Permissive zones for the centromere-binding protein ParB on the Caulobacter crescentus chromosome

Abstract Proper chromosome segregation is essential in all living organisms. In Caulobacter crescentus, the ParA–ParB–parS system is required for proper chromosome segregation and cell viability. The bacterial centromere-like parS DNA locus is the first to be segregated following chromosome replication. parS is bound by ParB protein, which in turn interacts with ParA to partition the ParB-parS nucleoprotein complex to each daughter cell. Here, we investigated the genome-wide distribution of ParB on the Caulobacter chromosome using a combination of in vivo chromatin immunoprecipitation (ChIP-seq) and in vitro DNA affinity purification with deep sequencing (IDAP-seq). We confirmed two previously identified parS sites and discovered at least three more sites that cluster ∼8 kb from the origin of replication. We showed that Caulobacter ParB nucleates at parS sites and associates non-specifically with ∼10 kb flanking DNA to form a high-order nucleoprotein complex on the left chromosomal arm. Lastly, using transposon mutagenesis coupled with deep sequencing (Tn-seq), we identified a ∼500 kb region surrounding the native parS cluster that is tolerable to the insertion of a second parS cluster without severely affecting cell viability. Our results demonstrate that the genomic distribution of parS sites is highly restricted and is crucial for chromosome segregation in Caulobacter.

, or R104A were restruck on PYE + xylose to induce the expression of the wild-type untagged ParB, or on PYE + glucose to repress the expression of the wild-type untagged ParB, or on PYE + glucose + vanillate to repress the expression of the wild-type untagged ParB while expressing the FLAG-tagged ParB WT, G101S, or R104A. The FLAG-tagged version of wild-type ParB is functional and can complement the depletion of wild-type untagged ParB while the spreading mutant ParB (G101S) or ParB (R1014A) cannot. (B) ChIP-seq profiles of FLAG-ParB (WT), FLAG-ParB (G101S), and (R104A) (using α-FLAG antibody), of CFP-ParB (using α-GFP antibody), and of ParB (using polyclonal α-ParB antibody). Note: the red dagger ( †) symbol on the CFP-ParB ChIP-seq profile indicates the genomic region where sequencing reads were missing. This is because CFP-ParB ChIP-seq reads were mapped to the wild-type Caulobacter reference genome instead of to the genome of parB::cfp-parB strain. Surface Plasmon Resonance (SPR) was used to measure binding affinity of ParB WT, ParB (G101S) and ParB (R104A) at 200 nM to a 24-bp double-stranded DNA that contains parS site 4. The level of ParB variants binding to DNA was expressed as a percentage of the theoretical maximum response, Rmax, assuming a single ParB dimer binding to one immobilized double-stranded DNA oligonucleotides. This normalization process enabled the various responses to be readily compared, irrespective of the quantity and length of the DNA tethered on an SPR chip surface. (C) Immunoblot analysis of FLAG-tagged ParB WT vs. G101S and R104A. Cells were depleted of wild-type untagged ParB for 5 hours, then vanillate was added for an additional hour to allow for expression of FLAG-tagged ParB. Equal amount of total protein was loaded on each well of the SDS-PAGE. The relative immunoblot intensity is indicated below each band.    Interactions between ParB and partners were assessed on a solid MacConkey agar or by β-galactosidase assay. Three biological replicates were performed for each pair of interacting partners. A negative control (T25 fragment alone) and a positive control: T25-ZIP and T18-ZIP were also included. (B) ChIP-seq profiles of T18-ParB at parS site 4, site 6 and site 1 in an E. coli heterologous host. T18-ParB protein was expressed by addition of 500 µM IPTG for an hour before fixing with formadehyde for ChIP-seq. DNA bounds to T18-ParB was immunoprecipitated using α-T18 conjugated sepharose beads. A scrambled parS site 3 was also inserted at the ybbD locus to serve as a negative control. ChIP-seq signals were reported as the number of reads at every nucleotide along the genome (RPBPM value).  Wild-type (black) or Δsmc (red) Caulobacter cells were mutagenized with parS + or parStransposon, and the number of insertions was binned to 10-kb segments along the Caulobacter chromosome. The ratio between insertion frequency for parS + transposon and that of parStransposon was calculated and plotted as a log 10 scale against genomic position.

