Supporting InformationSmith et al. 10.1073/pnas.1407862111SI MethodsBacterial Strains, Plasmids, and Culture Conditions. Table S1 lists thestrains and plasmids used in this work. All experiments wereperformed with derivatives of Caulobacter crescentus strainCB15N (1) grown to midexponential phase. Plasmids were mo-bilized from Escherichia coli to C. crescentus by conjugation usingE. coli strain S17-1 (2). CB15N strains were grown in peptone–yeast extract (PYE) medium or minimal glucose (M2G) mediumat 30 °C (2). Where indicated, growth media were supplementedwith glucose (0.02%) or xylose (0.03%, 0.05%, or 0.3%) to re-press or induce, respectively, expression from the xylX promoter(3). E. coli cloning strains were grown in Luria broth at 37 °C,and solid and liquid media were supplemented with antibiotics asdescribed (4). PCR products were cloned into the pGEMT-Easyvector (Promega) and sequenced before being subcloned intodestination vectors. The chimeric protein, α1–RD+15, wasconstructed by using overlap extension PCR (5). Site-directedmutagenesis was performed by using the QuikChange protocol(Stratagene). Sequences of primers used for amplification orsequence modification are available upon request.

Protein Purification. The proteins ClpP–His6, His6–CtrA, His6–CtrA3, His6–CtrA–DD, His6–RcdA, His6–ClpX, and His6–PopAwere expressed from the pET vectors indicated in Table S1.His6–SciP was expressed from pHIS–sciP (6). His6–Smt3–CpdRwas expressed from pET28b. All proteins were overexpressed inE. coli Tuner cells grown to an OD600 = 0.6 at 37°C in TerrificBroth (7). Isopropylthiogalatoside was added to a final concen-tration of 0.4 mM, and cells were incubated overnight at 18 °C.Cells were harvested by centrifugation at 6,000 × g for 5 min, andpellets were frozen in liquid nitrogen and stored at −80 °C untiluse. Proteins were purified by the following protocol withmodifications where indicated. Cell pellets were thawed andresuspended in Standard Lysis Buffer (SLB; 50 mM Tris·HCl,pH 8.2, 100 mM KCl, 1 mM MgCl2, 10% glycerol, 2 mM 2-mercaptoethanol). CtrA variants were purified by using SLBcontaining only 50 mM KCl and no MgCl2. ClpP-expressingcells were lysed in P-buffer [50 mM sodium phosphate, pH 8.0,1 M NaCl, 5 mM imidazole, 10% (vol/vol) glycerol]. Cells werelysed by 1-h treatment with 1 mg/mL lysozyme and 40 units ofBenzonase nuclease (Novagen) on ice, followed by sonication.Lysates were cleared by three rounds of centrifugation at20,000 × g. Imidazole was added to a concentration of 15 mM, ex-cept when purifying ClpP–His6, and cleared lysates were incubatedwith 1 mL of Ni–nitrilotriacetic acid (Ni-NTA) agarose (Qiagen)that had been preequilibrated with the lysis buffer specific for theprotein to be purified. The agarose was applied to a gravity col-umn and washed three times with the appropriate lysis buffer. Inall cases, the second of the three washes was supplemented with300 mM NaCl to remove nonspecifically bound proteins.Unless otherwise indicated, the desired protein was eluted from

Ni-NTA agarose by using the appropriate lysis buffer supple-mented with 300 mM imidazole. ClpP-bound resin was washedusing P-buffer with 20 mM imidazole before elution of ClpP usingP-buffer with 500 mM imidazole. ClpX and RcdA were removedfrom Ni-NTA by overnight cleavage with 20 units of thrombin(EMD/Novagen). After elution with imidazole, CpdR was in-cubated overnight with small ubiquitin-related modifier (SUMO)protease (Life Sensors) to remove the His6–Smt3 tag, and asecond round of Ni-NTA purification was used to remove boththe His6–Smt3 peptide and the His6-tagged SUMO protease.Unless indicated below, all proteins were further purified by ion-

exchange chromatography using HiTrap Q HP columns (GE).Elution was achieved with a gradient of increasing KCl con-centration in SLB supplemented with 1 mM DTT. SciP wasnot subjected to further purification after the Ni-NTA step. Allproteins were exchanged into PD buffer [25 mM Hepes–KOH,pH 7.6, 5 mM MgCl2, 15 mM NaCl, 10% (vol/vol) glycerol]supplemented with 100 mM KCl and 1 mM DTT before freezingin liquid nitrogen and storage at −80 °C.For fluorescence-based proteolysis assays, GFP–CtrA–RD+15

(6), CpdR, CpdR–D51A, ChpT, CckA (8), and GFP–ssrA (9)were purified as described.

