snapshot of general supercoiling trends. If supercoiling is averaged in bins that are too small, the results are likely to be less reliable.\nThe presentation of data throughout the manuscript could be improved. As it stands, the manuscript is mainly for aficionados in bacterial transcription field i.e. not accessible for a wider readership. For example, in Figure 1f, the x-axis should be labeled using the conventional +1, -10, and -35 nomenclature, rather than sequential positions from 1 to 60. It would be helpful to annotate, for example the -7 position as well. I recommend adopting the labeling style used in Henry et al. (PNAS, 2020; reference 6).\nWe have made the requested changes, now using both numbering (with respect to +1) and labelling of the key elements. The -7T position is highlighted by an asterisk and this is noted in the figure legend.\nIn Figure 3d (left panel), some CarD-associated promoters are upregulated following novobiocin treatment, rather than downregulated as one might expect. These cases are intriguing: what are these promoters, and what are their distinguishing features? A deeper analysis could yield additional insights into the relationship between CarD and supercoiling, yet this was not discussed/investigated in the manuscript.\nWe have done substantial further experiments with the carD promoter that is subject to repression by CarD (Figures 5, 6 and S6). We have also defined motifs for CarD-bound promoters activated and repressed by DNA relaxation (Figures 3f and 3g). There is surprisingly little difference. However, we do show that DNA supercoiling levels can determine if CarD acts as a repressor or activator (Figure 5b). We also show that improving CarD repressed promoters gradually negates the ability of CarD to repress transcription.\nFinally, in Figure 3c, the manuscript refers to changes in ChIP-seq peak height as “upregulated” or “downregulated.” I assume these terms are reserved for transcriptomic data?\nWe have altered the wording to instead describe increased or decreased binding.\nAlso, eliminate spaces between numbers and percent signs (e.g., use “50%” instead of “50 %”).\nThese spaces have been removed.\nReviewer #2 (Remarks to the Author):\nThis study offers compelling observations on the roles of CarD and DNA supercoiling in transcriptional regulation in R. sphaeroides. However, several aspects of data interpretation require closer alignment with existing mechanistic and structural work. In particular, a more thorough analysis of promoter architecture—including the -35 and extended -10 motifs—alongside consideration of non-canonical initiation mechanisms such as post-termination complexes (PTCs), is essential. The integration of ChIP-seq, Psoralen-seq, RNA-seq, and cappable-seq represents a significant strength, enabling a genome-wide view of regulatory dynamics. Together, these approaches provide complementary insights into promoter activity, DNA topology, and protein-DNA interactions. With the revisions and clarifications outlined above, this work could make a substantial contribution to our understanding of transcriptional regulation in bacteria. This work presents exciting observations but the interpretations need to be grounded more firmly in the mechanistic literature. A more rigorous analysis of promoter architecture (especially the -35 and extended -10 motifs), consideration of non-canonical initiation via PTCs, and improved data presentation will significantly strengthen the manuscript.\nMajor Points for Revision:\n1. -35 element and promoter assignment (fig. 1):\nThe lack of -35 element analysis is a puzzling. Why is this not included in the assignment of bona fide promoters? R. sphaeroides appears to have canonical -35 elements and lacks RbpA (PMID: 33168725), so their inclusion in promoter analysis is essential.\nWe are a little confused here and apologise if we have confused the reviewer. The entire promoter (including the -35 region) was analysed and the -35 element is evident (see original Figure 3f below, with additional annotation of key features for clarity). We wonder if the reviewer is referring to the fact that the -35 element seems poorly conserved or incomplete? If so, this is always the case when promoters are analysed in this way, regardless of the bacterial species (e.g. PMID: 27806702). There are three well-known reasons for this.\na. The promoters are aligned based on TSS position. As the distance between the TSS and -35 element is not the same for every promoter (because of variation in promoter spacer and discriminator length) the final sequence logo does not show a perfect -35.\nb. The -35 element, in general, is poorly conserved compared to the -10 element and TSS. This is because many promoters have either a poor -35 element, or no -35 at all, and instead rely on transcriptional activator proteins to recruit RNA polymerase to the DNA.\nc. The σ factor makes base specific interactions with the -35 element at positions 1,2,3 and 5 (PMID: 11931761). Hence, the upstream half of the -35 element is much better conserved.\nIn summary, there is nothing unusual about the -35 analysis in our paper. However, to try and avoid confusion, we’ve updated Figure 1 so that the individual promoter components are aligned individually rather than the whole promoter being aligned based on TSS position. This makes the -35 element easier to see.\n2. clarification of classification in cappable-seq:\nIt is unclear whether the cappable-seq approach filters out transcript reads without recognizable promoter elements.\nCappable-seq is completely unbiased and simply identifies triphosphoryalted RNA 5' ends, regardless of the DNA sequence adjacent to the site where the RNA 5' end maps.