The DUF1013 Protein Trcr Tracks with RNA Polymerase to Control the Bacterial Cell Cycle and Protect Against Antibiotics

The DUF1013 protein TrcR tracks with RNA polymerase to control the bacterial cell cycle and protect against antibiotics

Marie Delabya,1, Lydia M. Varesiob, Laurence Degeorgesa, Sean Crossonc, and Patrick H. Violliera,2

aDepartment of Microbiology and Molecular Medicine, Faculty of Medicine/Centre Médical Universitaire, University of Geneva, 1211 Genève 4, Switzerland; bCommittee on Microbiology, University of Chicago, Chicago, IL 60637; and cDepartment of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824

Edited by Graham C. Walker, Massachusetts Institute of Technology, Cambridge, MA, and approved December 23, 2020 (received for review May 25, 2020) How DNA-dependent RNA polymerase (RNAP) acts on bacterial cell Transcriptional control is not only used for stress adaptation cycle progression during transcription elongation is poorly inves- but also to orchestrate changes in gene expression systemically, tigated. A forward genetic selection for Caulobacter crescentus cell for example, during cell cycle progression in the free-living cycle mutants unearthed the uncharacterized DUF1013 protein alphaproteobacterium Caulobacter crescentus and in its symbi- (TrcR, transcriptional cell cycle regulator). TrcR promotes the accu- otic relative Sinorhizobium meliloti (14–18). Many alphaproteo- mulation of the essential cell cycle transcriptional activator CtrA in bacteria divide asymmetrically (Fig. 1A) (19) and use conserved late S-phase but also affects transcription at a global level to pro- transcriptional regulatory proteins to synchronize gene expres- tect cells from the quinolone antibiotic nalidixic acid that induces a sion changes at discrete stages in the cell cycle (17). In the case multidrug efflux pump and from the RNAP inhibitor rifampicin of C. crescentus, division yields two progeny cells that have dis- that blocks transcription elongation. We show that TrcR associates with promoters and coding sequences in vivo in a rifampicin- tinct polar appendages, transcriptional programs, and develop- dependent manner and that it interacts physically and genetically mental fates (15, 17, 20). The capsulated stalked (St) cell engages with RNAP. We show that TrcR function and its RNAP-dependent in DNA replication (DNA Synthesis phase [S-phase]) and cell chromatin recruitment are conserved in symbiotic Sinorhizobium division. By contrast, the swarmer (Sw) daughter cell lacks a MICROBIOLOGY sp. and pathogenic Brucella spp. Thus, TrcR represents a hitherto capsule, but it is motile, piliated, and in a (G1-like) nonreplicative unknown antibiotic target and the founding member of the state. This state is transient, and cell division requires the Sw cell DUF1013 family, an uncharacterized class of transcriptional regu- to first differentiate into a St cell. During this differentiation, the lators that track with RNAP during the elongation phase to pro- pili and flagellum that are located at the old pole of the Sw cell are mote transcription during the cell cycle. replaced by a polar stalk while replication competence is acquired. This polar remodeling and chromosomal replication are orches- TrcR | RNA polymerase | Caulobacter | transcription | Brucella trated by a genetic circuit comprising conserved transcriptional repressors and activators, acting in pairs to sequentially activate ontrol of transcription is key to ensure correct cell survival cell cycle–controlled promoters (17). Cand proliferation. By modulating gene expression, bacteria adapt in response to environmental change and also undergo Significance developmental processes in the absence of an exogenous trigger. The main component of the transcription machinery is the Control of transcription is fundamental to cell cycle progression multisubunit DNA-dependent RNA polymerase (RNAP) (1, 2). and the generation of dispersal cells in bacteria. Several cell While promoter DNA sequences, subunits of RNAP, small li- cycle regulatory proteins controlling transcription initiation gands, and transcription factors influence transcription at various have been identified in the alphaproteobacterium Caulobacter stages, the main regulatory step resides in promoter recognition crescentus, a model system for cell cycle studies. Our screen for and the initiation of transcription by the RNAP holoenzyme C. crescentus cell cycle mutants unearthed an uncharacterized (3–8). In order to initiate transcription, RNAP core enzyme must protein, TrcR, as the founding member of the conserved associate with a sigma factor that will direct RNAP to specific DUF1013 family of proteins. TrcR and its orthologs associate promoters and then promote the transition into the open (un- with initiating and elongating RNA polymerase (RNAP), and wound) promoter-RNAP complex (9). In most bacteria, during the association with elongating RNAP is prevented upon ad- normal growth conditions, this role is fulfilled by the house- dition of the RNAP inhibitor rifampicin. TrcR-deficient cells keeping sigma factor, such as σ70 of Escherichia coli, which rec- suffer from pleiotropic cell cycle defects, including an insuffi- ognizes most promoters. Alternatively, imbalances arising from ciency in the master cell cycle regulator CtrA, and antibiotic stress exposure may be remedied by alternative sigma factors sensitivity that are suppressed by mutations in RNAP. (10), redirecting RNAP to promoters of stress genes. Transcription initiation can also be modulated by other fac- Author contributions: M.D., L.M.V., S.C., and P.H.V. designed research; M.D., L.M.V., and L.D. performed research; M.D. and L.M.V. contributed new reagents/analytic tools; M.D., tors. Specific DNA sequences upstream or within the RNAP L.M.V., S.C., and P.H.V. analyzed data; and M.D., L.M.V., S.C., and P.H.V. wrote the paper. binding site may be recognized by other types of transcription The authors declare no competing interest. σ70· factors that subsequently recruit or stabilize the RNAP com- This article is a PNAS Direct Submission. plex. Recently, other RNAP-associated proteins such as RbpA Published under the PNAS license. (11) and CarD from Mycobacterium tuberculosis have been shown 1Present address: Département de microbiologie, infectiologie et immunologie, Faculté to control housekeeping transcription initiation by binding and de Médecine, Université de Montréal, Montréal, QC H3T 1J4, Canada. stabilizing the RNAP holoenzyme open complex (12, 13). In 2To whom correspondence may be addressed. Email: [email protected]. E. coli, DksA together with the conserved alarmone (p)ppGpp This article contains supporting information online at https://www.pnas.org/lookup/suppl/ also impacts open complex stability on many promoters, ultimately doi:10.1073/pnas.2010357118/-/DCSupplemental. causing a global change in transcription patterns (7, 8). Published February 18, 2021.

PNAS 2021 Vol. 118 No. 8 e2010357118 https://doi.org/10.1073/pnas.2010357118 | 1of11 Downloaded by guest on September 24, 2021 D A 107-140 HvyA NS264 NS245 or NS133 G1 Sw 237 aa G1 G1 S S DUF1013 S S St HTH 107-140 20 aa Capsule E 1 2 1231. WT 020406080 100 120140160 (min) 2. ΔtrcR 3. NS245 (trcR::Tn) 456 TrcR NS133 NS264NS264 4. WT pMT335-empty 5. ΔtrcR pMT335-empty 6. NS245 pMT335-empty CtrA 789 3 7. WT pMT335-trcR 8. ΔtrcR pMT335-trcR B DNA content 9. NS245 pMT335-trcR 1,5k F WT NS245 ΔtrcR WT ΔtrcR NS245 1,0k (trcR::Tn)

Count 500

0 1n 2n 1n 2n 1n 2n C 1 2 3 1 2 3 1. WT TrcR CtrA 2. ΔtrcR 3. NS245 PilA MreB (trcR::Tn) NS264 NS133 ΔtrcR ΔtrcR CC_0164 rpoBP642L rpoBP575S

