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Molecular Cell, Volume 43

Supplemental Information

Regulatory Cohesion of Cell Cycle and Cell

Differentiation through Interlinked

Phosphorylation and Second Messenger Networks Sören Abel, Peter Chien, Paul Wassmann, Tilman Schirmer, Volkhard Kaever, Michael T. Laub, Tania A. Baker, and Urs Jenal

Supplemental Experimental Procedures Strains, plasmids, and media

The bacterial strains and plasmids used in this study are listed in Supplemental Table S1. Caulobacter crescentus strains were grown at 30°C in one of the following media as indicated: peptone yeast extract (PYE), M2 minimal medium supplemented with 0.1% glucose (M2G) and/or 0.1% xylose (M2X/M2GX) (Ely, 1991) or Hutner base-imidazole-buffered-glucose-glutamate (HIGG) medium with 0.2 mM (high phosphate) or 30 µM PO4

3- (low phosphate). Escherichia coli strains were grown at 30°C or 37°C in Luria Broth (LB) or MacConkey base medium supplemented with 1% maltose. When appropriate, media were supplemented with antibiotics at the following concentrations: (solid/liquid media for E. coli; solid/liquid media for C. crescentus; in µg/ml): ampicillin (100/50; n.a./n.a.), gentamycin (20/15; 5/0.5), kanamycin (50/30; 20/5), nalidixic acid (n.a./n.a.; 20/n.a.), oxytetracycline (12.5/12.5; 5/2.5), spectinomycin (100/50; 50/25), and streptomycin (50/50; 5/5). For synchronization experiments, newborn swarmer cells were isolated by Ludox density gradient centrifugation as described before (Jenal and Shapiro, 1996). If necessary, inducible promoters were spiked three hours prior to the density gradient centrifugation. Plasmids were transformed into electro- or TSS-competent E. coli (Chung et al., 1989). Strain DH5α was used for cloning and plasmid propagation, strain S17-1 was used for mobilization of plasmids into C. crescentus by bacterial conjugation (Ely, 1991), strain BL21 (DE3) pLys was used for protein overexpression, and strain MG1655 cyaA::frt was used for bacterial two hybrid assays. In frame deletions were constructed by allelic exchange as described before (Jenal and Shapiro, 1996). Detailed protocols of strain and plasmid construction are available on request. Motility agar plates

Motility of C. crescentus was scored on semi-solid (0.3%) PYE agar plates. After three days of growth, the plates were scanned with a ScanMaker i800 scanner (Microtek, Germany) in transparency mode and the area of colonies was quantified using the “analyze particle” function of imageJ software. Motile suppressors of a C. crescentus pdeA mutant were isolated on semisolid agar plates (motility plates) as spontaneous motile suppressors originating from a non-motile colony.

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Attachment assays and holdfast staining To quantify the ability of C. crescentus to attach to inorganic surfaces, cells were

inoculated in PYE and cultivated in 96-well microtiter plates at 200 rpm on a rocking platform. After 24 hours planktonic cells were discarded and bacteria attached to the polystyrene surface were stained with crystal violet (0.3% crystal-violet, 5% isopropanol, 5% methanol). After washing with water, the dye retained was dissolved in 20% acetic acid and quantified in a photospectrometer (Genesys6, Thermo Spectronic, USA) at 600 nm. C. crescentus holdfast was stained with Oregon Green-conjugated wheat-germ agglutinin (0.2 mg/ml, Invitrogen, USA) and visualized by fluorescence microscopy. Immunoblotting and antibody production

Purified PdeA (Christen et al., 2005) and DgcB (see Experimental Procedures) were injected into rabbits for polyclonal antibody production (Laboratoire d’Hormonologie Marloie, Belgium). Immunoblotting was carried out using rabbit polyclonal antisera against PdeA (1:1,000), PleD (1:5,000), CtrA (1:5,000), DgcB (1:10,000), FliF (1:10,000), Clp (1:10,000), and ClpX (1:10,000). Detection was performed with secondary antibodies directed against rabbit antibodies and conjugated to horseradish peroxidase (1:10,000; Dako, Denmark). After incubation with ECL chemiluminescent substrate (Perkin Elmer, USA), Super RX X-ray films (Fuji, Japan) were used to detect luminescence. Band intensities were quantified using the integrated density tool from imageJ after scanning the exposed X-ray films. Di-guanylate cyclase activity assay