\nHow are genic and intergenic reads defined by the authors?\nGenic reads are those where the RNA 5' end (and so TSS) maps to the coding sequence of a gene. Intergenic reads are RNA 5' ends mapping between genes.\n3. Interpretation of intergenic/nongenic transcription in context of post-termination complexes (PTCs):\nWe appreciate the reviewer’s interest in PTCs and have considered the comments carefully. For the reasons outlined below, we don’t think we infer much about PTCs with the data available. Even so, we’ve tried to make some changes to the text to acknowledge the reviewer comments where we can. We stress that PTCs are not the subject of our study and hope the reviewer can appreciate our reasoning.\nMuch of the cappable-seq signal appears intergenic.\nThis is partially correct, but the majority of TSSs are intragenic, consistent with observations for other bacteria.\nThere is a growing body of literature showing that RNAP can initiate transcription without sigma via post-termination complexes (PTCs) (PMIDs: 39779919, 37406096, 37459525). These initiate at -10-like elements without -35 motifs and may recruit sigma and CarD (PMID: 31974358). The authors must consider:\nWe would revise this statement. In our view, there is evidence that post-termination complexes can be formed in vitro (PMID: 31974358) and if conditions are correct core RNA polymerase can subsequently initiate transcription (PMIDs: 31974358 & 39779919). The degree to which this might occur in vivo is unknown. However, available evidence strongly suggests cells take precautions to remove PTCs from the DNA and that a failure to do so is toxic (PMIDs: 39779919 & 37406096). As such, we think the consensus view of the field is that the vast majority, if not all, site specific transcription initiation in vivo is dependent on canonical promoter:RNAP interactions. As an aside, we also note that PMID: 39779919 reports PTC reinitiation “independent from... conserved promoter elements” rather than from -10 elements without -35 motifs. Hence, whether certain DNA sequences are associated with PTC reinitiation remains an open question.\n-Whether some of the observed TSSs originate from PTCs rather than bona fide promoters.\nThis is an interesting question, but it is impossible to use our data to differentiate between bona fide promoters and any TSSs due to PTCs (further details below). As outlined above, it seems very unlikely PTCs give rise to significant transcription initiation in vivo. All the available evidence is that cells go to great lengths to prevent this. Indeed, this is discussed in the paper the reviewer cites (PMID: 39779919) that notes “evolved strategies to suppress promoter-independent transcription appear manifold”. We have added text explaining this at the end of the first results section.\n-The presence of bidirectional symmetry and absence of -35 motifs as features of PTC-derived initiation.\nWe have done additional analyses and see no evidence for subclasses of TSSs (intragenic, intergenic, bidirectional, or otherwise) that consistently lack -35 elements. Of course, as mentioned above, the caveat is that individual promoters often lack a good match to the -35 element and rely either on activator proteins or alternative promoter elements (TG motifs, UP elements, AT-rich spacers) to recruit RNA polymerase. As such, the absence of a -35 is a common feature of canonical promoters and cannot be used as a simple marker of PTC derived initiation in vivo.\n-That PTCs are highly sensitive to supercoiling (PMID: 39779919).\nWe agree that this is true, but PTCs are not the subject of our paper and we don’t feel that we can add anything to the wider literature about PTCs using our data. Indeed, as noted above, PTCs are likely to be very transient inside cells as multiple mechanisms exist to remove them.\n-That R. sphaeroides appears to encode a RapA-like factor (WP_119001171.1), which clears PTCs.\nWe agree that this is true and argue this makes it even more likely that TSSs due to PTCs are likely to be very rare, if detected at all. We now briefly mention mechanisms to remove PTCs from the DNA in the results section.\nThat CarD and sigma association with intergenic TSSs may reflect stabilization of PTC bubbles rather than standard promoter interactions.\nWe do appreciate this point, and do acknowledge this is theoretically possible. However, all the available evidence, and the current position of the field as a whole, is that the vast majority of chromosomal sites, associated with σ and CarD, are likely to be promoters. Consistent with this, we find strong conservation of promoter elements at these locations.\n4.Missing -35 and extended -10 motif analysis:\nExplicit analysis of these motifs would help determine which TSSs are likely sigma-dependent versus PTC-derived. Without it, many TSSs may be misclassified.\nWe have modified Figure 1f to specifically align the various promoter elements. Even so, we respectfully disagree with the reviewer’s logic here. As outlined earlier in our response, it is not at all unusual to find promoters that lack a good -35 element. Indeed, this is very common because of the role played by transcriptional activators. For example, the promoter motif below is taken from our prior paper (PMID: 21398630) and the list of 554 E. coli promoters used to make the motif is described by PMID: 12907708. Importantly, this list pre-dates the genomic era and represents well-defined promoters for mRNAs. Compared to the -10 hexamer, the -35 is very poorly conserved. Hence, a missing -35 cannot be used to identify TSSs due to PTCs, which current data indicate must be short lived in vivo. Furthermore, there is no consensus view that a -10 element, in the absence of a -35, is a marker of PTC derived reinitiation as the reviewer suggests. For example, PMID: 39779919 reports PTC-like reinitiation by core RNAP without any promoter elements at all. Overall, we do not think it is possible to identify PTC-derived TSSs from our data and, even if it were, the expectation is that these would be very rare in wild type cells.