Fig. 1. Identification and phenotypic characterization of trcR mutant cells. (A) Schematic of C. crescentus division and morphogenesis along cell cycle and immunoblots showing steady-state levels of CtrA and TrcR during cell cycle progression of a synchronized WT population (time in minutes refers to the release of purified swarmer [G1] cells into M2G). During the Caulobacter cell cycle, capsulation is negatively regulated through the expression of hvyA that prevents capsulation in the Sw cell and is under the control of the transcriptional regulator CtrA. TrcR abundance peaks at the G1- to S-phase transition just prior the accumulation of CtrA. (B) DNA content (FL1-A channel) quantification, determined by FACS analysis, was performed during exponential growth phase in PYE. ΔtrcR and trcR::Tn populations show a strong decrease in G1 cell number (pink arrow) and cells that accumulate more than two chromosomes compared to WT cells (orange arrow). (C) The immunoblots on the left show the loss of the TrcR protein in ΔtrcR and trcR::Tn (NS245) cells. The immunoblots on the right show the reduction of CtrA and PilA in trcR mutant cells. MreB actin and CC_0164 serve a loading control for immunoblots. (D) Domain organization predicted for TrcR: DUF1013 is indicated in light blue, the putative helix-turn-helix domain within residues 107 through 140 is indicated in dark blue, and the predictive tertiary structure of this region based on structure homology modeling is shown on the right. Identified Tn-insertions in strains NS133 (NS245) and NS264 are indicated in red. (E) Motility (0.3% agar) plates inoculated with WT, ΔtrcR, and trcR::Tn (NS133, NS264, and NS245) (Left) and derivatives harboring empty vector (pMT335) or pMT335-trcR (Right). (F) Phase contrast light microscopy images of WT, ΔtrcR, trcR::Tn cells, and suppressor mutants during exponential growth in PYE. (Scale bar, 2 μm.)

CtrA is an essential cell cycle transcriptional regulator that analysis revealed three mutants carrying a HyperMu Tn insertion promotes the G1-state and directly regulates ∼180 promoters (NS264 and NS133/NS245) in the same uncharacterized gene, (21–24). CtrA is present and active during G1-phase, then pro- CCNA_03401 (CC_3292, named here trcR, Fig. 1 D, Left). The teolytically removed during the G1→S transition and reac- trcR gene is predicted to encode a cytosolic protein of 237 resi- cumulates as the cell prepares for division (25) (Fig. 1A). CtrA dues within a single DUF1013. The DUF1013 is highly con- directly activates promoters of many cell cycle–regulated tran- served among the alphaproteobacteria, and it has been described scripts (14, 16), for example, those encoding flagellar and pilus as one of the signature proteins for the alphaproteobacterial subunits, as well as an inhibitor of capsulation (HvyA) in G1- lineage (29). HHpred (30) analysis predicts a putative helix- phase (20). Mutations that perturb cell cycle progression, in- turn-helix in the center of the DUF1013 from residues 107 to cluding those that reduce CtrA activity or abundance, typically 140 in TrcR (Fig. 1 D, Right). While the Tn insertion in NS264 cause a reduction in the number of G1 cells in the population maps to codon 72 (Fig. 1D), the Tn inserted at codon 150 in and therefore also curb the overall motility of the population on mutant isolates NS245/NS133. Immunoblotting experiments with motility (soft agar) plates (21, 23, 26, 27). polyclonal antibodies to TrcR revealed that the trcR gene Here, we report the identification of an uncharacterized product is no longer present in trcR::Tn cells (Fig. 1 C, Left)orin alphaproteobacterial “signature” protein (dubbed TrcR [tran- cells in which we inactivated trcR by an in-frame deletion (ΔtrcR). scriptional cell cycle regulator]) that regulates cell cycle and The motility defect of trcR mutants is restored upon expression of motility in C. crescentus through its domain of unknown function TrcR from the vanillate-inducible Pvan promoter on a plasmid 1013 (DUF1013). Our studies show that loss of TrcR leads to (pMT335–trcR,Fig.1E). Furthermore, immunoblotting of extracts global reduction in transcript levels, diminished abundance of from synchronized wild-type (WT) cells showed that TrcR is CtrA, and crippled promoter activity of the ctrA gene and many present throughout the cell cycle, peaking in abundance during the CtrA targets. We find that TrcR tracks with active RNAP, unless G1→S transition or shortly thereafter (Fig. 1A), consistent with perturbed with rifampicin (Rif), and that mutations in rpoB com- the surge in trcR transcript levels and TrcR translation (14, 16, 31). pensate for the loss of TrcR, including an associated sensitivity to TrcR abundance begins to drop as CtrA levels rise (Fig. 1A)(25), Rif and first-generation quinolone antibiotics. Thus, TrcR inte- raising the possibility that TrcR is involved in regulating events in grates cell cycle and antibiotic stress control with the general early S-phase when cells undergo major transcriptional and transcriptional machinery, a function that is conserved from non- translational reprogramming. pathogenic to pathogenic alphaproteobacterial genera. Pleiotropic Defects of trcR Mutant Cells. Comparison of WT and Results trcR mutant cells grown in peptone yeast extract (PYE) broth A Screen for Cell Cycle Mutants Identifies TrcR, an Uncharacterized revealed that trcR mutant cells grow slower than WT cells (SI DUF1013 Protein. To uncover unknown cell cycle regulators of C. Appendix, Fig. S1A), accumulate multiple chromosomes (deter- crescentus, we probed our transposon (Tn) library of motility mined by FACS analysis, Fig. 1B), and form elongated and ab- mutants (28) by fluorescence-activated cell sorting (FACS) for errantly pinched chains of cells by phase contrast microscopy defects in the production of G1 cells (Fig. 1B). This secondary (Fig. 1F). Based on the finding that the G1 cell and motile

2of11 | PNAS Delaby et al. https://doi.org/10.1073/pnas.2010357118 The DUF1013 protein TrcR tracks with RNA polymerase to control the bacterial cell cycle and protect against antibiotics Downloaded by guest on September 24, 2021 population (Fig. 1 B and E) is reduced in trcR mutant cultures direct control of CtrA (Fig. 2B), it also revealed a general effect and the aforementioned reduced overall motility on soft agar of the ΔtrcR mutation on transcript levels, changing the steady- plates, we considered the possibility that the master regulator of state levels with >500 transcripts at least twofold, with 220 the G1 phase is affected by mutation of trcR. Indeed, immuno- transcripts being down-regulated and 329 up-regulated in ΔtrcR blotting revealed reduced CtrA levels in trcR mutants (Fig. 1C) versus WT cells (Fig. 2A and Supplementary Dataset 1). Thus, and diminished expression of genes that are directly activated by TrcR functions as a global regulator of transcript levels in C. CtrA, such as the gene encoding the PilA pilin subunit (Fig. 1C) crescentus. or the HvyA negative regulator of capsule formation as determined by promoter probe assays (SI Appendix, Fig. S1 B and C). TrcR Protects against Antibiotics and Functionally Interacts with Normally, G1 cells are noncapsulated and can therefore be in- RNAP. Since previous transposon mutagenesis followed by deep fected by the S-layer–specific phage ϕCr30 (20), but trcR mutant sequencing (Tn-Seq) analysis had concluded that trcR is essential cells are predominantly (constitutively) capsulated and resistant for viability in C. crescentus (32), we wondered if ΔtrcR cells to ϕCr30 as determined by phage spot and cellular buoyancy might be sensitive to antibiotics and thus unable to grow on assays (SI Appendix, Fig. S1 B and C and Supplementary Results). plates containing the quinolone antibiotic nalidixic acid (Nal), To probe for a global transcriptional defect in the absence of which was used to counter select E. coli donor cells delivering the TrcR, we compared the transcriptome between exponentially Tn for the Tn-Seq analysis. It should be noted that the trcR::Tn growing WT cells and ΔtrcR cells by RNA deep-sequencing mutant clones isolated in this study arose from Tn mutagenesis (RNA-Seq) (Fig. 2A). While this analysis confirmed the robust delivered by electroporation (28) without Nal on selective media. down-regulation of several G1-specific transcripts that are under Although our trcR mutant cells clearly grow slower than WT cells

