RNA polymerase-mediated regulation of intrinsic antibiotic resistance and bacterial cell division

RNA polymerase, Transcription, Division, Peptidoglycan, Cell wall biosynthesis, 26 Rifampicin, MtrAB Regulon, WalKR regulon, Metabolomics, Transcriptomics, Rifampicin 27 Resistance, Antimicrobial Resistance, Antibiotics, Two -Component Regulatory System, β -28 Lactams. 29 Abbreviations: Tuberculosis (TB), Mycobacterium tuberculosis (Mtb), Multi-drug resistance - 30 (MDR), Extensive-drug resistance (XDR), Total-drug resistance (TDR), RNA Polymerase (RNAP), 31 Rifampin (RIF), Rifampin-sensitive (rifS), Rifampin-resistant (rifR), UDP -GlcNAc (UG), 32 Peptidoglycan (PG), Tricarboxylic acid (TCA) 33 34 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 21, 2025. ; https://doi.org/10.1101/2025.07.20.665812doi: bioRxiv preprint 2 ASBTRACT 35 Rifampin is a frontline antibiotic that inhibits the RNA polymerase of Mycobacterium tuberculosis 36 (Mtb), the causative agent of tuberculosis (TB). Unlike most antibiotics, rifampin has an unusual 37 ability to shorten the duration of treatment needed to cure TB that is not simply explained by its 38 antimicrobial potency. We sought specific secondary effects of rifampin’s inhibition of Mtb RNA 39 polymerase that may mediate this activity. We discovered that rifampin elicited a cell division 40 arrest that was mediated through its inhibition of RNA polymerase. This arrest resulted in a 41 downstream inhibition of the MtrAB two -component regulatory system, a mediator of intrinsic 42 antibiotic resistance in Mtb. This inhibition is broadly conserved in other bacteria and represents 43 a novel form of antimicrobial activity, termed adjunctive sensitization, that can mediate synergy 44 and may contribute to rifampin’s unusual treatment shortening activity. 45

46 Rifampin (RIF) is an essential component of all current frontline treatments for TB, the leading 47 cause of deaths not only due to an infectious but also curable, disease(1). This essentiality is a 48 unique product of both its potent in vitro activity against Mycobacterium tuberculosis (Mtb)(2), 49 the causative agent of TB, and its clinical ability to shorten the duration of treatment needed to 50 achieve a durable, relapse-free cure(3, 4). However, resistance to rifampin, a defining feature of 51 multi- (MDR), extensively- (XDR), and totally-drug (TDR) resistant TB, is on the rise, prompting 52 a need for therapeutically equivalent alternatives(1). 53 Biochemical, structural, and genetic evidence has demonstrated that rifampin specifically binds 54 the beta-subunit of bacterial RNA polymerase (RNAP) and blocks the exit tunnel of nascent 55 transcripts to prevent their extension beyond a trinucleotide length and that this binding is 56 required for antimicrobial activity(5) (Fig 1A). Knowledge of whether this antimicrobial activity is 57 mediated or accompanied by specific secondary physiologic effects or is a non -specific 58 consequence of inhibiting transcription initiation as a whole however remains unaddressed. 59 Studies by Mitchison, for example, specifically showed evidence of continued incorporation of 60 3H uridine and recoverable Mtb mRNA following prolonged exposure to bactericidal 61 concentrations of rifampin in vitro (6) while phenotypic studies revealed specific synergies 62 between rifampin and β -lactam, but not protein synthesis, antibiotics (7). Mutations in RNAP 63 conferring resistance to rifampin have been similarly reported to lead to changes in cell wall 64 structure and bacterial morphology (Table S1). 65 Here, we provide evidence that rifampin -mediated inhibition of RNA polymerase in Mtb elicits 66 specific physiologic changes in cell wall metabolism and division that contribute to its 67 antimycobacterial activity. We further show that these effects are phylogenetically conserved in 68 other bacterial species. These results thus reveal a previously unrecognized, but evolutionarily 69 conserved, functional interaction between transcription and cell division that expands our 70 understanding of the antimicrobial mode-of-action of a well validated molecular target. 71 72 73 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 21, 2025. ; https://doi.org/10.1101/2025.07.20.665812doi: bioRxiv preprint 3