Enzymatic experiments were performed in a 20 mM Tris-HCl pH 8.0, 100 mM NaCl buffer with 26 µM purified DgcB. The reaction was started by the addition of 500 µM GTP and stopped by heat inactivation of the protein at 95°C for 1 min. Heated samples were filtered (Ultrafree-MC, 0.22 µm, Millipore, USA) and diluted 1:10 in running buffer (5 mM NH4HCO3). Products of the enzymatic reaction were analyzed by FPLC-anion exchange chromatography on a ResourceQ column attached to an ÄKTApurifier (GE Healthcare, UK). Elution was accomplished with a shallow NH4HCO3 gradient ranging from 5 to 1,000 mM. C-di-GMP concentrations were determined by integration of the area under the peak and comparison to c-di-GMP standards with known concentrations. Cyclic di-GMP measurements

C-di-GMP was quantified according to Spangler et al. (Spangler et al., 2010). In brief, C. crescentus cells were grown to mid-exponential phase in PYE. The growth medium was removed and c-di-GMP was extracted by acetonitril/methanol/water (40/40/20). The solvent was evaporated at 40°C under nitrogen gas stream and analyzed by reversed phase-coupled HPLC-MS/MS (Perkin Elmer Instruments, USA; Applied Biosystems Inc, USA). Extractions were performed from three independent bacterial cultures and normalized to the optical density of the culture. Size exclusion chromatography coupled multiangle light scattering (SEC-MALS)

SEC-MALS experiments were carried out using the ÄKTApurifier (GE Healthcare, UK) connected to a Superdex200 HR10/300 column (GE Healthcare, UK), a three-angle static light scattering detector (miniDAWN, Wyatt Technology Corporation, USA) and a refractive index detector (Optilab rEX, Wyatt Technology Corporation, USA). Bovine serum albumin (Sigma, USA) was used for normalization of the SEC-MALS hardware. Measurements were performed at flow rates of 0.7 ml/min. Concentrations of the eluted protein were monitored by the differential

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refractive index, which in combination with the light scattering signal was used for molecular weight calculations using the program ASTRA5.3 (Wyatt Technology Corporation, USA). Pulse-chase and chloramphenicol run-off experiments

To determine the stability of PdeA, C. crescentus cultures were grown in M2G medium to mid-exponential phase. Cells were pulse labeled with 20 μCi/ml L-[35S]-methionine/cysteine labeling mix (EasyTag EXPRE35S, NEN Perkin Elmer) for 4 min, followed by a chase with 1 mM unlabeled methionine, 20 μM cysteine and 0.2% trypton. Samples were taken at intervals during the chase period and cells were lysed in 10 mM Tri-HCl pH 7.5, 1% SDS, 1 mM EDTA and boiled for 2 min. The bacterial lysate was diluted 1.3 fold with low salt washing buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 50 mM EDTA, 1% Triton X-100) and pretreated with protein-A beads (Roche, Switzerland). The cell lysate was then incubated with protein A-agarose loaded with α-PdeA antibodies. Beads were washed once with low salt washing buffer and twice with high salt washing buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 50 mM EDTA, 1% Triton X-100) and proteins released by boiling samples in SDS-sample buffer (2% SDS, 50 M Tris-HCl pH 6.8, 0.1 M dithiothreitol, 0.1% bromphenol blue, 10% glycerol). The protein samples were separated on a SDS-PAGE, the gels were fixed in 30% methanol, 10% acetic acid, dried, exposed to a phosphoimager screen (Molecular Dynamics, USA) and quantified using imageJ software. The stability of CtrA was assayed after blocking protein synthesis in mid-exponential growth phase of C. crescentus with chloramphenicol (10 µg/ml final concentration). After adding the antibiotic samples were harvested every 15 min and CtrA levels were detected with specific antibodies in immunoblots.