A ΔtrcR vs WT RNA-Seq B 250 gene ID gene Name log2(FC)

200 CCNA_02061 CC_1982 -2.755425 CCNA_00835 fljN -2.699880 150 CCNA_02931 flgE -2.525345 100 CCNA_00166 hvyA -2.135979 -log10(FDR) CCNA_00094 hfsJ -1.912278

50 MICROBIOLOGY CCNA_00948 sciP -1.308740 0 CCNA_03043 pilA -1.008382 -6 -4 -2 0 2 4 Fold Change (log2) D pMT335- PYE + Gent + Van C PYE PYE + Nal20 empty WT WT trcR

ΔtrcR empty ΔtrcR trcR::Tn trcR 10-fold dliution series

NAL empty WT NOR UBN trcR 20 CIP

empty + Nal ΔtrcR trcR WT ΔtrcR trcR::Tn (NS245) 10-fold dliution series E PYE PYE + Nal20 F 0.3% agar a: WT ab b: ΔtrcR

c: ΔtrcR rpoBP642L cd

d: Δ trcR rpoBP575S 10-fold dliution series G WT ΔtrcR ΔtrcR rpoB P642LΔtrcR rpoB P575S

Novobiocin Rifampicin 15 μg 30 μg

Fig. 2. TrcR regulates transcriptome and antibiotic resistance. (A) Volcano plot showing the global changes in transcript levels of ΔtrcR cells versus WT cells grown in PYE in exponential phase. The abundance of 553 transcripts is affected more than twofold in the ΔtrcR cells compared to WT. All transcripts retained during the analysis are plotted. Each circle represents one transcript. The log2 fold change in ΔtrcR versus WT is represented on the x-axis. The y-axis shows the −log10 of the false discovery rate (FDR) value. Red dots show the genes that have an FDR value of 0.05, and gray lines indicate twofold changes in gene expression. (B) Table showing specific CtrA-dependent transcripts down-regulated in the ΔtrcR cells as determined by RNA-Seq (Supplementary Dataset 1). (C) EOP assay of WT and trcR mutant cells on plates with and without Nal (20 μg/mL). The plates below show antibiotic sensitivity tests by antibiotic disk diffusion assays with discs containing the quinolones Nal (NAL), flumequine (UBN), norfloxacin (NOR), and ciprofloxacin (CIP). The scheme on the Left shows the arrangement of the discs. (D) EOP assays with WT and ΔtrcR cells harboring pMT335–empty or pMT335–trcR on PYE plates supplemented with gentamycin and vanillic acid with or without Nal (20 μg/mL). (E) EOP assays of two ΔtrcR Nal-resistant suppressor mutants harboring mutations in rpoB (rpoBP642L and rpoBP575S), encoding the β-subunit of the RNAP, compared to WT and ΔtrcR cells on PYE plates and PYE with Nal (20 μg/mL). (F) Motility assays on 0.3% agar with WT, ΔtrcR, ΔtrcR rpoBP642L, and ΔtrcR rpoBP575S cells. (G) Antibiotic susceptibility test by disks diffusion assays with discs containing rifampicin (30 μg) and novobiocin (15 μg) in WT and mutant cells embedded in 6 mL soft (0.3%) PYE agar overlaid on a PYE plate. The scheme on the left shows the arrangement of the discs on the plate.