74 Metabolic impact of RNAP inhibition 75 We previously demonstrated that, despite its bulk effect on RNA synthesis, rifampin elicited a 76 specific set of metabolic changes that included a combination of some that were shared by other 77 antibiotics and others that were specific to rifampin (8). We hypothesized that those specifically 78 associated with the antimycobacterial activity of rifampin might serve as a biochemical window 79 into its treatment shortening activity. We therefore compared the metabolic profiles of isogenic 80 rifampin-sensitive (rifS) and -resistant (rifR) Mtb strains during the pre-lethal phase of exposure 81 to rifampin (Fig 1A, S1A). Pathway enrichment analysis of such activity -specific metabolites 82 revealed a statistically significant over -representation of metabolites associated with amino 83 acids, nucleotides, peptidoglycan (PG) biosynthesis, and the tricarboxylic acid (TCA) cycle (Fig 84 1B, S1B, S1C). We validated the functional association of these changes with the inhibition of 85 RNAP by profiling the metabolic response s of rif S and rifR strains of Mtb to two additional 86 structurally and mechanistically distinct inhibitors of its RNAP, fidaxomicin (FDX) and 87 myxopyronin B (MYXO), at similar levels of antimycobacterial activity (Fig 1B, S1D), as well as 88 following partial transcriptional silencing of the  (rpoB) and ’ (rpoC) subunits of RNAP (Fig 1C, 89 1D, S2, S3). We further showed that these changes were not carbon source specific (S4A) and 90 observed in both laboratory-adapted and clinical isolates (S4B). Metabolic tracing studies of Mtb 91 following treatment with rifampin (S5) more specifically revealed accumulations of UDP-GlcNAc, 92 GTP, ATP, and UTP indicative of an increase in PG turnover and arrest of de novo PG 93 biosynthesis (Fig 1E). 94 Transcriptomic impact of RNAP inhibition 95 Seeking further evidence of a specific physiologic impact of rifampin, we analyzed the 96 transcriptomes of rifS and rifR Mtb during the same pre-lethal phase of exposure to rifampin (Fig 97 2A, S6A). Despite its expected bulk inhibition of de novo transcription, treatment with rifampin 98 elicited both increases and decreases in transcript abundance (Fig 2A). We next overlaid the 99 transcriptional response of Mtb to rifampin with that of FDX , a structurally and mechanistically 100 distinct inhibitor of RNAP to define a biologically more specific transcriptional signature of RNAP 101 inhibition. Using an absolute cutoff (log2 fold>1 and pcorr <0.05), we identified an activity-specific 102 transcriptional signature that consisted in an accumulation of 537 and depletion of 344 103 transcripts (Fig 2A, S 6B). Functional enrichment analysis revealed a depletion of genes 104 associated with de novo PG biosynthesis (S6C), consistent with Mtb’s observed metabolic 105 response. Further analysis of this signature revealed a n enrichment of genes encoding and 106 belonging to the mtrAB regulon that was not observed in rifR strains exposed to rifampin (Fig 107 2B, 2 C). The mtrAB regulon consists of a two -component sensor kinase and cognate DNA -108 binding response regulator that was previously shown to regulate peptidoglycan remodeling 109 enzymes required for cell growth and division and reported to mediate both drug tolerance and 110 intrinsic drug resistance (Fig 2D)(9-13). Targeted qPCR analysis of mtrAB and a representative 111 subset of its regulon demonstrated that this repression was specific to RNAP -targeting 112 antibiotics (Fig 2E, S7) and carbon source independent (S8). This repression was additionally 113 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 21, 2025. ; https://doi.org/10.1101/2025.07.20.665812doi: bioRxiv preprint 4 observed in Mtb whose replication had been slowed by hypoxia, acidic pH , or nutrient (PBS) 114 starvation (S9). 