\n[REDACTED]\n5.Mechanistic interpretation of CarD and supercoiling (including Fig. 4C):\nRecent findings in M. tuberculosis suggest that CarD’s effects are inhibited on negatively supercoiled DNA (PMID: 40060575). The authors should:\nWe would describe the pre-print differently. Rather than showing “CarD’s effects are inhibited on negatively supercoiled DNA” the pre-print provides an example of a promoter that switches from being activated to repressed by CarD when DNA is negatively supercoiled. As expanded on below, this was also known from prior work and fits perfectly with our model. We covered the original paper (PMID: 37075846), reporting a similar result to PMID: 40060575, in our Discussion. We now also cite the newer work.\n-Discuss this preprint in more detail and reconcile the discrepancy with their findings in a more mechanistic way.\nWe now cite the pre-print (since published in JBC) but do not think there is a discrepancy with our findings. Indeed, we had already explained how this exact phenomenon (reported by PMID: 37075846, cited in the original text) fits within the framework of our model. This is in the Discussion section and the updated text is pasted below for clarity.\n-Clarify that novobiocin reduces negative supercoiling, likely destabilizing RPo indirectly, thus complicating their conclusions.\nWe probably don’t describe this clearly enough. Briefly, we don’t think this complicates our conclusions, rather this is exactly what we propose. To expand, CarD activates transcription by stabilising otherwise unstable open complexes. Negative DNA supercoiling does the same, albeit by a different mechanism. Hence the co-dependence of many promoters on negative DNA supercoiling and CarD. As the reviewer correctly notes, novobiocin reduces negative supercoiling, and so RPo stability. As CarD is part of the open complex formed by RNAP and the DNA, if this becomes less stable, the CarD binding signal will decrease. We now reiterate this model throughout the text.\n-Consider the potential impact of novobiocin on PTC activity.\nWe do appreciate the reviewer’s interest in PTCs but, as outlined above, genuinely don’t feel we can comment on this issue using the data we have.\n-Quantify transcription in all lanes in Fig. 4C to compare CarD+ vs. CarD– at increasing negative supercoiling levels.\nThis has been done and the revised figure shows fold activation by CarD (i.e. the CarD+/CarD- signal is plotted).\n-Discuss why RNAI is used as a control and provide its promoter sequence—CarD has no apparent effect here, which needs explanation.\nRNAI is widely used as a control for in vitro transcription assays and has been for many decades (e.g. PMIDs: 15126488, 9218807 & 10542171, but there must be 100s of papers). The RNAI is derived from the colE1 plasmid replication origin (where its biological function is to control plasmid copy number). Plasmids used as templates for in vitro transcription assays invariably contain colE1. As promoter driving RNAI production is constitutive, RNAI has become a ubiquitous control.\nWe note that others also found no impact of CarD on RNAI transcription (PMID: 33168725) so this is not unusual. Presumably, as the promoter is constitutive, there is simply no dependence on CarD. It’s also worth noting the RNAI promoter is not native to the Rhodobacter system. The promoter has the sequence 5’-tcTTGAAGtggtggcctaactacggcTACACTagaaggAca-3’\nWe haven’t altered the text, since we had already described RNAI has being derived from the plasmid replication origin, but hope this provides sufficient further explanation for the reviewer.\n-More clearly quantify results from Figs. 4 and 5 to support conclusions about the effects of -10 element mutations.\nThe quantification has been added. Although these figures are now largely new data.\n6. Analysis and rigor (Figs. 4 and 5):\n-Indicate the number of replicates for each experiment.\nThere are three replicates each for Figures 4 and 5. This detail has been added.\n-Quantify transcription in all lanes in Fig. 4C to compare CarD+ vs. CarD– at increasing negative supercoiling levels. The current quantification in 4c is just CarD. Also where is the error bar in the red bar?\nThe reviewer also requested this change earlier in the review, and we have made the alteration, but please see our prior response for the details. There was no error shown for the red bar because this lane was set to 100 % for each replicate, allowing for normalisation between experiments. An error bar is now included because we show activity +/- CarD, rather than % activity, in the revision.\n-Provide P-values and statistical significance.\nAdded where appropriate.\n-In the supplement, label each gel lane and replicate clearly (e.g., replicate 1, 2, 3).\nThis has been done.\n7. Mechanisms (Fig. 3D and Line 143):\n-The statement that CarD depletion causes \"open complex collapse\" should also consider that it might promote faster escape into elongation.\nWe agree that this could be the case for promoters repressed by CarD, where the open complex is over stabilised. However, most promoters we detect are activated by CarD. In these cases, loss of CarD results in less transcription, which isn’t consistent with a faster rate of promoter escape. Note that, for Figure 3D, faster escape into elongation does not fit the data, which shows decreased transcription. Additionally, the section has been rewritten and no longer mentions open complex collapse.