Microscopy. Exponential-phase cells were immobilized on 1%(wt/vol) agarose pads made with M2/0.2% glucose/0.2% xylosemedium. Images were acquired by using a Nikon Eclipse 80imicroscope with a PlanApo 100×, 1.40-numerical-aperture ob-jective and a Cascade 512B camera (Roper Scientific). EnhancedYFP was imaged by using Chroma filter set 41028. Images wereacquired by using Metavue software (Universal Imaging).

Protein Degradation After Chloramphenicol Treatment. StrainsKR3510 and KR3512 express CtrA or CtrA3, respectively, fromthe chromosomal xylX locus as the only allele of ctrA. Exponential-phase cultures were treated with 100 μg/mL chloramphenicol,after which samples of equal volume were withdrawn at the in-dicated times. Samples were analyzed by SDS/PAGE andWestern blotting with anti-CtrA antiserum (1:10,000) andhorseradish peroxidase (HRP)-conjugated anti-rabbit antibodies.Western blots were visualized by using Western Lightning (Perkin-Elmer) on a Bio-Rad Gel Doc XL and quantified by usingImagelab (Version 4.0). The data were fitted to single-exponentialcurves to calculate protein half-lives.

Fluorescence-Based Proteolysis Assays.Degradation of GFP–CtrA–

RD+15 was monitored as the loss of fluorescence over time.Reactions were initiated by adding the ATP regeneration system(75 μg·mL−1 creatine kinase, 4 mM ATP, 5 mM creatine phos-phate) to prewarmed reaction mixtures containing the indicatedconcentrations of protease and accessory factors. Reactions wereperformed at 30 °C in H-buffer [20 mM Hepes–KOH, pH 7.5,100 mM KCl, 10 mM MgCl2, 10% (vol/vol) glycerol, 5 mMβ-mercaptoethanol] and monitored with a Spectramax M2(Molecular Devices) plate reader with excitation and emissionwavelengths of 420 and 520 nm, respectively.To assay phosphorylation dependence of CtrA degradation,

CpdR (20 μM) was preincubated (40 min at 30 °C) with a CckA/ChpT-based phosphorelay and 5 mM ATP. The entire mix wasthen diluted 80-fold into a degradation reaction containing2 μM GFP–CtrA–RD+15, 1 μM PopA, 20 μM cyclic diguanylate(cdG), 0.4 μM ClpX6, and 0.8 μM ClpP14. As a control, a non-phosphorylatable variant of CpdR (CpdR–D51A) was subjectedto same experimental conditions.

In Vivo Stability of Truncated CtrA Variants. CB15N cells harboringa plasmid expressing the wild-type CtrA–RD+15 or an aminoacid variant from the xylX promoter were grown in M2Gmedium. Xylose (0.3%) was added to exponential-phase cul-tures for 2 h before the cells were harvested by centrifugationfor 15 min at 13,000 × g. Swarmer cells were isolated by dif-ferential centrifugation as described (4). Swarmer cells werereleased into M2G medium without xylose to halt transcrip-tion from xylX, and samples of equal volume were removed atintervals during the first half of the cell cycle. Samples were

Smith et al. www.pnas.org/cgi/content/short/1407862111 1 of 8

analyzed by SDS/PAGE and Western blotting with anti-CtrAantiserum (1:10,000; ref. 10) and HRP-conjugated anti-rabbitantibodies. Western blots were visualized with Western Lightning

(Perkin-Elmer) on a Bio-Rad Gel Doc XL and quantified byusing Imagelab (Version 4.0). The data were fitted to singleexponential curves to calculate protein half-lives.

1. Evinger M, Agabian N (1977) Envelope-associated nucleoid from Caulobactercrescentus stalked and swarmer cells. J Bacteriol 132(1):294–301.

2. Ely B (1991) Genetics of Caulobacter crescentus. Methods Enzymol 204:372–384.3. Meisenzahl AC, Shapiro L, Jenal U (1997) Isolation and characterization of a xylose-

dependent promoter from Caulobacter crescentus. J Bacteriol 179(3):592–600.4. Reisinger SJ, Huntwork S, Viollier PH, Ryan KR (2007) DivL performs critical cell cycle

functions in Caulobacter crescentus independent of kinase activity. J Bacteriol189(22):8308–8320.