Delaby et al. PNAS | 3of11 The DUF1013 protein TrcR tracks with RNA polymerase to control the bacterial cell cycle https://doi.org/10.1073/pnas.2010357118 and protect against antibiotics Downloaded by guest on September 24, 2021 (SI Appendix, Fig. S1A), this defect likely does not explain the (E. coli) RNAP that recognize the RpoB and RpoC subunits essentiality of trcR mutant cells predicted from Tn-Seq analyses (Fig. 3B). Since RpoB and RpoC indeed copurify with GFP-TrcR (32). We previously determined that WT C. crescentus cells are but not with GFP pulled down from extracts of WT cells in a resistant to Nal owing to a natural mutation in the gene (gyrA) control experiment, we concluded that TrcR resides in a firm and encoding Gyrase A(33), but mutants have been identified that do specific complex with RNAP. Indeed, sodium dodecyl sulfate not grow well in the presence of Nal despite this natural muta- polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the tion in gyrA (33). We therefore considered the possibility that Tn GFP-TrcR and GFP pull-down samples (SI Appendix,Fig.S3A) insertions in trcR are similarly conditionally lethal on plates with revealed a very prominent protein species of high molecular mass Nal, thus causing their elimination from the Tn pool that had (approximately 150 kDa) in the former. Mass spectrometry anal- been sequenced in the Tn-Seq experiment (32). Indeed, antibi- ysis of these samples identified peptides of RNAP subunits otic disk diffusion assays revealed that ΔtrcR cells are highly (RpoB, RpoC, RpoA, and RpoD) as the most abundant species in sensitive to Nal and the first-generation fluoroquinolone flu- the GFP-TrcR pull-down sample (SI Appendix,Fig.S3B)apart mequine compared to WT cells (Fig. 2C). Moreover, efficiency from GFP-TrcR itself. While we cannot exclude that an unde- 6 of plating (EOP) assays revealed a strong 10 -fold reduction in tected bridging factor is required to directly link TrcR to RNAP, the ability of ΔtrcR cells to form colonies on PYE agar supple- our suppressor and pull-down experiments support the conclusion mented with Nal (20 μg/mL, Fig. 2C) compared to WT cells that TrcR physically and robustly associates with RNAP. 4 (Fig. 2C). In the absence of Nal, a 10 -fold reduction in plating To probe for an interaction between TrcR and RNAP in vivo, efficiency is also observed, but both plating defects are restored we conducted chromatin immunoprecipitation followed by deep- when TrcR is expressed from Pvan on pMT335 (Fig. 2D). Thus, sequencing (ChIP-Seq) aimed at determining whether TrcR loss of TrcR curbs the fitness of cells and sensitizes them to Nal tracks with RNAP. We used polyclonal antibodies to TrcR to and flumequine. precipitate TrcR-bound chromatin from exponentially growing Δ Next, we isolated five independent spontaneous trcR sup- WT and ΔtrcR cells (Fig. 3C) and subjected the precipitated pressor mutants all able to grow and exhibiting an improved chromatin to deep sequencing. Bioinformatic analysis revealed μ plating efficiency on PYE agar containing Nal (20 g/mL, Fig. that TrcR has a broad occupancy on DNA in WT cells, with 524 2E). Whole genome sequencing of these five suppressor mutants statistically significant peaks called by the MACS2 software revealed two different single missense mutations in the rpoB (comparing the peaks between WT and ΔtrcR cells, Fig. 3D and gene, encoding the β (RpoB) subunit of RNAP, each of which Δ Supplementary Dataset 3). While these peaks are primarily ob- can mitigate the defect of trcR cells. One mutation, present in served at promoter sequences, TrcR also occupies coding se- four of the suppressor mutants, changes the conserved proline P642L quences (CDSs [i.e., transcription units], Fig. 3 B, C, and E). For residue at position 642 into a leucine (rpoB , SI Appendix, comparison, we also conducted ChIP-Seq analysis of RNAP Fig. S2A). P642 corresponds to residue T635 of E. coli RpoB that (Fig. 3 C, E, and F and Supplementary Dataset 2) and RpoD (SI is located near an interaction surface with RpoC (SI Appendix, Δ Appendix, Fig. S4 A and B and Supplementary Datasets 2 and 4). Figs. S1 and S2 A and B). In the remaining trcR suppressor In these experiments, we immunoprecipitated chromatin from mutant, the proline codon at position 575 is converted to a serine P575S WT and ΔtrcR cells with monoclonal antibodies to (E. coli) codon (rpoB ). E. coli RpoC also has a proline at the position 70 RNAP and σ , respectively. As expected, we found that RNAP (P567) corresponding to P575 and this residue faces D514 of σ70 and σ70 both bind prominently to promoter regions. Moreover, (RpoD) and the nascent transcript (SI Appendix, Fig. S2C) (34). unlike the sharp and promoter-centered peaks of σ70, the peaks Surprisingly, both suppressor mutants exhibited near WT of RNAP resemble those of TrcR in their broad disposition and swarming motility (Fig. 2F) and substantially improved CtrA- their tailing pattern into and throughout the CDSs, indicating dependent promoter activity (SI Appendix, Fig. S1B) compared that TrcR and RNAP occupy CDSs (Fig. 3 C and E and SI to ΔtrcR mutant cells. Moreover, cell division control was re- Appendix, Fig. S4 B and D). The congruency in the occupancy stored in the suppressor mutants compared to ΔtrcR parental cells (Fig. 1F). Since both mutations reside in rpoB, we deter- pattern between RNAP and TrcR is highlighted in the ChIP-Seq mined the sensitivity of WT, ΔtrcR, ΔtrcR rpoBP642L, and ΔtrcR trace across the ctrA gene (Fig. 3F and SI Appendix, Fig. S4D), rpoBP575S cells to the RNAP inhibitor Rif. Antibiotic disk dif- but also in other traces (SI Appendix, Fig. S5A). Of the 922 Δ RNAP peaks detected (log2 fold enrichment >2), 380 were also fusion assays revealed that trcR cells were more sensitive to Rif > compared to WT, ΔtrcR rpoBP642L, and ΔtrcR rpoBP575S cells occupied by TrcR (Fig. 3D), suggesting that 40% of the RNAP population is loaded with TrcR. Even though this value is de- (Fig. 2G). Δ In summary, TrcR protects cells from sensitivity to certain duced from comparison of the ChIP-Seq signal in WT and trcR antibiotics including the quinolones Nal and flumequine as well cells, it is possible that we underestimate the fraction of RNAP as the RNAP inhibitor Rif. The sensitivity to Rif, along with the containing TrcR, possibly due to the limited efficiency of anti- genetic interaction between trcR and rpoB described above, bodies in immunoprecipitation and/or owing to a consequence of support the view that TrcR and RNAP act in the same pathway. the width of the peaks that may restrict their identification (calling) by the automated peak assignment function in the TrcR Tracks with RNAP. Knowing that TrcR and RNAP interact software. Alternatively, the epitope recognized by the antibodies genetically, we probed for a physical association among them to TrcR may be masked by other proteins bound to RNAP in using coimmunoprecipitation experiments. In these experiments, certain complexes. we first pulled down RNAP from WT cells expressing a tandem To explore if a common DNA-binding motif underlies pro- affinity purification (TAP)-tagged variant of the β’ (RpoC) subunit moter binding of TrcR, we subjected a 300 nucleotide–long se- (RpoC-TAP) and then probed for the presence of various tran- quence centered on the 60 most prominent TrcR peaks to analysis scriptional regulators by immunoblotting. While TrcR, CtrA, and using the Multiple Expectation maximizations for Motif Elicita- the RNAP-binding cell cycle regulator GcrA (35, 36) all copurified tion (MEME) algorithm (37), but no consensus sequence emerged with RpoC-TAP (Fig. 3A), the FljK flagellin and MreB actin (both that could be attributed to TrcR. Further support for the con- soluble proteins) did not (Fig. 3A). To confirm these results, we clusion that TrcR does not bind specific DNA sequences came conducted an inverse experiment in which we pulled down green from in vitro experiments by electrophoretic mobility shift assay fluorescent protein (GFP)-tagged TrcR (GFP-TrcR) from lysates (EMSA) using TrcR purified from recombinant E. coli cells (SI of ΔtrcR pXGFPN-4-trcR cells and then probed by immunoblot- Appendix,Fig.S5B). We did not observe any preference for the ting for the presence of RNAP using monoclonal antibodies to four target promoters used as probes in EMSAs and found no

4of11 | PNAS Delaby et al. https://doi.org/10.1073/pnas.2010357118 The DUF1013 protein TrcR tracks with RNA polymerase to control the bacterial cell cycle and protect against antibiotics Downloaded by guest on September 24, 2021 A 1: Beads 2: WT 3: rpoC-TAP B FT Elution 1 2 12 * * 150 * * kDa * 1: WT pXGFPN-4 2: ∆trcR pXGFPN-4-trcR 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 TrcR CtrA GcrA FljK MreB RNAP C

WTT PYE (1) WTT PYE+Riff30(2)(2) ∆trtrcRcR PYEPYE ((3)3) 121 2 TrcRTrcRR Rel. ChIP-seq reads for TrcR 0 1 243 01313240 24 Chromosome position (Mbp) D WT PYE WT PYE+Rif30

144 380 542 WT PYE 922 RNAP 524 TrcR 524 TrcR WT PYE MICROBIOLOGY significant peaks significant peaks Rel. ChIP-seq 01313240 24 reads for RNAP Narrow ChIP-Seq peaks Chromosome position (Mbp) Chromosome position (Mbp) (>2 fold-enrichment) E TrcR RNAP F 450 ldpD ctrA 03131 WTWT T+RifRif ΔtrcR WT+ Rif WT 100 400 WT RNAP + Rif

350

80 WT RNAP 300

60 250

200 40 150 WT TrcR for TrcR & RNAP 100 Rel. ChIP-Seq reads 20 Rel. ChIP-seq reads

50 WT TrcR + Rif ΔtrcR TrcR ATG ATG ATG ATG ATG 0 (rpm) 3278000 3279000 3280000 Centered on ATG Chromosome position (bp)

Fig. 3. TrcR tracks and associates with RNAP in vivo. (A) RpoC-TAP pull-down samples were assessed for the presence of TrcR, CtrA, and GcrA by immunoblotting. FljK and MreB are used as negative control, as they are not known to associate with the transcriptional machinery. Specificity was assessed using extracts from untagged WT cells subjected to TAP-based pull-down. Asterisks indicate the expected migration position for the marker proteins detected by the antibodies. (B) GFP-TrcR pull-down sample probed for the presence of the RNAP by immunoblotting. (C) Genome-wide occupancies of TrcR (Upper)and

RNAP (Bottom) in different conditions (PYE and PYE Rif30) in WT and ΔtrcR cells as determined by ChIP-Seq. The x-axis represents the nucleotide position on the genome (Mbp), whereas the y-axis shows the normalized ChIP-Seq read abundance in reads per million (rpm). Control ChIP-seq in the ΔtrcR cells (orange graph on the right) shows only one unspecific peak. The inset shows the TrcR steady-state levels in the presence or absence of Rif as determined by immunoblotting. (D) Venn diagram comparing the 524 TrcR significant (narrow) promoter peaks to the significant 922 RNAP promoter peaks as determined by ChIP-Seq analysis. (E) Heatmap of TrcR and RNAP ChIP-Seq reads aligned on the translation initiation codon (ATG) of Coding DNA Sequence in the center. The alignment covers an 800-bp range around the translation initiation codon in WT and ΔtrcR cells treated or not with Rif and sorted with reference to the relative abundance of reads compared to WT (highest read abundance at the top). ChIP-Seq signals are normalized in read per million (rpm) and the color bar (intensity) indicates the enrichment in rpm. Gray arrows indicate the presence or absence of binding downstream of the ATG. (F) Comparison of TrcR and RNAP ChIP-Seq traces, including the narrow promoter peak at the ctrA locus in WT cells treated or not with Rif. Coding sequences are represented as boxes on the upper part of the graph. ChIP-Seq traces of ΔtrcR cells are shown as a control.