\n-Novobiocin treatment results in both upregulation and downregulation of transcription—even at CarD-associated promoters—this duality needs discussion. It is currently ignored.\nThe new Figures 3e, 3f, 5 and 6 now deal with this topic. We compare the two classes of promoter both in vivo, using genome scale data, and in vitro using biochemical assays.\n- Different promoters may respond to CarD in different ways depending on their kinetic properties (PMID: 37075846); this should be acknowledged.\nWe have added this point to the discussion.\n8. Discussion:\nThe discussion should incorporate:\n-The broader literature on CarD function, supercoiling effects, initiation kinetics, and promoter escape.\nWe have added extra details, partly in response to a previous question that the reviewer raised. We also compare CarD activated and repressed promoters, as well as the different impact of CarD in M. tuberculosis, and T. thermophilus.\n-The likely contribution of topoisomerases and DNA topology to transcription across bacteria.\nWe did already touch on this (e.g. supercoiling control of rRNA transcription in B. subtilis, and supercoiling changes in starved E. coli).\n-A critical evaluation of whether all observed TSSs represent bona fide promoters.\nWe’ve probably answered this question in our responses above; we think the vast majority of, if not all, observed TSSs represent bona fide promoters. Of course, there is also the issue of what a “bona fide promoter” really is. Do these have to be promoters in intergenic regions? Do these include DNA sequences that resemble a promoter but don’t generate a useful mRNA? Does there have to be a certain level of activity for a promoter to be considered real? These are quite broad philosophical questions that we have discussed at length in prior work. However, we are not sure there is much to add in the current paper; there isn’t really a logical place to introduce this sort of ideas. However, should the paper be accepted, our detailed discussions above will also be available online for interested parties.\n-The roles of DksA seem less relevant and can be de-emphasized in favor of the points above.\nOnly two very short sentences mention DksA, and we think it’s important to compare strategies for coupling rRNA transcription to growth phase in different bacteria, so we would like to keep this detail in if possible (we are not restricted for space).\nReviewer #3 (Remarks to the Author):\nForrest et al is a well-designed study on the effects of the transcription factor CarD in regulating Rhodobacter sphaeroides promoters. The authors combine various high-throughput data, including ChIP-seq, RNA-seq, transcript start site mapping (cappable-seq), and negative supercoiling mapping (with psoralen sequencing) to define the CarD regulon and supercoiling-sensitive genes of R. sphaeroides. These authors then use biochemical experiments with plasmids of different negative supercoiling to demonstrate that the rrnBP1 promoter requires both CarD and negative supercoiling for activation, with this requirement bypassed by a -7A -> T transversion in the -10 sequence. The authors thus conclude that “an optimal -10 element sequence, negative supercoiling, and CarD all enhance promoter activity, and that maximal R. sphaeroides transcription requires only two of these stimulatory factors.”\nI found the global study technically sound and the biochemical experiment especially compelling. Although a relationship between CarD, supercoiling, and open complex formation has been reported, the high-throughput studies are novel in R. sphaeroides and the biochemical experiment clearly demonstrates the relationship between CarD and negative supercoiling at the rrnBP1 promoter. However, these studies are not sufficiently complementary, with the conclusions are not justified by the experiments. I believe that the authors are conflating multiple distinct classes of CarD-dependent promoters in this study. I also have many questions about the proposed model and experimental approaches. Either more experiments or significantly more limited interpretations of the data is warranted.\nWe agree with the reviewer. There are different classes of CarD regulated promoter and these could have been considered. To explain our initial reasoning, in the case of R. sphaeroides, most (~75%) CarD-associated promoters, detected in vivo, are relaxation repressed. Hence, we focused the original manuscript on activation due to interplay between CarD and negative supercoiling. We chose rrnBP1 for the in vitro work because of the promoter’s importance for cell physiology, as it had been used in the past to study CarD in R. sphaeroides, and since it is an example of a relaxation repressed promoter (discussed further below). We appreciate that we could have stated the intention of our focus more clearly. As requested, the new version of the paper includes different promoter classes.\nMajor concerns:\n1) As noted by the authors, CarD-dependent promoters can be repressive or stimulatory, but no effort is made to distinguish between these classes. Indeed, previous work in Mycobacteria suggests that CarD-mediated open complex formation could either promote promoter escape (activation) or inhibit it (repression) (Zhu et al, PMID: 31217290), suggesting that CarD/supercoiling could identically impact open complex formation at many promoters but cause vastly different transcriptional outcomes.