5. Higuchi R, Krummel B, Saiki RK (1988) A general method of in vitro preparation andspecific mutagenesis of DNA fragments: Study of protein and DNA interactions.Nucleic Acids Res 16(15):7351–7367.

6. Gora KG, et al. (2013) Regulated proteolysis of a transcription factor complex is criticalto cell cycle progression in Caulobacter crescentus. Mol Microbiol 87(6):1277–1289.

7. Lessard JC (2013) Growth media for E. coli. Methods Enzymol 533:181–189.8. Abel S, et al. (2011) Regulatory cohesion of cell cycle and cell differentiation through

interlinked phosphorylation and second messenger networks.Mol Cell 43(4):550–560.9. Chien P, Perchuk BS, Laub MT, Sauer RT, Baker TA (2007) Direct and adaptor-

mediated substrate recognition by an essential AAA+ protease. Proc Natl Acad SciUSA 104(16):6590–6595.

10. Quon KC, Marczynski GT, Shapiro L (1996) Cell cycle control by an essential bacterialtwo-component signal transduction protein. Cell 84(1):83–93.

* * * *

Fig. S1. Bioinformatic search for the receiver domain degradation signal in CtrA. Amino-terminal regions of CtrA receiver domains from each of 26 genera inthe α-proteobacteria were aligned by using Clustal Omega. Organisms whose genomes encode homologs of RcdA and CpdR are shaded light gray, and or-ganisms whose genomes lack these genes are unshaded. CtrA residues highly conserved in bacteria with homologs of rcdA and cpdR, but divergent in specieslacking these genes, are highlighted in yellow. Asterisks mark intervals of 10 residues. Species and National Center for Biotechnology Information genomeaccession numbers associated with the CtrA receiver domain sequences are as follows: Rhizobium etli CFN 42, NC_007761.1; Agrobacterium tumefaciensLBA2413, CP007225.1; Sinorhizobium meliloti SM11, CP001830.1; Mesorhizobium loti MAFF303099, BA000012.4; Brucella melitensis biovar Abortus 2308,NC_007618.1; Stappia aggregata, renamed Labrenzia aggregata IAM 12614, AAUW00000000.1; Bradyrhizobium japonicum USDA 6, NC_017249.1; Nitrobacterwinogradskyi Nb-255, CP000115.1, Rhodopseudomonas palustris BisB5, CP000283.1; Azorhizobium caulinodans ORS 571, NC_009937.1; Xanthobacter auto-trophicus Py2, CP000781.1; Methylobacterium extorquens HTCC2597, AAMO01000004.1; Roseobacter litoralis Och 149, CP002623.1; Roseovarius sp. 217,AAMV01000001.1; Rhodobacter sphaeroides 2.4.1, CP000143.2; Sagittula stellata E-37, AAYA01000004.1; Dinoroseobacter shibae DL12 = DSM16493,CP000830.1; Sulfitobacter sp. EE-36, AALV01000002.1; Loktanella vestfoldensis SKA53, AAMS01000005.1; Jannaschia sp. CCS1, NC_007802.1; Sphingomonaswittichii RW1, NC_009511.1; Paracoccus denitrificans, PD1222, NC_008687.1.

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CtrA

CtrA

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α1-RD+15min0 15 30 45 60 75 90 105 120

β1 α1CtrA MRVLLIEDDS ATAQTIELML KSEGFNVCzcR MRVLLIEDES EMANLIEITL ASEGIVC ********:* *: **: * ***:

A

α1 β2 α2S ATAQTIELML KSEGFNVYTT DLGEEGVDLG KIYS EMANLIEITL ASEGIVCDKA SVGVEGLRLG KVG* *: **: * ***: .: .:* **: ** *:

CtrACzcR

B

Fig. S2. Substitution of α-helix 1 from CtrA confers degradation on CzcR–RD+15. (A) Alignment of the first 43 amino acids of C. crescentus CtrA andRickettsia prowazekii CzcR. Predicted secondary structures are indicated above the alignment. Gray shading indicates residues that usually differ betweenα-proteobacteria containing and lacking rcdA and cpdR, but that are identical in C. crescentus and R. prowazekii, which lacks rcdA and cpdR. Yellow shadingindicates residues that differ between α-proteobacteria containing and lacking rcdA and cpdR, and which are also different in C. crescentus and R. prowazekii.Arrows flank the sequences from CtrA that were substituted into the corresponding region of CzcR–RD+15 to create α1–RD+15. (B) Swarmer cells of CB15Nexpressing CtrA–RD+15, CzcR–RD+15, or α1–RD+15 were isolated and allowed to progress synchronously through the cell cycle. Aliquots of equal volume wereremoved at the indicated time points, and samples were analyzed by SDS/PAGE and Western blotting with anti-CtrA antiserum. CzcR–RD+15 is thought toappear as a doublet due to an alternative methionine start site at position 12.