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Figure S1. Biochemical Characterization of DgcB, Related to Figure 1 (A) DgcB is a di-guanylate cyclase. Purified DgcB was assayed for the ability to convert GTP into c-di-GMP. GTP (solid line) and nucleotides after the enzymatic reaction (pointed line) were separated by FPLC. A c-di-GMP standard is indicated as control (dashed line). (B) Metal dependence of DgcB diguanylate cyclase activity. The catalytic activity of DgcB was characterized varying the concentrations of magnesium (full black rectangles) and calcium (in presence of 2 mM Mg2+) (open grey rectangles). The product quantity was normalized to the c-di-GMP amount produced by DgcB in presence of 5 mM Mg2+. (C) Characterization of DgcB di-guanylate cyclase activity as a function of pH. C-di-GMP produced by DgcB was quantified with FPLC. Different pH values were adjusted by exchanging the Tris-HCl in the reaction buffer by NaAcetate pH 5.5; MES pH 6.5; HEPES pH 7.2; Tris pH8.0 or 9.0 and CAPS pH 10.0 (100 mM each). (D) Analysis of DgcB oligomerization state. SEC MALS experiments were performed with DgcB at a concentration of 26 µM. The analysis includes monitoring of the UV signal (red stripped line), differential refractive index signal (black dotted line) and the light scattering signal (black line) in the range between the column void volume (V0) and the total column volume (Vt). The blue lines indicate the calculated molecular weight.

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Figure S2. DgcB and PleD Synergistically Promote Stalk Elongation, Related to Figure 1 Box plot representation of stalk length measurements. C. crescentus strains were either grown in balanced HIGG medium containing 0.2 mM PO4

3- (A) or in low phosphate HIGG medium containing 30 µM PO4

3- (B) to mid-exponential growth phase. The length of at least 100 stalks was determined; cells without stalk were omitted from the analysis. The box plot function of the R language for statistical computing (Ihaka, R., and R. Gentleman. 1996. R: a language for data analysis and graphics. J. Comput. Graph. Stat. 5:299–314.; www.r-project.org) was used for data analysis. Big middle lines indicate the median of the sample. The box indicates the interquartile range and the whiskers include all data points not considered as outliers. Circles mark individual outliers.

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Figure S3. PdeA and DgcB Localize to the Cell Poles in Swarmer and Predivisional Cells, Related to Figure 2 Analysis of VEN-PdeA (A) or PdeA-YFP (B) fusion protein distribution throughout the cell cycle. Cells from a synchronous population of C. crescentus ΔpdeA expressing ven-pdeA (A) or pdeA-yfp (B) were analyzed microscopically. DIC images and corresponding YFP channels are shown. In parallel, immunoblot analysis of C. crescentus ΔpdeA expressing ven-pdeA (A) or wild-type expressing pdeA-yfp (B) with an anti-PdeA antibody was used to document cell cycle-fluctuations of the fusion proteins (panels above micrographs). Cell cycle progression is depicted

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schematically above the corresponding graphs. White arrows highlight polar PdeA foci. The grey bar underneath the micrographs indicates the time window during which PdeA is localized to the cell pole. (C) Time-lapse microscopy with cells expressing a DgcB-GFP fusion protein. White arrows indicate polar clusters of DgcB. The progression of cells through the cell cycle is shown schematically above the micrographs (see also Movie S2).

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Figure S4. Complementation Assays with pdeA, dgcB and pleD, Related to Figures 1, 2, and 3 Quantification of motility on semi-solid agar plates and attachment to surfaces of strains expressing (A) DgcB, (B) PleD or (C) PdeA or the indicated fusions to fluorescent proteins. The data have been normalized to the wild type and compared to the deletion mutant, both containing the appropriate control vectors. However, for simplicity only the wild-type and mutant stain without vector are depicted. The mean of at least seven independent colonies for attachment and of three colonies for motility assays are shown. Error bars indicate the standard error of the mean.

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Figure S5. PdeA Localization Is Not Dependent on ClpXP, Related to Figure 3 The wild type, as well as ClpX and ClpP depletion strains expressing GFP fused to the C-terminus of PdeA were grown in the presence (M2X) or absence (M2G) of xylose for 8 h. The absence of xylose blocked ClpX and ClpP expression in the depletion strains. The localization of PdeA was investigated under the fluorescence microscope and DIC as well as GFP channels of the same region are shown. The fraction of cells containing fluorescent foci and the total number of cells counted are indicated below the corresponding pictures. As control for the absence of ClpX or ClpP, respectively, the same samples used for microscopy were tested in immunoblots with antibodies directed against ClpP, ClpX and PdeA.