Delaby et al. PNAS | 5of11 The DUF1013 protein TrcR tracks with RNA polymerase to control the bacterial cell cycle https://doi.org/10.1073/pnas.2010357118 and protect against antibiotics Downloaded by guest on September 24, 2021 evidence for sequence-specific binding by TrcR in the presence of Appendix, Fig. S4C), again consistent with the increase in RNAP competitor DNA. promoter occupancy in the presence of Rif. In fact, neither σ70 On the basis of these results, we speculated that the disposi- nor GcrA (SI Appendix, Fig. S4C note the absence of binding tion of TrcR on chromatin is driven by association with RNAP signal downstream of the ATG start codon for both σ70 and holoenzyme rather than by RNAP-independent binding to a GcrA ChIP-Seq heat maps) are associated with CDSs. More- special target sequence within or around the core promoter. This over, CtrA and CdnL that both bind RNAP and promoters (23, model is supported by our findings that 1) the TrcR ChIP-Seq 38, 39) also do not occupy CDSs (Fig. 3A and SI Appendix, Fig. profile mirrors that of RNAP, 2) TrcR is pulled down with RpoC S4 C and D). We conclude that 1) the chromosomal occupancy and vice versa, 3) the rpoBP642L mutation suppresses the ΔtrcR of TrcR at CDSs is dissimilar to that of other transcriptional phenotypic defects, 4) there is a global defect on transcript levels factors that associate with σ70·RNAP at promoters and that 2) in ΔtrcR cells versus WT cells, and 5) ΔtrcR cells are more the association of TrcR within CDSs is dependent on tran- sensitive to Rif than WT cells. scription elongation (and RNAP activity) and likely represents a conserved property of the DUF1013 (see below). Rifampicin Dislodges TrcR from Elongating RNAP. If TrcR indeed Our ChIP-Seq experiments suggest that TrcR enhances the tracks with RNAP, then the disposition of TrcR on chromatin recruitment of σ70 to promoters or the stability of σ70·RNAP– might be altered in the presence of Rif. We tested this possibility initiating complex at the promoter. A comparison between the by conducting ChIP-Seq experiments using antibodies to TrcR to occupancy of σ70 on chromatin from WT, ΔtrcR single mutant, precipitate chromatin prepared from WT cells before or after a and ΔtrcR rpoBP642L double mutant cells revealed that 90% ± 14 very short (10 min) pulse with Rif (30 μg/mL). These experi- of the 250 top σ70 peaks seen in WT cells (Supplementary ments revealed that the characteristic binding pattern of TrcR Dataset 3 and SI Appendix, Fig. S4) are also present in ΔtrcR within the CDSs is lost and the peak intensity is generally sub- mutant cells. However, the peak intensity is reduced in ΔtrcR stantially reduced (Fig. 4 C, E, and F and Supplementary Dataset cells and therefore fewer peaks are called by the MACS2 soft- 2) after exposure to Rif, although TrcR is still abundantly ware, because some peaks no longer pass the threshold for peak expressed (Fig. 3 C, Inset). In fact, quantification of the residual calling in the absence of TrcR (only 589 significant peaks are peaks revealed a 90% reduction in TrcR occupancy compared to detected in ΔtrcR cells compared to 1108 peaks in WT cells, the control condition without antibiotics (Supplementary Data- Supplementary Dataset 3). Remarkably, σ70 occupancy is increased set 2, percentage of TrcR occupancy). However, the residual to 181% ± 17 in ΔtrcR rpoBP642L double mutant cells, and MACS2 binding of TrcR to promoters is still discernible, albeit weakly, called 999 relevant peaks (Supplementary Dataset 3), indicating that upon comparing the TrcR peaks in Rif-treated WT cells to the the compensatory mutation in RpoB acts by improving σ70·RNAP background ChIP-Seq signal from ΔtrcR cells. In control ChIP- promoter association in the absence of TrcR. Seq experiments conducted with antibodies to RNAP and to σ70, we found that the occupancy of RNAP at promoters increases in Antibiotic-Inducible Recruitment of TrcR to the Promoter of a Drug the presence of Rif, while the association within the CDSs is lost Efflux Locus. To examine if TrcR is recruited to a promoter (Fig. 3 C, E, and F), indicating that Rif interferes with tran- when transcription is induced, we conducted ChIP-Seq experi- scription elongation. Unlike TrcR, the chromosomal occupancy ments in cells treated with Nal, since this quinolone antibiotic 70 of the σ -binding transcriptional activator GcrA is increased at induces the promoter (PacrA) driving expression of the acrAB– promoters in the presence of Rif compared to without Rif (SI nodT operon encoding a tripartite drug efflux system of the

A B significant peaks WT WT PYE PYE+Nal20 580 TrcR WT 469 Nal

PYE 55 111 acrA2 WT 524 TrcR for TrcR significant peaks

0123401234 Narrow ChIP-Seq peaks Rel. ChIP-Seq reads Chromosome position (Mbp) (>2 fold-enrichment)

C D 200 acrB2 acrA2 tipR 853 TrcR - WT 25 acrA2 promoter ds 150 * TrcR - WT Rif 20 rea bacterioferritin q * TrcR - WT Nal e 15 -associated 100 dnaJ-like -S TrcR - ∆trcR ferredoxin

10 hIP Ratio djlA * RNAP - WT

C 50 * * σ70 - WT 5 l. *

Re Ti - WT 0 0 01234 925000 926000 927000 928000 929000 TrcR Nal/TrcR Control Chromosome position (Mbp) Chromosome position (bp)

Fig. 4. Recruitment of the TrcR·RNAP complex to the promoter of the acrAB–nodT operon induced by Nal. (A) Genome-wide ChIP-Seq profile of TrcR chromosomal occupancy in WT cells before (Left) and after a 10-min treatment with Nal (20 μg/mL, Right). The acrAB–nodT locus is indicated as acrA2 (corresponding to its annotation in the genome) to avoid confusion with another annotated acrA gene. (B) Venn diagram showing comparison of genes containing significant promoter (narrow) peaks of TrcR binding as determined by ChIP-Seq analysis from WT before and after exposure to Nal. (C) Ratio of normalized TrcR ChIP-Seq profiles between untreated and Nal-treated conditions. (D) Comparison of TrcR, RNAP, and RpoD ChIP-Seq traces at the acrAB–nodT locus between WT cells treated or not with antibiotics (Nal, Rif) or in ΔtrcR cells. Total input (Ti) chromatin DNA (prior pull-down) was sequenced as a control for sequence bias, and the corresponding traces are shown as control. Genes encoded are represented as boxes on the upper part of the graph (annotations or CCNA gene annotation numbers of the sequenced NA1000 reference genome).