YFP

DIC

CtrA-RD+15 CtrA3-RD+15 CtrA6-RD+15

Fig. S3. CtrA3 is diffuse, whereas CtrA6 aggregates in Caulobacter. Expression of the indicated YFP fusion proteins was induced for 4 h during exponentialgrowth phase. Cells of each strain were concentrated, immobilized on agarose pads, and visualized with differential interference contrast and fluorescencemicroscopy. White arrows indicate polar foci of YFP–CtrA–RD+15 in swarmer and predivisional cells. White arrowheads indicate randomly placed fluorescentaccumulations of YFP–CtrA6–RD+15. (Insets) A rare instance of polar localization of YFP–CtrA3–RD+15.

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0.1

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CtrA3CtrA3

half-life (min)14.38.326.819.723.9

Fig. S4. CtrA3 is degraded more slowly in vivo than wild-type CtrA. Mixed, exponential-phase cultures of cells lacking the native ctrA allele and expressingeither CtrA (KR3510) or CtrA3 (KR3512) from the xylX locus were grown in PYE medium containing 0.5% xylose. Chloramphenicol was added to a finalconcentration of 100 μg/mL to block translation, and samples withdrawn at the indicated times were analyzed by SDS/PAGE and Western blotting with anti-CtrA antiserum.

CtrA + ClpXP

CtrA + AllCtrA3+ ClpXP

CtrA3 + Alltime (min) 5 15 25 350 10 20 30

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CtrA + AllCtrA3 + ClpXP

CtrA3 + Alltime (min) 5 25 55 1200 15 40 90

A

B

no addition of DNA or SciP

with PpilA DNA and SciP

Fig. S5. Accessory proteins and cdG stimulate CtrA proteolysis in vitro. (A) Representative gels corresponding to Fig. 4A. (B) Representative gels correspondingto Fig. 4B.

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CpdR (CtrA substrate)CpdR (CtrA3 substrate)

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Fig. S6. Stability of accessory factors during CtrA proteolysis. (A) Quantification of CpdR band intensities from SDS/PAGE gels corresponding to Fig. 4A. (B)Quantification of RcdA band intensities from SDS/PAGE gels corresponding to Fig. 4A. Error bars indicate SDs. (C) Quantification of PopA band intensities froma CtrA degradation experiment containing 1 μM CtrA, 1 μM RcdA, 2 μM CpdR, 1 μM PopA, 20 μM cdiGMP, 0.4 μM ClpX6, and 0.8 μM ClpP14, and an ATPregeneration system.

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Fig. S7. The accessory factors do not accelerate GFP–ssrA proteolysis. Degradation of GFP–ssrA was monitored by following loss of fluorescence. Reactionscontain 2 μM substrate, 0.4 μM ClpX6, 0.8 μM ClpP14, and an ATP regeneration system. In addition to the basic protease components, reactions labeled All alsoinclude 1 μM RcdA, 2 μM CpdR, 1 μM PopA, and 20 μM cdG. Graph illustrates the fraction of fluorescence remaining over time.

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Table S1. Strains and plasmids used in this study

Number Description Source

C. crescentus strainsCB15N Synchronizable derivative of wild-type CB15 Ref. 1KR476 CB15N + pKR146 Ref. 2KR547 CB15N + pKR155 Ref. 2KR563 CB15N + pEJ146 Ref. 2KR2841 CB15N + pSS17 This workKR2904 CB15N + pAK36 This workKR2905 CB15N + pAK29 This workKR3178 ctrA::spec xylX::ctrA-3xFLAG This workKR3179 ctrA::spec xylX::ctrA3-3xFLAG This workKR3205 CB15N + pKZ8A This workKR3566 CB15N + pDW13 This workKR3571 CB15N + pSS73 This workKR3573 CB15N + pSS90.1a This workKR3510 ctrA::spec xylX::ctrA This workKR3512 ctrA::spec xylX::ctrA3 This workLS4379 hfq::hfq-m2 Ref. 3