115 Effect of RNAP on divisome activity 116 Given the foregoing data, we sought to probe the functional relationship between RNAP activity 117 and cell wall metabolism in replicating Mtb. Prior studies reported a robust but mechanistically 118 unexplained phenotypic synergy between rifampin and cephalosporin antibiotics against Mtb(7). 119 We found that this synergy is also extended to a panel of cell wall-targeting a β-lactam antibiotics 120 (cephradine, cefadroxil, meropenem, and faropenem) that selectively inhibit FtsI ( PbpB or 121 Rv2163c)(14-20), the transpeptidase required for septal peptidoglycan biosynthesis during cell 122 division(21, 22), but not inhibitors of other peptidoglycan biosynthesis, including vancomycin and 123 cycloserine (Fig 3A). 124 Previous work had demonstrated that MtrB , the sensor kinase, localized to the cell septum via 125 interaction with divisome components (FtsI and FtsZ) and that this interaction was required for 126 activation of mtrA and its regulon(23, 24). We, therefore, sought to test if the impact of rifampin 127 on mtrAB regulon activity might be a downstream consequence of impaired septal Z -ring 128 formation (Fig 3B). To do so, we first tested the effect of inhibiting Z-ring formation on rifampin 129 susceptibility by transcriptionally silencing the expression of ftsZ to levels that only mildly slowed 130 but did not arrest growth ( S10). This revealed a selective sensitization to rifampin, but not 131 isoniazid, upon ftsZ silencing (Fig 3C, S10). Similar effects were observed upon mtrA knockdown 132 (Fig 3C, S11). We further found that treatment of Mtb with FtsI inhibitors resulted in increased 133 levels of UDP-GlcNAc (S12). 134 Seeking more direct evidence of the effect of rifampin on septal Z-ring assembly, we conducted 135 single-cell time-lapse microscopy of reporter strains that either expressed fluorescent fusions of 136 ftsZ and rpoB and were exposed to defined pulses of rifampin (S1 3). Owing to resource 137

and a high degree of conservation of both FtsZ and RpoB in Mtb and M. smegmatis 138 (89.61% amino acid identity/94% similarity and 99% amino acid identity/95% similarity, 139 respectively), we conducted these experiments in M. smegmatis(25, 26) (S14 and S15). These 140 studies demonstrated that rifampin selectively inhibited Z-ring formation in cells that had recently 141 or not yet initiated cell division while allowing the remainder to complete the process of 142 chromosomal segregation and cytokinesis (Fig 3D and E, upper panels). In contrast, treatment 143 with isoniazid revealed no such effect on Z-ring formation (Fig 3D and E, lower panels), though 144 both isoniazid and rifampin slowed cell elongation (Fig 3E, 3F). We further quantified the fraction 145 of cells with clear FtsZ-mCherry2B foci at different stages of cell division and found that rifampin 146 exposure almost completely collapsed nascent Z -ring formation within 2 hours of exposure, 147 whereas isoniazid permitted continued Z-ring assembly (Fig. 3F). 148 Conservation of RNAP-MtrAB interaction 149 The biological centrality and evolutionary conservation of both RpoB and FtsZ across bacteria 150 taxa further led us to wonder if the foregoing effects of RNAP inhibition might also be conserved 151 in other bacterial species (S1 5). Focusing specifically on bacteria genera annotated to encode 152 orthologs (WalKR/YycFG/VicKR) of the MtrAB two -component regulatory system (Fig 4A and 153 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 21, 2025. ; https://doi.org/10.1101/2025.07.20.665812doi: bioRxiv preprint 5 B), we characterized the molecular and phenotypic effects of rifampin against Bacillus subtilis 154 (BS) and Staphylococcus aureus (SA). Both organisms encode orthologs of rpoB and ftsZ with 155 greater than 55% amino acid identity and 75% similarity (S15) and MtrAB orthologs exhibiting 156 greater than 45% identity and 66% similarity (S16) with similar roles in cell wall remodeling, 157 division, and antibiotic resistance (27-31) (Table S2). Moreover, exposure to rifampin, 158 fidaxomicin and myxopyronin B in both organisms demonstrated evidence of increased levels of 159 UDP-GlcNAc (Figure 4C, S17, S18), repression of their respective mtrAB orthologs (Figure 4D), 160 and phenotypic synergy with cephalosporin and carbapenem antibiotics (Figure 4E). 161