6of11 | PNAS Delaby et al. https://doi.org/10.1073/pnas.2010357118 The DUF1013 protein TrcR tracks with RNA polymerase to control the bacterial cell cycle and protect against antibiotics Downloaded by guest on September 24, 2021 resistance–nodulation–division family in C. crescentus (33). PacrA B. ovis cells, we engineered a conditional mutant harboring an in- is negatively regulated by TipR, a TetR-like transcriptional re- frame deletion of trcR on the B. ovis genome and a WT version pressor that is antagonized by Nal (33). ChIP-Seq analysis of the TrcR coding sequence under the control of an IPTG- revealed that TrcR does not occupy PacrA abundantly in the inducible promoter on the replicating plasmid pSRK (42). In absence of Nal (Fig. 4 A and D). However, within 10 min after the absence of IPTG, the resulting cells displayed evidence of a WT cells were exposed to Nal (Figs. 4A and 5B), we observed a division defect and abnormal (elongated and lemon-shaped) major peak of TrcR at PacrA (>20-fold induced, Fig. 4 C andD morphology (Fig. 5 D and E). In general, the size of TrcR-depleted and Supplementary Dataset 3) at or near the binding site of σ70· B. ovis cells (measured as two-dimensional cell area) is increased RNAP at this (divergent) promoter that controls transcription of and more irregular compared to control cells (Fig. 6 E, Left). As is acrAB–nodT and the cis-encoded tipR gene (Fig. 4D). TrcR was the control, two principal shape modes were discernable under also recruited to a few other loci such as the locus encoding the conditions of TrcR depletion. However, TrcR-depleted cells were DnaJ-like protein DjlA (>4-fold induced, Fig. 4 B and C and longer, and cell size was more variable along both the long and Supplementary Dataset 3), albeit far less prominently than for short cell axes in both shape modes (Fig. 5 E, Right). Importantly, PacrA. Thus, TrcR is recruited to new sites of transcription, spe- the plating efficiency (Fig. 5F) was strongly reduced in the absence cifically forming a prominent peak at PacrA that is otherwise in- of TrcR, and cells are rendered more susceptible to Nal or Rif. We active during growth in PYE in the absence of Nal. This finding conclude that the ability of TrcR to associate with chromatin under further supports the conclusion that recruitment of TrcR to conditions of active transcription elongation and its role in pro- promoters depends on transcriptionally active RNAP (i.e., that moting proper cell division cycle and antibiotic stress protection is TrcR does not associate with promoters that do not fire). conserved across multiple genera of alphaproteobacteria.

TrcR Function Is Conserved. To determine if the functional inter- Discussion action between TrcR and RNAP in cell cycle and antibiotic stress Mechanism of Transcriptional Control by TrcR. The alphaproteo- control is maintained during evolution, we tested if the TrcR/ bacterial signature protein TrcR controls the cell cycle globally at DUF1013 ortholog from the obligate intracellular pathogens the transcriptional level but in a distinct fashion to other known Ehrlichia chaffeensis and Rickettsia prowazekii can substitute for cell cycle transcriptional regulators of alphaproteobacteria that C. crescentus TrcR. First, we asked if ehrlichia_duf1013, rick- simply target a subclass of cell cycle–regulated promoters by ettsial_duf1013, and bovis_1678 can compensate the pleiotropic recognizing a dedicated target sequence. Our ChIP-seq analyses defects of ΔtrcR cells (Fig. 5 A and B and SI Appendix, Fig. S6 A revealed that TrcR associates prominently with promoters and and B). Indeed, expression of these DUF1013 orthologs from a CDSs. This pattern matches that of RNAP, and it depends on MICROBIOLOGY plasmid improved the EOP of ΔtrcR cells compared to the empty elongating RNAP that can be inhibited with Rif. Moreover, vector (pMT335 or pSRK). Similarly, the DUF1013 ortholog suppressive mutations in the RpoB subunit of RNAP compen- from S. meliloti and Brucella ovis can slightly improve motility of sate for the general transcriptional defects caused by the absence ΔtrcR (Fig. 5A and SI Appendix, Fig. S6A). Next, we asked if of TrcR. TrcR has the hallmarks of a factor that binds core these DUF1013 orthologs can protect ΔtrcR cells against Nal RNAP (Figs. 3 A and B and 6) rather than a sigma subunit such (Fig. 5B and SI Appendix, Fig. S6B) and only observed a small as σ70 that does not associate with CDSs in ChIP-Seq experi- increase in plating efficiency on PYE plates containing Nal ments (Fig. 3 and SI Appendix, Fig. S4) and whose association compared to ΔtrcR harboring the empty vector. with promoters is not compromised in the presence of Rif. Other To determine if alphaproteobacterial TrcR also associates transcriptional factors, for example, GcrA that interacts with σ70, with chromatin, we conducted ChIP-Seq experiments using our is also not associated with CDSs, and binding is not compro- polyclonal antibodies to C. crescentus TrcR to precipitate TrcR mised by Rif. Thus, the properties of TrcR are unprecedented from chromatin prepared from S. meliloti cells (SI Appendix, Fig. and apparently reflect a hallmark of newly discovered DUF1013 S7A). Our analysis revealed a binding pattern resembling that of class of transcriptional regulators (Fig. 6). TrcR in C. crescentus, with ChIP signals occurring primarily at Rif binds RpoB in a cleft close to the active center of the main the S. meliloti promoter sequences, but also spanning CDSs (SI DNA/RNA channel in RNAP from E. coli (34, 43) and many Appendix, Fig. S7B). This typical binding profile was seen on all mutations that render E. coli resistant to Rif map to RpoB, in- three S. meliloti replicons: the chromosome and the two symbi- cluding to a region referred to as cluster II (34, 43). The residue otic plasmids, pSymA and pSym. Akin to what was observed with (P575) in rpoB that we identified as a suppressor site of ΔtrcR chromatin isolated with WT C. crescentus (Fig. 3 C and E), a cells is conserved in E. coli (P567). It is near the transcription short pulse of 10 min with Rif (100 μg/mL) on S. meliloti WT bubble and D514 of the σ70 loop in the DNA/RNA channel. chromatin leads to a loss of TrcR binding within the CDS, and Three and four residues upstream lie the mutations P563T and the promoter peak is substantially reduced in intensity on the S. T564P that also confer Rif resistance in E. coli, akin to the meliloti chromosome (SI Appendix, Fig. S7B). Interestingly, nearby position I572 (44). In C. crescentus, the P575S substitu- however, the addition of Rif had little effect on TrcR occupancy tion ameliorates all (known) defects of ΔtrcR cells including the with CDSs on the pSymB megaplasmid (SI Appendix, Fig. S7B) sensitivity to Rif and to Nal. Paradoxically, it was a selection for and no detectable effect on TrcR occupancy with CDSs on the suppressor mutants using the quinolone antibiotic Nal that pSymA (SI Appendix, Fig. S7B). Thus, TrcR associates with unearthed the rpoB mutations that also protect ΔtrcR cells toward chromosomal and episomal DNA in S. meliloti, possibly through Rif. Nal targets DNA gyrase A, but C. crescentus is naturally re- different RNAP populations specific to each episomal element sistant to Nal owing to natural polymorphism in the quinolone that respond differently when Rif is added at the concentrations resistance determining region of the gyrA gene (33). While Nal used here. induces expression of the AcrAB–NodT transenvelope complex As for C. crescentus, Tn-Seq analysis suggests that TrcR that promotes efflux of antibiotics, strong induction of AcrAB– orthologs are also important to control the cell division cycle of NodT can have deleterious effects in cell division mutants (33) for other alphaproteobacteria. While Tn insertions in gene RL4728 reasons that are unclear but likely related to destabilization of the encoding the Rhizobium leguminosarum TrcR ortholog were cell envelope. Since the RpoB suppressor mutations ameliorate reported to impair growth (40), no Tn insertions were found in the Nal sensitivity of ΔtrcR cells, we speculate that the muta- the gene encoding the TrcR ortholog in the pathogens Brucella tions act in Nal resistance by correcting the envelope or cell di- abortus and B. ovis (39, 41) (Fig. 5C and SI Appendix, Fig. S8). To vision (and other general) defects of ΔtrcR cells through general verify that trcR is essential for viability or efficient growth of WT augmentation of transcription.