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Figure S6. PdeA Degradation Is Stimulated by Addition of GTP and Dependent on Dephosphorylated CpdR, Related to Figure 4 (A) Degradation kinetics of PdeA as monitored by release of acid-soluble peptides. PdeA was added at the indicated concentrations to 0.2 µM ClpX, 0.4 µM ClpP and an ATP-regeneration system, either with 1 µM CpdR and 1 mM GTP (red line), with 1 µM CpdR and without GTP (blue line) or without CpdR and GTP (green line). Initial rates of CpdR stimulated PdeA degradation

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were fitted to the Michaelis-Menten equation (KM= 0.95 +/- 0.1 µM; turnover rate = 1.3 +/- 0.05 PdeA/ClpX6/min). (B) Reactivation of CpdR~P by a phosphatase-only mutant of CckA. Activation of PdeA degradation by unphosphorylated CpdR (solid square) is slightly inhibited by addition of the CckA phosphatase mutant (solid triangles). Phosphorylation of CpdR with acetyl-phosphate inhibits degradation of PdeA (open squares) but addition of the phosphatase reactivates CpdR. Replicate experiments in the presence of phosphatase are shown.

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Figure S7. In Vivo Stability of CtrA Determined after Block of Protein Biosynthesis, Related to Figure 6 De novo biosynthesis of proteins was blocked in the indicated strains by addition of chloramphenicol at time point zero. Every 15 min the cellular levels of the ClpXP substrate CtrA and stable control DgcB were analyzed by immunoblot analysis using CtrA and DgcB specific antibodies. A strain expressing FLAG tagged CtrA was used as control for intrinsic stabilized CtrA.

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Table S1. Strains and Plasmids, Related to the Results Caulobacter crescentus strains: Name Genotype Description Source or Reference

NA1000 CB15N Laboratory strain derived from CB15 (Evinger and Agabian, 1977)

CB15 CB15 C. crescentus wild type ATCC 19089 Caulobacter vibrioides LOT: 3967454

(Poindexter and Cohen-Bazire, 1964)

LS2382 NA1000 lon::Ω Disruption of the lon gene by a Strep/SpecR cassette in NA1000

(Wright et al., 1996)

LS4185 NA1000 rcdA::hyg Disruption of the rcdA gene by a HygR cassette in NA1000 by allelic exchange

(McGrath et al., 2006)

LS4218 NA1000 cpdRD51A NA1000 carring cpdRD51A instead of a wild type cpdR (Iniesta et al., 2006)

UJ134 NA1000 hslU::pSA33 Disruption of the hslU gene by pSA33 in NA1000 this study

UJ199 NA1000 clpP::Ω::pUJ174

Disruption of the clpP gene by a Strep/SpecR

cassette and integration of clpP at the xyl locus in NA1000

(Jenal and Fuchs, 1998)

UJ200 NA1000 clpX::Ω::pUJ175

Disruption of the clpX gene by a Strep/SpecR

cassette and integration of clpX at the xyl locus in NA1000

(Jenal and Fuchs, 1998)

UJ838 NA1000 clpA::Ω Disruption of the clpA gene by an Strep/SpecR

cassette in NA1000 by allelic exchange (Grunenfelder et al., 2004)

UJ945 NA1000 ftsH::Ω Disruption of the ftsH gene by an Strep/SpecR

cassette in NA1000 by allelic exchange (Fischer et al., 2002)

UJ1249 NA1000 pMO88 NA1000 containing pMR20 carrying clpXATP* under control of the xyl promotor

(Potocka et al., 2002)

UJ2827 NA1000 ∆popA markerless in frame deletion of popA in NA1000 by allelic exchange using plasmid pAD8

(Duerig et al., 2009)

UJ4373 NA1000 cpdR::tetR Disruption of the cpdR gene by a TetR cassette in NA1000 by allelic exchange

(Skerker et al., 2005)

UJ4449 NA1000 ∆dgcB markerless in frame deletion of dgcB in NA1000 by allelic exchange using plasmid pAD7

this study

UJ4450 NA1000 ∆pleD markerless in frame deletion of pleD in NA1000 by allelic exchange using plasmid pSA95

this study

UJ4454 NA1000 ∆pdeA markerless in frame deletion of pdeA in NA1000 by allelic exchange using plasmid pSA81

this study

UJ4462 CB15 ∆dgcB markerless in frame deletion of dgcB in CB15 by allelic exchange using plasmid pAD7

this study

UJ4463 CB15 ∆pleD markerless in frame deletion of pleD in CB15 by allelic exchange using plasmid pSA95