162 Owing to the biological centrality of transcription and well -defined molecular mechanism of 163 biochemical inhibition, studies of rifampin have historically focused on its quantitative impact on 164 rates of transcription initiation as a whole, rather than the specific physiologic consequences of 165 that inhibition(5). Our studies of both rifamycin and non-rifamycin inhibitors of RNA polymerase 166 against rifampin-sensitive and -resistant strains now shed new light on the latter. Despite its 167 genome-scale impact, we discovered that inhibition of transcription initiation, or genetic silencing 168 of the β - or ’ subunits of RNA polymerase, elicited physiologically specific and reversible 169 metabolic changes that tracked with its antimycobacterial activity, and were associated with both 170 transcriptional and phenotypic inhibition of the mtrAB two-component regulatory system (Fig 5). 171 This inhibition effectively sensitizes Mtb (not completely killed by inhibition of transcription) to a 172 number of secondary host - and drug-imposed stresses, and, in doing so, reveals a previously 173 unrecognized form of antimicrobial impact, adjunctive sensitization, that expands the scope of 174 rifampin’s antimicrobial mode-of-action. 175 Among currently approved TB drugs, rifampin is distinguished by its ability to shorten the 176 duration of treatment needed to achieve a durable, relapse-free cure(32), an activity not simply 177 explained by standard measures of its in vitro antimycobacterial potency(33, 34). This activity 178 has instead been ascribed to a combination of its ability to penetrate caseous or necrotic lesions 179 and kill the non - or slowly replicating Mtb subpopulations therein (35-37). However, the 180 mechanistic basis of this activity remains incompletely defined. That rifampin’s impact on mtrAB 181 was both carbon source independent and extended to non- or slowly replicating Mtb populations 182 highlights a novel mechanistic effect of high potential therapeutic importance. Treatment 183 shortening aside, the potential for this activity to inform the development of rational mechanism-184 based drug combinations warrants further study. 185 Translational potential notwithstanding, this work extends our understanding of the fundamental 186 biology of RNA polymerase. Previous studies of the mtrAB and walKR systems had 187 demonstrated its interaction with and regulation of genes involved in cell division and cell wall 188 metabolism, many of which include enzymes involved in peptidoglycan remodeling (10-12, 29, 189 38, 39). In Mtb and other bacteria, MtrAB or WalKR activity has been shown to depend on septal 190 localization and therefore be functionally associated with Z -ring formation and cell division (23, 191 24, 38-41). As discussed previously, mutations conferring resistance to RIF (in the RpoB subunit 192 of RNAP) in other microbes have been reported to changes in cell wall structure (42), bacterial 193 morphology(42-45), virulence(46) as well as resistance to various drugs targeting peptidoglycan 194 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 21, 2025. ; https://doi.org/10.1101/2025.07.20.665812doi: bioRxiv preprint 6 biogenesis (e.g., vancomycin and daptomycin) (47-50) while resistance to some β -lactams and 195 cephalosporins has conversely been reported to be associated with mutations in the rpoB and 196 rpoC subunits of RNAP(7, 42, 43, 51-54) (55, 56) (Table S1). Our work now extends this biology 197 further to include an even more upstream and evolutionarily conserved role of RNA polymerase 198 and its state of transcriptional competence as a potential checkpoint regulator of cell division. 