Delaby et al. PNAS | 7of11 The DUF1013 protein TrcR tracks with RNA polymerase to control the bacterial cell cycle https://doi.org/10.1073/pnas.2010357118 and protect against antibiotics Downloaded by guest on September 24, 2021 A B PYE Gent PYE Gent 0.3% agar pMT335 - Van Van + Nal + Van 50 μM 1= empty 2= trcR 1 5 3= ehrlichia-DUF1013 WT 2 6 4= rickettsia-DUF1013 empty 3 7 5= 6= trcR 4 8 7= ehrlichia-DUF1013 8= Δ trcR rickettsia-DUF1013 12 9 9= empty 10= trcR

10 WT 13 11= S. meliloti-DUF1013 12= empty 14 11 13= trcR

14= Δ trcR S. meliloti-DUF1013 CD10-fold dilution series 300 WT trcR trcR 200 /pSRK-EV ∆ /pSRK-

100 1 B. ovis # reads 0 2 300

200

100 # reads B. abortus 0 TA sites

genes trcR WT/pSRK-EV ∆trcR/pSRK-trcR 87.9% 87.4% E 2 WT/pSRK-EV 1.5 ∆trcR/pSRK-trcR Shape

mode 1 -2 s.d. -1 s.d. 1 mean 5.9% 4.8% 1 s.d. 0.5 2 s.d. Frequency Distribution

0 Shape 0.5 1 1.5 2 2.5 3 mode 2 F Area (μm2) 123 123 123 123 123 123 1) WT/pSRK-EV 2) WT/pSRK-trcR 3) ∆trcR/pSRK-trcR 10-fold dilution

– IPTG + IPTG – IPTG + IPTG – IPTG + IPTG No Abx Nal 20 Rif 0.025

Fig. 5. Conservation of TrcR activity in alphaproteobacteria and its essentiality for Brucella viability. (A) Motility assays on soft (0.3%) agar inoculated with WT and ΔtrcR containing the empty vector (pMT335-) or plasmids with trcR orthologs from two different rickettsiales (Upper) and S. meliloti (Bottom).

Complementation of the ΔtrcR mutant with trcR orthologs under control of the Pvan promoter on pMT335 improves the motility defect of the ΔtrcR mutant cells. Numbers shown correspond to the legend described in B.(B) EOP assay on PYE plates and PYE Nal (20 μg/mL) supplemented with gentamycin (1 μg/mL) and vanillate 50 μM in WT and ΔtrcR cells containing the empty vector (pMT335-) or plasmids with trcR orthologs from two different rickettsiales (Upper)and S. meliloti (Bottom). (C) Quantification of Tn miniHimar reads from the B. ovis trcR locus (locus ID BOV_RS08235) and from the B. abortus trcR locus (locus ID BAB_RS24265). DNA for sequencing was prepared from pooled B. ovis ATCC 25840 or B. abortus ATCC 2308 miniHimar mutant libraries. All possible TA dinucleotide miniHimar insertion sites at the locus are marked in vertical orange lines. Gene orientation is shown. (D) Phase contrast micrographs (63×)ofWT B. ovis carrying an IPTG-inducible empty plasmid vector (WT/pSRK-EV; Left) and B. ovis ΔtrcR carrying plasmid pSRK-trcR (ΔtrcR/pSRK-trcR; Right) were captured from cells harvested from agar medium lacking IPTG. Single cell contours were extracted from micrographs of WT/pSRK-EV (n = 721) and ΔtrcR/ pSRK-trcR (n = 931). (Scale bar, 5 μm.) (E, Left) Smoothed histogram showing marginal distribution of cell area (in μm2) for all WT/pSRK-EV (gray) and ΔtrcR/ pSRK-trcR (blue) single cell contours. (Right) Two principal shape modes account for over 90% of variance in each strain population. Percentage of variance accounted by each mode is shown. For each mode, mean shape ± 2 SD is shown. (Scale bar, 0.5 μm.) Cell contour extraction, cell area, and shape mode calculations were conducted using Celltool (61). (F) Antibiotic sensitivity assay for WT/pSRK-EV (1), WT/pSRK-trcR (2), and ΔtrcR/pSRK-trcR (3). Nal (20 μg/mL) or

Rif (0.025 μg/mL) was added to plates, and cells were plated in a log10 dilution series (starting at a top cell titer of OD [optical density]600 of 0.01). Plates images are inverted to enhance contrast and colony identification. IPTG was added at 1 mM final concentration where indicated.