this study

UJ4467 CB15 ∆pdeA markerless in frame deletion of pdeA in CB15 by allelic exchange using plasmid pSA81

this study

UJ4731 NA1000 pSA120 NA1000 containing plasmid pSA120 this study

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UJ4732 NA1000 ∆pdeA pSA120

NA1000 ∆pdeA containing plasmid pSA120 this study

UJ4736 NA1000 ∆popA pSA120

NA1000 ∆popA containing plasmid pSA120 this study

UJ4737 NA1000 rcdA::hygR pSA120

NA1000 rcdA::hygR containing plasmid pSA120 this study

UJ5780 NA1000 ∆dgcB ∆pdeA

markerless in frame deletion of dgcB in NA1000 ∆pdeA by allelic exchange using plasmid pAD7

this study

UJ5781 NA1000 ∆pleD ∆pdeA

markerless in frame deletion of pleD in NA1000 ∆pdeA by allelic exchange using plasmid pSA95

this study

UJ5783 CB15 ∆dgcB ∆pdeA markerless in frame deletion of dgcB in CB15 ∆pdeA by allelic exchange using plasmid pAD7

this study

UJ5784 CB15 ∆pleD ∆pdeA markerless in frame deletion of pleD in CB15 ∆pdeA by allelic exchange using plasmid pSA95

this study

UJ5786 CB15 ∆dgcB ∆pleD markerless in frame deletion of pleD in CB15 ∆dgcB by allelic exchange using plasmid pSA95

this study

UJ5787 CB15 ∆dgcB ∆pleD ∆pdeA

markerless in frame deletion of pleD in CB15 ∆dgcB ∆pdeA by allelic exchange using plasmid pSA95

this study

UJ5788 NA1000 pSA115 NA1000 containing plasmid pSA115 this study

UJ5790 NA1000 ∆dgcB ∆pleD

markerless in frame deletion of dgcB in NA1000 ∆pleD by allelic exchange using plasmid pAD7

this study

UJ5791 NA1000 ∆dgcB ∆pleD ∆pdeA

markerless in frame deletion of dgcB in NA1000 ∆pleD ∆pdeA by allelic exchange using plasmid pAD7

this study

UJ5792 NA1000 cpdR::tetR pSA120

NA1000 cpdR::tetR containing plasmid pSA120 this study

UJ5793 NA1000 pdeA::pSA122

Integration of pSA122 into the pdeA locus of NA1000. Fuses an flag tag to the 3` end of pdeA

this study

UJ5794 CB15 pdeA::pSA122 Integration of pSA122 into the pdeA locus of CB15. Fuses an flag tag to the 3` end of pdeA

this study

UJ5795 NA1000 ∆pdeA pSA127

NA1000 ∆pdeA containing plasmid pSA127 this study

UJ5796 CB15 ∆dgcB pdeA::pSA122

Integration of pSA122 into the pdeA locus of CB15 ∆dgcB. Fuses an flag tag to the 3` end of pdeA

this study

UJ5797 CB15 ∆pleD pdeA::pSA122

Integration of pSA122 into the pdeA locus of CB15 ∆pleD. Fuses an flag tag to the 3` end of pdeA

this study

UJ5798 CB15 ∆dgcB ∆pleD pdeA::pSA122

Integration of pSA122 into the pdeA locus of CB15 ∆dgcB ∆pleD. Fuses an flag tag to the 3` end of pdeA

this study

UJ5799 NA1000 ∆pdeA pSA148

NA1000 ∆pdeA containing plasmid pSA148 this study

UJ5801 CB15 ∆pdeA pSA148 CB15 ∆pdeA containing plasmid pSA148 this study

UJ5802 CB15 ∆pdeA pSA115 CB15 ∆pdeA containing plasmid pSA115 this study

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UJ5803 NA1000 ∆dgcB pAD17

NA1000 ∆dgcB containing pAD17 this study

UJ5804 NA1000 clpP::Ω::pUJ174 pSA115

NA1000 clpP::Ω::pUJ174 containing pSA115 this study

UJ5805 NA1000 clpX::Ω::pUJ175 pSA115

NA1000 clpX::Ω::pUJ175 containing pSA115 this study

UJ5806 NA1000 ∆dgcB pdeA::pSA122

Integration of pSA122 into the pdeA locus of NA1000 ∆dgcB. Fuses an flag tag to the 3` end of pdeA