199 Growing evidence has implicated a broader range of physiologic roles for RNA polymerase 200 beyond its activity as a bulk enzymatic catalyst of RNA synthesis(43, 51, 57). Once focused on 201 the primary drug-target interaction, modern drug development has expanded in scope to include 202 studies of specific secondary or downstream consequences of the drug-target interaction. Such 203 studies have helped increase knowledge of the mechanistic basis for drug activity, and in doing 204 so, reveal additional new potential targets whose inhibition could mimic the activity of the drug 205 itself and/or slow/prevent the emergence of resistance. Though less well recognized, such 206 studies have also created a window into the normal physiologic functions of the targets they 207 inhibit. 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 21, 2025. ; https://doi.org/10.1101/2025.07.20.665812doi: bioRxiv preprint 7 FIGURE LEGENDS 228 Figure 1: Inhibition of RNAP affects peptidoglycan metabolism of Mtb: (A) Experimental 229 scheme used to define Mtb’s pre -lethal response to RNAP inhibitors (RNAPi). The inset shows 230 the domain structure of RNAP highlighting the binding site of different RNAPi. Rifampin (RIF) 231 binds at the exit channel. Fidaxomicin (FDX) binds at the base of the RNAP clamp. Myxopyronin 232 B (MYXO) binds at the switch region of RNAP. (B) Heatmap showing statistically significant log2 233 fold changes (FC) in metabolite levels following a prelethal 10x MIC exposure to RNAP inhibitors 234 both in rifS and rifR (S450L and H445Y) strains for 24 hrs. (C) Schematic of CRISPRi-mediated 235 genetic inhibition of RNAP. sgRNA targeting rpoB, rpoC, and non -targeting (negative control) 236 were electroporated into Mtb -Erdman (rifS and rifR) and gene knockdown phenotype was 237 screened by growing mutants with and without anhydrotetracycline (ATc). Samples were 238 collected for metabolomic profiling. (D) Heatmap highlighting log 2 FC of metabolites upon 239 CRISPRi-mediated silencing of RNAP subunits (RpoB and RpoC). Partial depletion of rpoB and 240 rpoC was achieved using anhydrotetracycline (ATc) for 4 days, resulting in similar metabolic 241 signatures as observed upon RNAP inhibitor administration (S2, and S3). Lower row displays 242 matching metabolic changes of rifS-Mtb strain after RIF treatment. All results are representative 243 of biological triplicates and two independent experiments. (E) Diagrammatic illustration showing 244 the impact of RNA polymerase inhibition on peptidoglycan precursor and ribonucleotide pools in 245 Mtb (top panel). Impact of rifampin (10x MIC dose; 0.5 µg/ml for 24 hrs) on pool sizes of GTP, 246 UTP, GlcN-1P, and UDP-GlcNAc in Mtb that had been prelabeled with 13C and transferred to 12C 247 upon rifampin treatment. p-values were calculated using non-parametric t-test (for GlcN-1-P and 248 GTP) or two-way ANOVA mixed model (for UTP and UDP-GlcNAc). ****p<0.0001, and **p<0.01. 249 Error bars denote s.e.m. 250 Figure 2: Transcriptomic profiles of Mtb RNAP inhibition by rifampin and fidaxomycin: (A) 251 Venn diagram showing the differentially expressed and overlapping genes (in overlay) upon 252 exposure of 10x MIC dose of rifampin and fidaxomicin to rifS strain (Mtb-H37Rv) for 24 hrs. Cutoff: 253 log2 fold changes >1 and < -1; padj value <0.05. (B) Donut graph shows the functional enrichment 254 analysis of significant overlapping genes with known functions. Almost 27.8% of genes belong to 255 the cell wall-related mechanism and 2.8% of total genes belong to mtrAB regulon (see overhang) 256 (C) Heatmap displaying log2 fold changes of representative members of the mtrAB regulon upon 257 inhibition of RNAP both in rifS and rifR strains. Stars show the range of padj values for the most 258 significant differential expressions calculated using the Benjamini -Hochberg method. p values: 259 **< 0.05; ***<0.0005. (D) Depiction of the mtrAB regulon and its function in bacteria. 