\nIn the revised paper we have extended the scope of our in vitro analysis to include an additional relaxation repressed promoter (rpsL), a relaxation activated promoter (carD), and a series of chimeric promoters derived from rrnBP1 and the carD promoter (Figures 5, 6 and S6). We have also investigated both rrnBP1 and the carD promoter using mutagenesis (Figures 5 and 6). Furthermore, the genome-scale data have been subject to further analyses as suggested (Figures 3f and 3g). We feel the altered text better links the different parts of the story.\nThe details of the additional analyses and experiments are laid out in response to the various questions below.\na. Only the rrnBP1 promoter is tested biochemically and it is not made clear the relationship between this promoter and the high-throughput experiments. To what extent is rrnBP1 an appropriate model for the relaxation-repressed, CarD-bound promoter class proposed in this study? Is the rrnBP1 promoter bound by CarD in ChIP-seq and significantly downregulated in response to chromosome relaxation?\nWe have now made the relationship to the genomic data clear. Briefly, in our high-throughput experiments, binding of σ93 and CarD at rrnBP1 is indeed downregulated by novobiocin, consistent with this being an appropriate model. We can’t specifically comment on precise transcript levels, since RNA-seq experiments i) require rRNA depletion and ii) any reads detected are difficult to map because of the multiple rRNA operons. We also note the link to the prior study of CarD and rrnBP1, which informed our decision to use this promoter.\nWhat is the sequence of the rrnBP1 promoter and how does it relate to the predicted CarD-dependent promoters (see further comments regarding sequence logo in 1b, below)?\nBiochemically validating additional CarD-associated novobiocin-repressed promoters is essential to strengthen the conclusions presented here.\nThe sequence of rrnBP1 is now shown in the new Figure 5a. As requested in comments below, we have made new sequence logos (from the genome-scale data) for sites bound and unbound by CarD (Figure 1f). The frequent lack of position -7T is clear for the CarD bound promoters and this is consistent with the sequence of rrnBP1.\nAs suggested, we have also biochemically validated an additional CarD-activated novobiocin-repressed promoter, for the gene rpsL. The data resemble those obtained for rrnBP1 (see new Figure S6). We have also tested a series of chimeric rrnBP1 derivatives containing sections of PcarD (Figure 6). The chimeric promoters remain CarD activated (Figure 6), although sequence changes can reduce the stimulatory impact of supercoiling.\nIt is also clear from this study and the literature that there is a class of CarD-repressed promoters, with the prime example the CarD promoter itself (Henry et al, PMID: 34152199). Henry et al showed that an extended -10 element and other spacer sequences mediated repression. In this work, the authors have a set of CarD-dependent relaxation-activated promoters (Figure 3) that are overlooked.\nWe agree, and the responses below detail additional analysis of the genome-scale data (Figures 3f, 3g and S4) and in vitro experiments with the carD promoter (Figures 5 and 6). These include and comparisons with rrnBP1.\nDo these promoters have the same conserved sequences as those that are relaxation-repressed?\nWe have done additional analysis of the genome-scale data (Figures 3f, 3g and S4) to answer this question. Relaxation-activated and -repressed promoters, bound by CarD, are extremely similar (Figure 3f). Overall, the only significant (but still very small) difference is that relaxation activated promoters tend to have better -10 hexamers (Figure 3g). We conclude that there is no simple “hallmark” that defines one promoter class compared to another. This is further evidenced by comparing PcarD and rrnBP1 (Figures 5 and 6) in vitro. Hence, introducing the -7T mutation, a TG motif, and a more AT-rich discriminator (the latter two being features of PcarD) fail to convert rrnBP1 into a CarD repressed promoter, though the requirement for both CarD and negative supercoiling is removed.\nIs the CarD promoter supercoiling-dependent and is it repressed or activated?\nWe have now done these experiments (Figures 5, 6 and S6). Consistent with Henry et al., we find that the carD promoter is repressed by CarD. We show that this repression is very sensitive to supercoiling. In fact, on a relaxed DNA template, the role of CarD switches to that of a PcarD activator (Figure 5). This is consistent with the overall model that i) negative supercoiling and CarD both enhance DNA opening and ii) at some promoters CarD and negative supercoiling over stabilise DNA opening and the result is repression.\nAre the sequence features identified in Henry et al observed in any promoter classes? An ideal experiment would be to deplete CarD and reexamine transcription to distinguish between these promoter sets but some effort should be made to categorize and validate that the promoter features associated with different CarD/supercoiling regulation classes.\nAs noted above, we have compared promoter classes and there are no consistent sequence differences. Rather, CarD-repressed relaxation-activated promoters tend to have a slightly better match to the -10 element consensus (Figure 1f and 1g). We’ve also looked carefully at the Henry et al (PMID: 34152199) data. Consistent with our conclusion, the carD promoter is a better match to the promoter consensus than rrnBP1 (Figure 1a of Henry et al). Of even more interest is Henry et al Figure 4. As the reviewer notes above, the authors make a series of promoter mutations around the carD promoter -10 element and spacer region. None of these mutations allow CarD to activate instead of represses. However, the mutations do influence overall carD promoter activity and the degree of repression by CarD. Comparison of Figures 4c and 4d in Henry et al. is particularly striking, as there is a near perfect inverse correlation between overall promoter activity (Figure 4c) and fold-repression by CarD (Figure 4d). This is true despite the different mutations impacting different parts of the promoter. Overall, this seems fully consistent with our conclusion above (CarD repressed promoters tend to be better promoters, but no single feature identifies a CarD repressed promoter). We have also made chimeric promoters with parts of PcarD replacing the equivalent rrnBP1 region (Figure 6). Whilst these changes can increase rrnBP1 activity, and reduce the fold activation by CarD, a promoter subject to repression by CarD is not created.