Plasmids and Strains construction
All strains used are listed in Supplementary Table S1. All plasmids and primers used in strain and plasmid construction are listed in Supplementary Table S2. pMT675::flag-parB (WT) The coding sequence of ParB (CCNA_03868) was amplified from the Caulobacter genomic DNA by PCR using primers flag_parB_F_part1 and flag_parB_R. The PCR product was purified using a Qiagen PCR purification column and used as a template in a second PCR (primer: flag_parB_R and flag_parB_part2) to attach the sequence encoding a flexible linker (GGGS) to parB. The resulting PCR product was purified and used as a template in the third PCR (primer: flag_parB_R and flag_parB_part3) to attach the sequence of the FLAG tag to parB. The final PCR product was gel-purified and assembled to an NdeI-NheI-cut pMT675 (1) using a 2x Gibson master mix (NEB). Briefly, 2.5 µL of each DNA fragment at equimolar concentration was added to 5 µL Gibson master mix (NEB), and the mixture was incubated at 50°C for 60 minutes. 5 µL was used to transform chemically-competent E. coli DH5α cells.
Gibson assembly was possible due to a 23 bp sequence shared between the PCR fragment and the NdeI-NheI-cut pMT675 backbone. The resulting plasmid was sequence verified by Sanger sequencing (Eurofins, Germany).
pMT675::flag-parB (G101S) To introduce point mutation to the coding sequence of ParB, a pair of primers: parB_G101S_F and parB_G101S_R were used in a PCR to amplify around the pMT675::parB (WT) plasmid. DpnI (1 µL) was added to the 50 µL PCR reaction to remove circular template plasmid. The PCR product was precipitated and used to transform chemically-competent E. coli DH5α cells. The resulting plasmid was sequenced by Sanger sequencing (Eurofins, Germany) to confirm the successful introduction of the intended mutation.
pMT675::flag-parB (R104A) The same procedure as above was used to mutagenize arginine 104 to alanine, except that primers:parB_R104A_F and parB_R104A_R were used for PCR.
pET21b::ParB-(His)6, pET21b::ParB(G101S)-(His)6, pET21b::ParB(R104A)-(His)6 pET21b-ParB-(His)6 is a gift from Christine Jacobs-Wagner (2). To introduce point mutation to the coding sequence of ParB, a pair of primers: parB_G101S_F and parB_G101S_R (or parB_R104A_F and parB_R104A_R) were used in a PCR to amplify around the pET21b-ParB-(His)6 plasmid. DpnI (1 µL) was added to the 50 µL PCR reaction to remove circular template plasmid. The PCR product was precipitated and used to transform chemicallycompetent E. coli DH5α cells. The resulting plasmid was sequenced by Sanger sequencing (Eurofins, Germany) to confirm the successful introduction of the intended mutation. pET21b-Spo0J-(His)6 The gene encoding Spo0J was amplified by PCR from Bacillus subtilis genomic DNA using primers 21b-spo0J-F and 21b-spo0J-R. The final PCR product was gel-purified and assembled to an NdeI-HindIII-cut pET21b using a 2x Gibson master mix (NEB). Briefly, 2.5 µL of each DNA fragment at equimolar concentration was added to 5 µL Gibson master mix (NEB), and the mixture was incubated at 50°C for 60 minutes. 5 µL was used to transform chemically-competent E. coli DH5α cells. Gibson assembly was possible due to a 23 bp sequence shared between the PCR fragment and the NdeI-HindIII-cut pET21b backbone. The resulting plasmid was sequence verified by Sanger sequencing (Eurofins, Germany). pENTR::parB The coding sequence of ParB was amplified by PCR from Caulobacter genomic DNA using primers parB_entr_F and parB_entr_R. The backbone of pENTR plasmid was amplified by PCR using primers pENTR_gibson_backbone_F and pENTR_gibson_backbone_R from the pENTR-D-TOPO cloning kit (Invitrogen). The resulting PCR product was subsequently treated with DpnI to remove the methylated template DNA. The two PCR fragments were each gel-purified and assembled together using a 2xGibson master mix (NEB). Gibson assembly was possible due to 23 bp sequence shared between the two PCR fragments. These 23 bp regions were incorporated during the primer design to amplify parB. The resulting plasmid was sequence verified by Sanger sequencing (Eurofins, Germany). pENTR::yfp The same procedure as above was employed to assemble the YFP coding sequence to the pENTR backbone. The yfp gene was amplified by PCR using primers yfp_pentr_F and yfp_pentr_R, and pMT675 (1) as template. pML477::flag-yfp The yfp gene was recombined into a Gateway-compatible destination vector pML477 via LR recombination reaction (Invitrogen). For LR recombination reactions: 1 µL of purified pENTR::yfp was incubated with 1 µL of the destination vector pML477, 1 µL of LR Clonase II mastermix, and 2 µL of water in a total volume of 5 µL. The reaction was incubated for an hour at room temperature before being introduced to DH5α E. coli cells by heat-shock transformation. Cells were then plated out on LB agar + spectinomycin. Resulting colonies were restruck onto LB agar + spectinomycin and LB agar + kanamycin. Only colonies that survived on LB + spectinomycin plates were subsequently used for culturing and plasmid extraction.