260 Autophosphorylation or external stimuli (unknown) activates the MtrB sensor kinase which results 261 in the downstream phosphorylation -mediated activation of its cognate response regulator MtrA 262 and expression of almost 62 genes participating in PG remodeling, maintenance, cell shape, 263 elongation, division, and intrinsic antimicrobial resistance (AMR). (E) Real-time qPCR results 264 showing log 2 fold changes of some key genes from mtrAB regulon after treatment of different 265 RNAP inhibitors and antibiotics targeting cell wall or respiration. Centroid linkage hierarchical 266 cluster analysis was done using Gene Cluster 3.0 software. All results are representative of 267 biological triplicates and two independent experiments. 268 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 21, 2025. ; https://doi.org/10.1101/2025.07.20.665812doi: bioRxiv preprint 8 Figure 3: Impact of RNAP inhibition on divisome assembly: (A) Heatmap showing the most 269 synergistic area score of rifampin combined with antibiotics of differing modes of action. Most 270 synergistic area scores and Zero Interaction Potency (ZIP) scores were measured using 271 SynergyFinder (https://synergyfinder.fimm.fi/). Scale: less than -10: antagonistic, from -10 to 10: 272 additive, larger than 10: synergistic. All experiments were done in biological triplicates. (Table 273 S3). (B) Graphic showing the impact of localization of the divisome components FtsZ (1) and FtsI 274 (2) on the activation of MtrBA (3) system and their role in peptidoglycan biosynthesis and 275 remodeling. Numbers (1,2, and 3) show the sequence of molecular events. (C) Dose-response 276 graphs showing the minimum inhibitor concentrations of rifampin (RIF) and isoniazid (INH) upon 277 partial depletion of ftsZ and mtrA in Mtb -Erdman. Gene knockdown was achieved using 278 anhydrotetracycline (ATc) regulated CRISPRi constructs expressing respective sgRNA. (D-F) 279 Subcellular localization dynamics of FtsZ and RpoB following RIF and INH treatment. Dynamics 280 of FtsZ and RpoB were tracked in M. smegmatis mc2155 cells expressing fluorescent fusions of 281 RpoB (rpoB-msfGFP) and FtsZ (ftsZ-mCherry2B) (S13). Bacteria were grown for 9 hrs followed 282 by 4 hrs of drug treatment (20µg/ml RIF and 25µg/ml INH) and 2 hrs of recovery. Spatial-temporal 283 expression dynamics of the fluorescent proteins (in yellow color) were measured using Fiji and 284 customized Python scripts. (D) Representative microscopy images illustrating Z -ring dynamics 285 upon RIF or INH treatment. (E) Single bacteria kymographs highlighting differences in Z -ring 286 formation between RIF and INH treated cells. The upper left panel shows that RIF inhibits FtsZ 287 localization, whereas the right panel reveals that RIF can also halt FtsZ recruitment even after 288 cell division has initiated. (F) Bar charts showing the percentage of cells with detectable FtsZ foci 289 at each time point. Scale bar: 5µm. Results represent biological triplicates across two 290 independent experiments, except for time -lapse imaging, which represents two independent 291 experiments with biological duplicates. 292 Figure 4: Phylogenetic conservation of the RNAP -cell wall -division relationship: (A) A 293 phylogenetic tree of MtrA orthologs in different bacterial species was created using 294 https://itol.embl.de/. (B) Graphic presenting the conserved two -domain structure of MtrA and its 295 orthologs. Conserved aspartic acids on the N -terminal domain and tyrosine on the C -terminal 296 domain are targets of the sensor kinase (MtrB or WalK or YycF), while threonine on the C-terminal 297 domain is primarily targeted by a serine -threonine protein kinase (PknB or PrkC). (C) Heatmap 298 representing log 2 FC of key metabolites in B. subtilis and S. aureus upon treatment with RIF, 299 FDX, and MYXO for 20 min (S17). Respective metabolites from rifS-Mtb treated with RIF are also 300 presented for as reference. (D) Real-time qPCR estimation showing the levels of two-component 301 systems upon RIF treatment for B. subtilis and S. aureus walKR . A significant depletion is 302 consistent with changes in the corresponding MtrAB regulon of Mtb. (E) Distribution of most 303 synergistic area score showing the extent of synergy of RIF with different antibiotics both in B. 304 subtilis and S. aureus. Lower segment is showing the overlapping antibiotic synergy score from 305 rifS-Mtb strain. White cells indicate an absence of corresponding antibiotics. All results are 306 representative of biological triplicates and two independent experiments. 