\nThe authors should also consider biochemically testing CarD-associated novobiocin-induced promoters. In these cases, is CarD functioning in synergy or opposition to supercoiling? Without these experiments, the authors should be extremely cautious in generalizing their biochemical result to encompass the full range of possible CarD/supercoiling interactions.\nAs also noted above, we have done this for PcarD. Repression by CarD is synergistic with supercoiling as expected (Figure 5). Indeed, when the PcarD DNA is relaxed, CarD acts as an activator rather than a repressor. This is consistent with our overall model that both negative supercoiling, and CarD, stimulate DNA opening. Usually, this leads to activation. However, at some promoters (on average having a better -10) repression occurs because DNA opening is presumably too stable.\nb. The authors make an argument for -7T/-12T de-enrichment in CarD promoters. However, there are several issues with idea. In figure 1F, I cannot see the difference in the -7T/-12T as reported. The de-enrichment of -7T is well-supported by the literature (Henry et al, PMID: 33168725; 34152199) but T is still an enriched base in the sequence logo presented. The -12 T is clearly enriched as well. Is the interpretation based on pvalue? It does not make sense to quantitatively compare two p-values. I would suggest the bigger differences are that CarD-bound promoters lack -8T and a G at -33. Perhaps a quantitative analysis like in Henry et al (PMID: 33168725) would be more appropriate.\nWe agree with the reviewer, comparing motifs based on p-values could be misleading. We have redesigned the analysis to better identify individual promoter motifs (these are now aligned individually, rather than the entire promoter region being aligned based on TSS position). It is clear in the revised figure panel that CarD bound promoters are de-enriched for -7T. In the new sequence logos, based on the frequency at which different bases occur, rather than p-value, -12T is similarly prevalent in CarD bound and unbound promoters. Even so, we have still investigated the role of position -12T further, since it also has an important, but different, role in DNA opening. Whilst CarD can compensate for a missing -7T, the factor cannot correct promoter defects if -12T is mutated (Figure 5).\nMore worryingly, nothing is ever explored for -12T (or for any other promoter differences). Henry et al (PMID: 33168725) observed that -12T (without -7T) was conserved for 15 promoters activated by CarD in vitro. Further, the CarD promoter itself contains a -12T. What is the explanation for these discrepancies? What is the function, if any for these sequences? How do they relate to the different potential CarD/supercoiling promoter classes (1a, above)? Does -12T have any effect on CarD/negative supercoiling synergy?\nAs noted above, when assessing frequency of occurrence rather than p-value, -12T is similarly prevalent in CarB bound and unbound promoters. Even so, for completeness, we have examined the impact of mutating this position at both the carD promoter and rrnBP1 (Figure 5).\nAt the end of the work, I am convinced that for rrnBP1 both CarD and negative supercoiling are required for activation. However, I have many remaining questions about the overall model.\n2) Why is rrnBP1 be regulated in this fashion? I agree with the authors that synergy is logical, but this operates more like an “AND” logic gate, meaning the effects are not tunable. Is it to quickly shutoff ribosomal synthesis during unfavorable conditions? I feel this idea would be better-supported if there are additional examples of this regulation. Perhaps this lack of synergy is unique to rrnBP1.\nWe’re not sure we agree fully on this point, but we see why the reviewer asks this question; it’s perhaps a consequence of the way the result is presented. We think the data do show tunability. For example, Figures 4b and 4c show rrnBP1 activity across a range of negative supercoiling levels in the presence and absence of CarD. As negative supercoiling gradually increases, more transcript is progressively produced. The promoter responds to CarD levels in a similar way (e.g. Figure 2a of PMID: 33168725). Of course, drastic changes in supercoiling, that may be encountered in certain stress conditions, would allow the promoter to be quickly shut off, as the reviewer notes.\n3) How generalizable is this finding for all R. sphaeroides promoters? I also agree that “the combined impact of CarD and DNA topology must differ according to biological context”. Examining several more promoters and, ideally, in different regulatory classes (novo-activated, novo-inhibited, CarD-bound, CarD-free, etc) is essential for extending the experiments to infer CarD’s effects on individual promoters. This is especially true since CarD is itself is controlled by a negative feedback loop. Whether negative supercoiling at the CarD promoter increases or represses CarD expression will have consequences for the interpretation of the transcriptomic/ChIP data, especially if CarD is rapidly turned over.