this study

UJ5807 NA1000 ∆pleD pdeA::pSA122

Integration of pSA122 into the pdeA locus of NA1000 ∆pleD. Fuses an flag tag to the 3` end of pdeA

this study

UJ5808 NA1000 ∆dgcB ∆pleD pdeA::pSA122

Integration of pSA122 into the pdeA locus of NA1000 ∆dgcB ∆pleD. Fuses an flag tag to the 3` end of pdeA

this study

UJ5809 NA1000 pdeA::pSA144

Integration of pSA144 into the pdeA locus of NA1000. Exchanges the last 6 bp at the 3` end of pdeA from CGGGGT to GACGAC leading to PdeARG554DD

this study

UJ5811 CB15 ∆dgcB pAD17 CB15 ∆dgcB containing plasmid pAD17 this study

UJ5957 CB15 ∆pdeA CB15 ∆pdeA containing plasmid pSA237 this study

UJ5958 NA1000 ∆pdeA NA1000 ∆pdeA containing plasmid pSA237 this study

UJ5959 CB15 ∆dgcB CB15 ∆dgcB containing plasmid pSA162 this study

UJ5960 CB15 ∆pleD CB15 ∆pleD containing plasmid pSA164 this study

UJ5961 CB15 ∆pdeA CB15 ∆pdeA containing plasmid pSA168 this study

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Escherichia coli strains: Name Genotype Description Source or Reference

BL21 (DE3)

F- dcm ompT hsdS(rB-mB-) pLysS gal λ (DE3) [pLysS CAMR]

Used for protein overexpression from pET based plasmids

Stratagene

AB1768 MG1655 ΔcyaA::frt Used for bacterial two hybrid screen expressing pUT18, pUT18C and pKT25 based split cyaA fusion proteins

S. Steiner

S17-1 RP4-2, Tc::Mu, KM-Tn7

Used as donor strain in conjugations to transfer oriT containing plasmids to C. crescentus

(Simon et al., 1983)

DH5α (F-) F` endA1 hsdR17 (rK-mK plus) glnV44 thi1 recA1 gyr ∆(NalR) relA1 ∆(lacIZYA-argF)U169 deoR (Φ80dlac ∆(lacZ) M15)

Used for general cloning purposes (Woodcock et al., 1989)

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Plasmids: Name Description Source or Reference

pAD7 pNPTS138; used for inframe deletion of dgcB A. Dürig

pAD17 pMR20; dgcB 3` fused to egfp under control of the lac promotor A. Dürig

pAD44 pUT18C; clpX 3` fused to the T18 fragment (Duerig et al., 2009)

pAD53 pUT18C; cpdR 3` fused to the T18 fragment (Duerig et al., 2009)

pAD54 pKT25; cpdR 3` fused to the T25 fragment (Duerig et al., 2009)

pAD58 pUT18; clpX 5` fused to the T18 fragment (Duerig et al., 2009)

pAD62 pUT18; cpdR 5` fused to the T18 fragment (Duerig et al., 2009)

pcpdRD51A pNPTS138; carrying cdpRD51A and 500 bp upstream and downstream (Duerig et al., 2009)

pET21c::dgcB pET21c; dgcB, 3` fused to a his6 tag this study

pKT25-zip pKT25; region coding for the leucine zipper of GCN4 3` fused to the T25 fragment

(Karimova et al., 1998)

pRP90 pET11; pleD*D53N, 3` fused to a his6 tag (Paul et al., 2004)

pSA33 pBGS18T; carrying a 500 bp fragment of hslU A.Stotz

pSA67 pKT25; pleD 3` fused to the T25 fragment this study

pSA72 pUT18C; pleD 3` fused to the T18 fragment this study

pSA77 pUT18; pleD 5` fused to the T18 fragment this study

pSA81 pNPTS138; used for inframe deletion of pdeA this study

pSA95 pNPTS138; used for inframe deletion of pleD this study

pSA115 pMR10; pdeA 3` fused to egfp under control of the pdeA promotor this study

pSA116 pNPTS; 3` region of pdeA 5` fused to egfp this study

pSA118 pPHU281; 3` region of dgcB 5` fused to myfp this study

pSA120 pMR10; pdeA 3` fused to myfp under control of the pdeA promotor this study

pSA122 pNPTS138; carrying 300 bp of the 3` end of pdeA 3` fused to a flag tag this study