307 Figure 5: Generalized model of RNAP mediated regulation of cell wall biosynthesis and 308 bacterial division: Under normal growth conditions, bacteria maintain a state of metabolic 309 homeostasis that provides a continuous supply of nucleotides to sustain transcription and cell 310 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 21, 2025. ; https://doi.org/10.1101/2025.07.20.665812doi: bioRxiv preprint 9 wall biosynthesis. Perturbations of transcription (either antibiotic or genetic) result in diminished 311 turnover of nucleotides (UTP and GTP) that may affect de novo peptidoglycan biosynthesis and 312 septa formation. The resulting imbalance leads to septum delocalization, consequently 313 suppressing the regulon of the bacterial two component system (MtrAB/ YycFG/ WalKR or VicKR) 314 and disrupting cell wall biosynthesis, elongation, bacterial division, and intrinsic antimicrobial 315 resistance. 316 317 318 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 21, 2025. ; https://doi.org/10.1101/2025.07.20.665812doi: bioRxiv preprint 10 319 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 21, 2025. ; https://doi.org/10.1101/2025.07.20.665812doi: bioRxiv preprint 11 320 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 21, 2025. ; https://doi.org/10.1101/2025.07.20.665812doi: bioRxiv preprint 12 321 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 21, 2025. ; https://doi.org/10.1101/2025.07.20.665812doi: bioRxiv preprint 13 322 323 324 325 326 327 328 329 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 21, 2025. ; https://doi.org/10.1101/2025.07.20.665812doi: bioRxiv preprint 14 Figure 5 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 21, 2025. ; https://doi.org/10.1101/2025.07.20.665812doi: bioRxiv preprint 15

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We thank Jenny Zhaoying Xiang and Adrian Y Tan from the Genomics core of Weill 539 Cornell Medicine for their constant support for RNA sequencing and genomics analysis. We 540 acknowledge Dirk Schnappinger, and Jeremy Rock for providing CRISPRi strains of ftsZ and mtrA. 541 We also thank Michael DeJesus, Nicholas Poulton, Shuqi Li, Dr. Cara Boutte, Kristin Burns-Huang, 542 Allison Fay, Daniel Fitzgerald, David Alland, David Sherman, Murty Madiraju, and Dr. 543 Dhandayuthapani S. for their help with different clinical, lab, and drug -resistant strains. Thanks to 544 Jennifer Herrmann from Helmholtz Institute for Pharmaceutical Research Saarland, Germany for 545 generously providing Myxopyronin B. 546 Funding: 547 National Institutes of Health grant: U19 AI162584 548 Bill & Melinda Gates Foundation: BMGF INV-004709 549 Author contributions: 550 Conceptualization: V.S. and K.Y.R. 551 Methodology: V.S., K.Y.R., J.Z. 552 Investigation: V.S. and K.Y.R. 553 Visualization: V.S. and K.Y.R. 554 Funding acquisition: K.Y.R. 555 Project administration: K.Y.R. 556 Supervision: K.Y.R. 557 Writing – original draft: V.S. and K.Y.R. 558 Writing – review & editing: V.S., K.Y.R., Y.P, J.D.H., and E.J.R. 559 Competing interests: The authors declare that they have no competing interests. 560 Data and materials availability: Raw metabolomics data are available at Metabolomics 561 Workbench under the project ID PR002179. Additionally, the raw transcriptomics data are 562 deposited to the NCBI Short Read Archive with the BioProject ID PRJNA1181268. All code used 563 to generate cell kymographs and representative micrographs was deposited on GitHub and 564 available at https://github.com/jzrolling/FtsZ_kymographs. Other data are available in the 565 supplementary materials. 566 567 568 569 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 21, 2025. ; https://doi.org/10.1101/2025.07.20.665812doi: bioRxiv preprint