8of11 | PNAS Delaby et al. https://doi.org/10.1073/pnas.2010357118 The DUF1013 protein TrcR tracks with RNA polymerase to control the bacterial cell cycle and protect against antibiotics Downloaded by guest on September 24, 2021 WT σ70 RNA TrcR affects the cell cycle by promoting the accumulation of the RNAP TrcR essential cell cycle transcriptional regulator CtrA (Fig. 1C) which RNAP TrcR σ70 σ70 RNAPTrcR ++ retains cells in the motile G1-phase (21, 22). CtrA is degraded - 35 - 10 35 - 10- - 10 during the G1→S transition and then reaccumulates later in ∆trcR S-phase (25). As the steady-state levels of TrcR are highest σ70 RNAP during the G1→S transition (Fig. 1A), just preceding the accu- RNAP σ70 RNAP mulation of CtrA, we speculate that TrcR is needed for efficient σ700 + Δ - 10- 35 - 10- 35 - 10- - 10 resynthesis of CtrA in S-phase. However, knowing that trcR mutant cells exhibit a global effect on mRNA levels (Fig. 2A), σ70 ∆trcR rpoB* compromising swarming motility (i.e., curbing the G1-phase RNAP* RNAP* σ70 population) as well as cell division (Fig. 1 C and E), and the RNAP* σ70 ++ fact that TrcR levels peak at the onset of S-phase (Fig. 1A), we - 10- 35 - 10- 35 - 10- - 10 propose that TrcR enhances transcription for normal progres- Pre-initiation Initiation Elongation Transcriptional sion of the cell division cycle especially in S-phase when tran- Output scription of ctrA and other important genes (Fig. 6) surges. Fig. 6. Model for TrcR acting together with RNAP. TrcR forms a stable CtrA protein has been estimated at >20,000 molecules per cell complex with RNAP and is brought to promoters and coding sequence via (46). As CtrA is an unstable protein that is turned over and this interaction. TrcR association and tracking with the RNAP allow gene resynthesized each cell cycle by regulated proteolysis (25, 47) transcription and cell cycle progression. In absence of TrcR, transcription at (Fig. 1A), it is not surprising that a reduction in its synthesis 70 selected cell cycle–regulated promoters is impaired and σ occupancy is could potentially have dramatic effects on steady-state levels σ70 reduced. Compensatory mutations in rpoB* restore occupancy and per- and thus many CtrA-dependent functions that are cell cycle– mit adequate transcription of cell cycle genes, thus supressing the cell cycle and other defects, including the antibiotic sensitivity. regulated and dependent on CtrA. One of these events that has clear phenotypic consequences that are thus unambiguous to dissect is the CtrA-dependent expression of the capsulation in- An appealing model posits that TrcR binds RNAP, stabilizes hibitor HvyA in G1-phase (SI Appendix, Fig. S1 B and C). Both the RNAP holoenzyme open complex after σ70 recruitment and/ transcriptional and translational regulatory steps that are de- or promotes promoter escape, and then tracks with RNAP during pendent on CtrA have been described for HvyA expression, and the elongation phase (Fig. 6). In the absence of TrcR, transcrip- again, HvyA is a very unstable protein, explaining why reduced tion is compromised at least at certain transcription units and synthesis would have a strong effect on HvyA steady-state levels MICROBIOLOGY the mutations in RpoB compensate for this defect, perhaps (20). Consistent with this explanation, we found that constitutive strengthening the association with σ70 (Fig. 6 and SI Appendix,Fig. synthesis of HvyA from Pvan reverses the effect on capsule and S4B). Rif may destabilize the association of TrcR with RNAP by leads to concomitant change in cellular buoyancy (SI Appendix, Δ directly competing with TrcR for binding on the same region on Fig. S1B). Another CtrA-dependent synthesis defect of trcR RpoB or indirectly by promoting a structural change along the cells is a defect in expression of PilA (SI Appendix, Fig. S1D) which is required for pilus formation and infection by the pilus- polypeptide that subsequently impairs the association with TrcR. ϕ Interestingly, a recent Cryo-Electron Microscopy (Cryo-EM) specific bacteriophage CbK (48). We speculate that the steady- structure of the E. coli σ70·RNAP holoenzyme–promoter com- state levels of unstable cell cycle proteins are most likely to be plex reveals that the nonconserved region of σ70 interacts with affected by loss of TrcR. the promoter region upstream of the −10 element through the RNAP-Associated Proteins. TrcR may be functionally analogous to well-conserved arginine residue at position 157 which facilitates a small group of transcriptional regulators such as RbpA, DksA, the formation of stable RNAP open complex (45). While this and CarD that modulate the stability of open complexes (7, 12, residue is well conserved in betaproteobacteria, gammaproteo- 13, 49). While DksA (50) and CarD (CdnL) (38, 51) orthologs bacteria, epsilonproteobacteria, deltaproteobacteria, and chla- are encoded in the C. crescentus genome, RbpA is specific for σ70 mydiae (45), it is absent from alphaproteobacterial s. Perhaps actinobacteria, including the major human pathogen M. tuber- alphaproteobacteria depend on TrcR for a function provided by culosis and the model streptomycete Streptomyces coelicolor in σ70 s in other lineages. which RbpA was originally identified (11, 52–54). Mutations in RbpA cause an increased sensitivity to Rif compared to WT TrcR Is an Alphaproteobacterial Signature Protein and Cell Cycle actinobacterial cells, and RbpA has been reported to interact Regulator. The DUF1013 domain in TrcR is ubiquitous among with the RpoB and RpoC subunits of RNAP core enzyme (11, the alphaproteobacteria, including the obligate intracellular and 12, 52, 53, 55, 56). Thus, RbpA and TrcR may shield RNAP from pathogenic lineage of Rickettsiae, indicating a common ancestor Rif or impair Rif entry into the active center of RNAP. M. tu- for DUF1013 superfamily. The fact that DUF1013 from Ehrli- berculosis CarD works together with RbpA to directly interact Δ chia and Rickettsia improve the motility of the trcR mutant cells with RpoB (57) and associates with most σ70-dependent promoters indicates that TrcR function is conserved. Moreover, TrcR also in vivo (58). The peaks of CarD on chromatin as determined by exhibits a broad disposition on the chromosome and on symbi- ChIP-Seq are restricted to the promoter and are congruent to otic plasmids of S. meliloti, implying that TrcR transcriptional those seen for σ70 (58). Analogous cooperative interactions be- activity is conserved in this alphaproteobacterial branch. However, tween C. crescentus CarD (CdnL) and TrcR are conceivable, with the effect of Rif differs between chromosome versus mega- TrcR perhaps serving a similar role as RbpA. While the distribu- plasmids, raising intriguing questions about a differential effect of tion of RbpA in vivo remains to be determined, TrcR is clearly not Rif on TrcR on different genomic elements or replicons. Might just restricted to promoters as are σ70 and CarD (CdnL) (38). the conformational state of the replicon (chromosomes versus ChIP-Seq analyses of CarD (CdnL) and σ70 in C. crescentus reveal plasmids) confer different properties or requirements on RNAP that nearly all CarD (CdnL) binding sites are also occupied by σ70, depending on the state of DNA supercoiling/topology and/or but there are a substantial number (fourfold more) of σ70 binding nucleoid-associated proteins? sites that are apparently not associated with CarD (CdnL) in vivo We showed that TrcR is required for proper control of the cell (38). If CarD (CdnL) and TrcR functionally interact in alphapro- division cycle in the nonpathogenic bacterium C. crescentus and teobacteria, then C. crescentus cells lacking CarD (CdnL) may in the mammalian intracellular pathogen B. ovis.InC. crescentus, present similar phenotypes as trcR mutants. Interestingly, CarD

Delaby et al. PNAS | 9of11 The DUF1013 protein TrcR tracks with RNA polymerase to control the bacterial cell cycle https://doi.org/10.1073/pnas.2010357118 and protect against antibiotics Downloaded by guest on September 24, 2021 (CdnL) is also annotated as an essential gene in C. crescentus based When required, IPTG (1mM), kanamycin (50 μg/mL), rifampicin (0.025 μg/mL), on Tn-Seq analysis (32) even though ΔcdnL cells are viable with Nal (20 μg/mL), and sucrose (5%) were added. substantial growth and division defects (38, 51). This situation E. coli S17-1 λpir (60), EC100D (Epicentre Technologies, Madison, WI), and matches the findings reported here for ΔtrcR cells. Top10 strains were grown in LB and on LB plates supplemented with 1.5% In sum, our data suggest that TrcR function is conserved in the agar (Fisher Bioreagents) at 37 °C. Kan (50 μg/mL) and chloramphenicol alphaproteobacteria where it plays an important role in the (Chlor, 20 μg/mL) were supplemented when needed. The E. coli WM3064 control of cell cycle transcription but also in the response to strain was grown on LB agar plates or in LB, supplemented with dia- certain antibiotics. It may thus itself be an attractive target for minopimelic acid (DAP, 60 mM) and with Kan (50 μg/mL) or Chlor (10 μg/mL) drug development facilitated by future studies aimed at resolving when required. the tertiary structure of the DUF1013. Tn Libraries. The B. ovis ATCC 25840 and B. abortus ATCC 2308 Tn libraries Materials and Methods were prepared and analyzed as described previously (39, 41). Growth Conditions. C. crescentus NA1000 and derivatives were grown at 30 °C in PYE. Antibiotic concentrations used for C. crescentus include Additional Methods, Bacterial Strains, Plasmids, and Oligonucleotides. Addi- kanamycin (solid: 20 μg/mL; liquid: 5 μg/mL), tetracycline (1 μg/mL), genta- tional methods, bacterial strains, plasmids, and oligonucleotides used in this mycin (1 μg/mL), and Nal (20 μg/mL). When needed, sucrose was added at study are listed and described in SI Appendix, Tables S1–S3). 0.3% final concentration and vanillate at 50 μM final concentration. Swarmer cell isolation, electroporations, biparental matings, and bacterio- Data Availability. Sequence data have been deposited in the Gene Expression phage ϕCr30-mediated generalized transductions were performed as pre- Omnibus (GSE148654). viously described (26, 59). Plasmids for β-galactosidase assays were introduced into C. crescentus by electroporation. ACKNOWLEDGMENTS. We thank Gaël Panis for help with ChIP-Seq analysis S. meliloti Rm2011 was grown at 30 °C in lysogeny broth (LB, Fisher Bio- and EMSAs and Nicolas Kint for help with immunoblotting of GFP-TrcR pull- reagents) supplemented with CaCl2 2.5 mM and MgSO4 2.5 mM. down samples. This work was supported by Swiss National Science Founda- B. ovis was grown on tryptic soy agar (Difco Laboratories) supplemented tion Grant 31003A_182576 (to P.H.V.) and the NIH R01AI107159 and

with 5% sheep blood (Quad Five) at 37 °C with 5% CO2 supplementation. R35GM131762 grants (to S.C.).

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