pUTC18::parB parB was recombined into a Gateway-compatible bacterial-two hybrid destination vector pUTC18-DEST (3) via LR recombination reaction (Invitrogen). For LR recombination reactions: 1 µL of purified pENTR::parB was incubated with 1 µL of the destination vector pUTC18-DEST, 1 µL of LR Clonase II master mix, and 2 µL of water in a total volume of 5 µL. The reaction was incubated for an hour at room temperature before being introduced to DH5α E. coli cells by heat-shock transformation. Cells were plated out on LB agar + carbecicilin. The resulting colonies were restruck onto LB agar + carbenicilin and LB agar + kanamycin. Only colonies that survived on LB agar + carbenicilin were subsequently used for culturing and plasmid extraction.
pKT25::parB parB was amplified by PCR using primers KT25-parB-F and KT25-parB-R, and pMT675::flag-parB as template. The PCR product was gel-purified and assembled to a BamHI-EcoRI-cut pKT25 (Euromedex) using a 2x Gibson master mix (NEB). Gibson assembly was possible due to a 23 bp sequence shared between the PCR fragment and the BamHI-EcoRI-cut pKT25. These 23 bp regions were incorporated during the primer design to amplify parB. The resulting plasmid was sequence verified by Sanger sequencing (Eurofins, Germany).
pKTN25::parA parA was amplified by PCR from Caulobacter genomic DNA using primers KTN25-parA-F and KTN25-parA-R. The PCR product was gel-purified and digested with BamHI and HindIII. The digested PCR product was further purified using a Qiaquick PCR purification column (Qiagen) before being ligated to BamHI-HindIII-cut pKTN25 (Euromdex) using T4 DNA ligase (NEB). The ligation reaction was composed of 1 µL of BamHI-HindIII-pKTN25, 8 µL of BamHI-HindIII-cut parA PCR product, 1 µL of T4 ligase buffer, and 0.5 µL of T4 ligase enzyme (NEB). The ligation was incubated at room temperature for an hour. 5 µL was used to transform chemically-competent E. coli DH5α cells.
pKTN25::mipZ mipZ was amplified by PCR using primer KTN25-mipZ-F and KTN25-mipZ-R, and Caulobacter genomic DNA as template. The PCR product was gel-purified and assembled to a BamHI-HindIII-cut pKTN25 (Euromedex) using a 2x Gibson master mix (NEB). Gibson assembly was possible due to a 23 bp sequence shared between the PCR fragment and the BamHI-HndIII-cut pKTN25. These 23 bp regions were incorporated during the primer design to amplify mipZ. The resulting plasmid was sequence verified by Sanger sequencing (Eurofins, Germany).
pMT687::parS site 1, 2, 3, 4, 5, 6 and 7 To clone parS site 1 into pMT678 (1) , we first used primers parS_site1_F and pMT687_cir_R to amplify the backbone of pMT687 by PCR. Since the 24 bp sequence of parS site 1 was incorporated in the primer parS_site1_F, the PCR generated a linear DNA fragment that contains both the backbone of pMT687 and parS site 1. DpnI (1 µL) was added to the 50 µL PCR reaction to remove circular methylated template plasmid. The PCR product was then gel-purified, and subsequently phosphorylated using T4 PNK enzyme (NEB). The phosphorylated DNA fragment was religated using T4 DNA ligase (NEB) to regenerate a circular plasmid. 5 µL was used to transform chemically-competent E. coli DH5α cells. The resulting plasmid was sequenced by Sanger sequencing (Eurofins, Germany) to confirm the incorporation of parS site on pMT687 plasmid.
To clone parS site 2 into pMT687, we employed the same procedure as above but using primers parS_site2_F and pMT687_cir_R instead.
To clone parS site 3 into pMT687, we employed the same procedure as above but using primers parS_site3_F and pMT687_cir_R instead.
To clone parS site 4 into pMT687, we employed the same procedure as above but using primers parS_site4_F and pMT687_cir_R instead.
To clone parS site 5 into pMT687, we employed the same procedure as above but using primers parS_site5_F and pMT687_cir_R instead.
To clone parS site 6 into pMT687, we employed the same procedure as above but using primers parS_site6_F and pMT687_cir_R instead.
To clone parS site 7 into pMT687, we employed the same procedure as above but using primers parS_site7_F and pMT687_cir_R instead.