\nAs suggested earlier in the review, we have now explored the impact of negative supercoiling at an additional relaxation-repressed CarD-activated promoter (PrpsL) and at a relaxation-activated CarD-repressed promoter (PcarD). In all cases, the promoters are supercoiling sensitive, in the expected way (Figures 5 and S6).\nWe have also constructed and tested chimeric promoters based on rrnBP1 (see the new Figure 6). In these experiments, we have swapped parts of the CarD-repressed relaxation-activated PcarD promoter for the equivalent region in rrnBP1. As eluded to above, there is no hallmark sequence change that can convert rrnBP1 into a carD repressed promoter. However, introducing the various PcarD sequences gradually improve the overall activity of rrnBP1 and negates the need for CarD. Importantly, although the changes alter the combinatorial impacts of CarD and supercoiling, these are never removed. Hence, we think it’s reasonable to conclude that most CarD bound promoters are supercoiling sensitive.\nWith respect to the genome-scale experiments, and as expected, novobiocin treatment does indeed lead to increased carD transcription. In the same conditions, transcription from most CarD associated promoters decreases, consistent with our overall conclusion that CarD is primarily and activator during normal growth. Indeed, the impact of supercoiling must be acute if CarD activated promoters are downregulated, despite cells producing more CarD.\n4) How does CarD/RNAP together sense negative supercoiling? Figures 2-3 show that CarD-bound sites are more negatively supercoiled than random sites. However, are these CarD-bound sites more negatively supercoiled than σ93-bound sites?\nWe have now done this analysis and the answer is yes, sites bound by CarD and σ93 are significantly more negatively supercoiled than those bound by σ93 alone. See the new Figure S3.\nOne would imagine that any active TSS must be negatively supercoiled since negative supercoiling promotes promoter melting and RNAP escape.\nWe agree, although suggest adjacent transcription units are also likely to influence this. For example, a promoter between tandem genes could be impacted by positive supercoiling from the upstream transcription unit. Overall, σ93 bound regions, whether associated with CarD or not, are more negative supercoiled than random genomics regions.\nIf CarD-free σ93-bound sites are more negatively supercoiled, CarD could perhaps compensate for the absence of negative supercoiling at promoters rather than require it for binding, which is the model presented here.\nWe did not intend to imply that negative supercoiling is required for CarD binding (at least not in the sense to which we think the reviewer refers). Rather, we think open complex stability (and so stability of the entire DNA-RNAP-CarD ternary complex) is sensitive to supercoiling. Hence, when negative supercoiling decreases, it is not only the binding of CarD that decreases but also σ93. We suspect we did not make this point clearly enough, as reviewer 1 made a similar comment. The text has been altered in several places.\nThis analysis should also be performed again in the novobiocin-treated dataset, as it would be interesting to know if σ93/RNAP remains more bound when CarD is present/absent, which would support the hypothesis that CarD stabilizes open complexes.\nAs noted above, we probably weren’t clear enough on this issue, when CarD binding decreases so does binding of σ93 (e.g. see Figure 3e).\nRelatedly, for Figure 6B, because CarD is associated with σ93 (housekeeping) promoters, this set should be naturally enriched in metabolism and central dogma processes. I’d be curious to see the comparison to the remaining σ93-dependent non-CarD promoters, which are also likely enriched for these functions. Significant differences between these two sets would be more compelling than the comparison to random promoters, which are likely enriched for alternative sigma factor promoters that regulate stress response genes.\nThis is a really good point, and we have done the suggested analysis (see the new Figure 7). When compared to promoters bound by σ93 alone, promoters also bound by CarD are enriched for adjacent gene functions related to central dogma processes. In particular, promoters for genes encoding tRNAs and ribosomal proteins. We appreciate this comment as it allows us to make a more nuanced conclusion.\n5) Based on the Methods, it is not clear how the σ93 and CarD overlapping promoters were selected. Further definition of what “overlap” constitutes would be helpful.\nWe have updated the methods to make this clearer.\nI also wonder where CarD binds at these promoters (e.g. around TSS, -10, or hundreds of bases upstream) and if this information could be used to refine the models for CarD/supercoiling behaviors.\nThe spatial resolution of ChIP-seq, related to the length of sonicated DNA fragments (a few hundred base pairs) is insufficient to distinguish between CarD binding at TSSs compared to -10 elements. Even so, we have quantified the distances between CarD binding peaks and TSSs. The majority are within 100 bp of each other. I.e. the locations are essentially indistinguishable.\n6) Is there a relationship between CarD and the bidirectional promoters noted in Figure 1? Is the hypothesis that CarD should activate these promoters and it should activate both? Is CarD bind to these bidirectional promoters?\nBidirectional promoters are slightly more likely to be CarD associated (see new Figure S2d) and we would predict activation by CarD in both orientations.