E. coli strainsKR30 BL21 + pKR14 This workKR1838 BL21 Tuner + pJT20 This workKR2096 BL21 Tuner + pJT31 This workKR2278 BL21 Tuner + pES53 Ref. 4KR2609 BL21 Tuner + pES118 This workKR2854 BL21 Tuner + pSS39 This workKR2861 BL21 Tuner + pSS44 This workKR3569 BL21 Tuner + pSS142 This workKR3574 BL21 Tuner + pKZ13b This workKR3419 BL21 Tuner + pHIS–sciP Ref. 5

PlasmidspJS14 Broad-host-range, high-copy vector derived from pBBR1MCS, chlorR Ref. 6pMR10 Broad-host-range, low-copy vector, kanR Ref. 7pMR20 Broad-host-range, low-copy vector, tetR Ref. 7pXGFPC4 Integration vector for gene expression from xylX Ref. 8pKR146 pJS14–Pxyl::CzcR–RD+15 Ref. 2pKR155 pJS14–Pxyl::CtrA–RD+15 Ref. 2pEJ146 pMR10–Pxyl::YFP–CtrA–RD+15 Ref. 2pDW13 pMR10–Pxyl::YFP–CtrA3–RD+15 This workpAK29 pMR10–Pxyl::YFP–CtrA6–RD+15 This workpAK36 pJS14–Pxyl::CtrA–RD+15 (Q14K) This workpSS73 pJS14–Pxyl::CtrA–RD+15 (Q14K, K21A) This workpKZ8A pJS14–Pxyl::CtrA3–RD+15 (A11T, Q14K, K21A) This workpSS17 pJS14–Pxyl::CtrA–RD+15 (S10A, A11T, K21A, E23A) This workpSS90.1a pJS14–Pxyl::αRD+15 This workpKR14 pET28a–His6–ClpX This workpJT20 pET29–ClpP–His6 This workpJT31 pET28a–His6–PopA This workpES53 pET28a–His6–RcdA Ref. 4pES118 pET28a–His6–EnvZ HK This workpSS39 pET33b–His6–CtrA This workpSS44 pET33b-His6-CtrA-DD This workpSS142 pET28-His6-Smt3-CpdR This workpKZ13b pET42-His6-CtrA3 This workpHIS-sciP pET-His6-sciP Ref. 5pKT1 pXGFPC4-xylX::ctrA This workpSS129 pXGFPC4-xylX::ctrA-3xFLAG This workpSS133 pXGFPC4-xylX::ctrA3-3xFLAG This workpSS135 pXGFPC4-xylX::ctrA3 This work

1. Evinger M, Agabian N (1977) Envelope-associated nucleoid from Caulobacter crescentus stalked and swarmer cells. J Bacteriol 132(1):294–301.2. Ryan KR, Judd EM, Shapiro L (2002) The CtrA response regulator essential for Caulobacter crescentus cell-cycle progression requires a bipartite degradation signal for temporally

controlled proteolysis. J Mol Biol 324(3):443–455.3. Iniesta AA, Hillson NJ, Shapiro L (2010) Cell pole-specific activation of a critical bacterial cell cycle kinase. Proc Natl Acad Sci USA 107(15):7012–7017.4. Taylor JA, Wilbur JD, Smith SC, Ryan KR (2009) Mutations that alter RcdA surface residues decouple protein localization and CtrA proteolysis in Caulobacter crescentus. J Mol Biol

394(1):46–60.

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5. Gora KG, et al. (2010) A cell-type-specific protein-protein interaction modulates transcriptional activity of a master regulator in Caulobacter crescentus. Mol Cell 39(3):455–467.6. Kovach ME, Phillips RW, Elzer PH, Roop RM, II, Peterson KM (1994) pBBR1MCS: A broad-host-range cloning vector. Biotechniques 16(5):800–802.7. Roberts RC, et al. (1996) Identification of a Caulobacter crescentus operon encoding hcrA, involved in negatively regulating heat-inducible transcription, and the chaperone gene grpE.

J Bacteriol 178(7):1829–1841.8. Thanbichler M, Iniesta AA, Shapiro L (2007) A comprehensive set of plasmids for vanillate- and xylose-inducible gene expression in Caulobacter crescentus. Nucleic Acids Res 35(20):e137.

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