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pSA127 pXVENN-4; pdeA 5` fused to ven under control of the xyl promotor this study

pSA144 pNPTS138; carrying 300 bp of the 3` end of pdeA with an exchange of the last 6 bp at the 3` end of pdeA from CGGGGT to GACGAC leading to PdeARG554DD

this study

pSA148 pMR10; pdeA 5` fused to ven under control of the pdeA promotor this study

pSA149 pNPTS138; used for integration of a ven tag between the promotor of pdeA and the pdeA ORF. Fuses ven to the 5` end of pdeA

this study

pSA162 pMR20; dgcB under control of the dgcB promotor this study

pSA164 pMR10; pleD under control of the pleD promotor this study

pSA168 pMR10; pdeA under control of the pdeA promotor this study

pSA179 pKT25; pdeA 3` fused to the T25 fragment this study

pSA180 pUT18; pdeA 5` fused to the T18 fragment this study

pSA181 pUT18C; pdeA 3` fused to the T18 fragment this study

pSA182 pKT25; dgcB 3` fused to the T25 fragment this study

pSA183 pUT18; dgcB 5` fused to the T18 fragment this study

pSA184 pUT18C; dgcB 3` fused to the T18 fragment this study

pSA225 pKT25; clpX 3` fused to the T25 fragment this study

pSA237 pBBR1-MCS2; pdeA 5` fused to ven under control of the pdeApromotor

this study

pUT18C-zip pUT18C; region coding for the leucine zipper of GCN4 3` fused to the T18 fragment

(Karimova et al., 1998)

pMR10 RK2 based KanR low copy number and broad host range vector with oriT

(Roberts et al., 1996)

pMR20 RK2 based TetR low copy number and broad host range vector with oriT

(Roberts et al., 1996)

pNPTS138 KanR, suicide vector with sacB gene and oriT D. Alley

pXVENN-4 GentR, suicide vector with oriT, integration at the xyl locus, used for N-terminal VEN fusions

(Thanbichler et al., 2007)

pPHU281 TetR, suicide vector with oriT (Hubner et al., 1993)

pBGS18T KanR, suicide vector with oriT (Alley, 2001)

pKT25 KanR, pSU40 derivative, used for fusions to the C-terminus of the T25 fragment of CyaA

(Karimova et al., 1998)

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pUT18 AmpR, pUC19 derivative, used for fusions to the N-terminus of the T18 fragment of CyaA.

(Karimova et al., 1998)

pUT18C AmpR, pUC19 derivative, used for fusions to the C-terminus of the T18 fragment of CyaA.

(Karimova et al., 1998)

pSUMO-His6

PdeA AmpR, pET23b derivative; pdeA 5` fused to a his6 tagged SUMO domain

this study

pSUMO-His6

CpdR

AmpR, pET23b derivative; cpdR 5` fused to a his6 tagged SUMO domain

this study

pSUMO-His6

CpdRD51A

AmpR, pET23b; cpdRD51A 5` fused to a his6 tagged SUMO domain this study

pET28b-CCX pET28b; clpX 5` fused to a his6 tag followed by a thrombin cleavage site

(Chien et al., 2007)

pQE70-CCP pQE70 ; clpP 3` fused to a his6 tag; under control of a lactose inducible promotor

(Chien et al., 2007)

pTRX-chpT pTRX-HIS-DEST; chpT 5` fusion to a his6 tag (Biondi et al., 2006)

pCckAK-P+ p-HIS-DEST; cckAH322A 3` fused to a his6 tag (Chen et al., 2009)

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Thanbichler, M., Iniesta, A.A., and Shapiro, L. (2007). A comprehensive set of plasmids for vanillate- and xylose-inducible gene expression in Caulobacter crescentus. Nucleic Acids Res 35, e137. Woodcock, D.M., Crowther, P.J., Doherty, J., Jefferson, S., DeCruz, E., Noyer-Weidner, M., Smith, S.S., Michael, M.Z., and Graham, M.W. (1989). Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res 17, 3469-3478. Wright, R., Stephens, C., Zweiger, G., Shapiro, L., and Alley, M.R. (1996). Caulobacter Lon protease has a critical role in cell-cycle control of DNA methylation. Genes Dev 10, 1532-1542.