\n7) Line 147: Is the idea here that more CarD-bound genes are downregulated than CarD-independent? Can this be explained more clearly (e.g. more CarD-bound genes were downregulated)? There also appears to be more genes upregulated for CarD-independent promoters, is this correct?\nThis part of the paper has been substatially reworked. Briefly, a greater percentage of CarD bound promoters were downregulated, but the absolute number is higher for CarD free promoters (as there are more of them). We have revised the figure to make space for the comparison of relaxation-repressed and relaxation-activated promoters. In doing so we have taken out the volcano plots for CarD-independent promoters, since there is potential for confusion.\n8) Line 151-154: I did not understand the data for “Novobiocin predominantly downregulates transcription and this is more pronounced for CarD bound genes”. Is the idea that more CarD-bound genes are downregulated upon treatment? To my eye, it appears that there are more genes downregulated for CarD-free promoters. I also suggest the statement “novobiocin caused significant DNA relaxation, loss of both CarD and σ93 binding, and lower gene expression” should be revised. Novobiocin-induced/DNA relaxation-induced genes have been observed in diverse organisms, and there are many genes (red) that are induced upon DNA relaxation in Figure 3D.\nThe answer here is similar to the above; a greater percentage of CarD bound promoters were downregulated, but the absolute number is higher for CarD free promoters. As noted in response to the prior question, this part of the paper has been reworked.\nMinor comments:\n1) The authors should carefully check over their referencing and reference the primary literature as often as possible:\na. Line 27-28: a review here would be more appropriate, as reference 2 (Zhang et al) were only able to visualize domains 2-4 and do not discuss the role of sigma domains in depth. Reference 3 also does not the primary source to show sigma and -10/-35 contacts.\nb. Line 30: Instead of references 2-4, which are not the references that show sigma domain 2 interacting with -7T/-11A, please cite Feklistov and Darst (PMID: 22136875), Zhang et al (PMID: 23086998), and Zuo and Steitz (PMID: 25866247).\nWe have re-referenced the section related to the above two points.\nc. Line 108: The authors may also want to cite Lal et al (PMID: 27025941), the first report of psoralen use genome-wide in bacteria.\nWe now cite this paper.\n2) Line 46-47: The authors are correct that lack of rotation of RNAP increases supercoiling. However, simple duplex DNA melting by RNAP also leads to torsional strain and consequent twisting of DNA.\nWe have added the word “primarily” to ensure we don’t exclude other processes.\n3) Line 75: Is there some way to distinguish between “active” and “infrequently used” promoters from cappable-seq? Perhaps the studies would provide better motif resolution if the highest-active promoters are selected.\nThis is perhaps not as straightforward of a distinction as it may seem. For instance, there will be many intrinsically poor promoters that give strong signals (i.e. are very active) because activator proteins intervene. Similarly, many intrinsically strong promoters will be infrequently used because of repressors. It’s also not unusual for promoters, occurring by chance, inside genes, to be more active than the canonical promoter upstream of the gene. The adjacent motifs are derived from the top or bottom 10% of promoters, according to cappable-seq signal strength. The motif differences are surprisingly modest, given the differences in promoter activity. This is particularly true for promoters that occur inside genes.\n4) Please renumber the logo (indicating +1) in Figure 1F and mark the correct bases to examine to ease the burden on the reader.\nThe figure panel has been redesigned to also include this suggestion.\nWe than the reviewers again for their supportive comments. Our response to the final concern of reviewer 2 is provided below.\nReviewer #1 (Remarks to the Author):\nThe authors have address my concerns, no further comment. Thank you.\nReviewer #2 (Remarks to the Author):\nThe authors have satisfactorily addressed my previous comments. My only remaining concern is the use of the word “flaw” throughout the manuscript (including the Abstract and Introduction) to describe the lack of consensus among promoter elements. I recommend replacing this term with more precise language—for example, explicitly stating that certain promoters deviate from the canonical −10 consensus or lack specific consensus features such as the −7T. The term “flaw” implies a subjective judgment, whereas a more descriptive formulation would be more precise.\nTo explain our rational, particularly for the abstract, we want to get to the key point quickly. Hence, we only say that CarD aids -10 opening and, in R. sphaeroides, this is particularly important because there is an inherent -10 element defect. If we were to get more specific, and describe the defect, we must also explain that different -10 element bases have different roles, introduce the importance –7T, and discuss base flipping. This is probably too much for the abstract, which is already on the longer side. In the main body of the text, we agree, and were mostly careful, every time this was mentioned, to specify that the defect is missing -7T base. However, we found one incidence, on page 3, where we hadn’t done this and have added extra text.\nReviewer #3 (Remarks to the Author):\nI commend the authors on the extensive and thorough revision. All the new experiments and analyses greatly improve the manuscript, and I have no further comments. Congratulations on the fine work."}, "author_response": ""}]

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