To clone a scrambled parS site 3 into pMT687, we employed the same procedure as above but using primers parS_scrambled_site3_F and pMT687_cir_R instead.
pMCS5-parS 3+4 at +200kb, +1000 kb and +1800kb For insertion of a 260 bp sequence containing Caulobacter parS sites 3 and 4 at +200 kb on the Caulobacter genome, primers label200-NdeI-F and label200-SacI-R were used to amplified a ~500 bp fragment by PCR from the Caulobacter genomic DNA. This fragment was 5' phosphorylated by T4 PNK (NEB) before being blunt-end ligated to a Smal-cut pUC19 (Fermentas). The resulting construct was sequence verified by Sanger sequencing (Eurofins, Germany). The NdeI-SacI-ended insert was then liberated from the pUC19-based plasmid by NdeI and SacI double digestion before being cloned into the same sites on pMCS5::parS 3+4 (Tran et al., 2017). The construction of pMCS5::parS 3+4 at +1000 kb were carried out essentially as above, except the pair of primers used were: label1000-NdeI-F and label1000-SacI-R. The construction of pMCS5::parS 3+4 at +1800 kb was reported previously in Tran et al (2017) (6).
pMCS1-Tn5-ME-R6Kγ-kan R -ME The transposon delivery plasmid was constructed by Gibson assembling three PCR products (fragment 1 to 3) together. To generate fragment 1: the backbone of pMCS1 (1) was amplified by PCR using primers ampMCS1-F, ampMCS1-R, and pMCS1 as template. The resulting PCR product was treated with 1 µL of DpnI enzyme (NEB) at 37 o C for an hour to remove circular methylated template DNA. The PCR product was further purified by gel extraction. To generate fragment 2: the Tn5 transposase-encoding gene together with its promoter was amplified by PCR using primers amp_Tn5_F, amp_Tn5_R, and pIT2 (a gift from Colin Manoil) as template. The resulting PCR product was purified by gel extraction. To generate fragment 3: the transposon cassette (ME-R6Kγ origin-kanamycin R -ME) was amplified by PCR using primers amp_jumpF, amp_jumpR, and Ez-Tn5 (EpiCentre) as template. The resulting PCR product was purified by gel extraction. To assemble three fragments together, 1.7 µL of each fragment (1-3) at equimolar concentration was added to 5 µL Gibson master mix (NEB), and the mixture was incubated at 50°C for 60 minutes. 5 µL was used to transform chemically-competent E. coli DH5α cells. Gibson assembly was possible due to 23 bp sequence shared among PCR fragments. These 23 bp regions were incorporated during the primer design. The resulting plasmid was sequence verified by Sanger sequencing (Eurofins, Germany).
pMCS1-Tn5-ME-R6Kγ-kan R -parS 3+4+5 -ME To insert 660 bp sequence containing parS sites 3, 4, and 5 into the Tn5 transposon cassette, we first amplify around the pMCS1-Tn5-ME-R6Kγ-kan R -ME plasmid by PCR using primers amp_cirF and amp_cirR. At the end of the PCR, 1 µL of DpnI enzyme (NEB) was added, and the reaction was incubated at 37 o C for an hour to remove circular methylated template DNA. Subsequently, the PCR product was purified by gel extraction. To generate DNA fragment containing parS sites 3, 4, and 5, PCR was used to amplify a 660 bp fragment from the Caulobacter genomic DNA using primers amp_parS_3sites_F and amp_parS_3sites_R. The resulting PCR product was purified by gel extraction. The two DNA fragments were assembled together by Gibson assembly. 2.5 µL of each fragment at equimolar concentration was added to 5 µL Gibson master mix (NEB), and the mixture was incubated at 50°C for 60 minutes. 5 µL was used to transform chemically-competent E. coli DH5α cells. Gibson assembly was possible due to 23 bp sequence shared among PCR fragments. These 23 bp regions were incorporated during the primer design. The resulting plasmid was sequence verified by Sanger sequencing (Eurofins, Germany).

Strain TLS1628
Electro-competent Caulobacter cells were electroporated with pMT675::flag-parB (WT) plasmid to allow for a single integration at the vanA locus. The correct integration was subsequently verified by PCR.

Strain TLS1634
Electro-competent Caulobacter cells were electroporated with pML477::flag-yfp and plated out on PYE + spectinomycin. Resulting colonies were re-struck out on PYE + spectinomycin twice to purify the strain.
Strains TLS1635, TLS1636, and TLS1619 Electro-competent Caulobacter cells were electroporated with plasmids pMCS5::parS 3+4 at +200 kb, pMCS5::parS 3+4 at +1000 kb, or pMCS5::parS 3+4 at +1800 kb to allow for a single integration at the site of interest. The correct integration was verified by PCR using a primer specific to the parS 3+4 and another primer upstream of the ~500 bp homologous region used to drive integration. Strains TLS1637 to TLS1650 We use Lambda Red (4) to insert a cassette consisting of 24-bp Caulobacter parS site and an apramycin antibiotic resistance gene aac (3)IV at the ybbD locus on the E. coli chromosome. An apramycin resistance cassette was first amplified by PCR using primer apramycinR_F and apramycinR_R, and pSET152 (a gift from Lucy Fouston) as template.
The resulting PCR product was purified by gel extraction and further used as a template in a second round of PCR to attach a 24 bp parS site to the beginning of the apramycin resistance cassette. Pairs of primers that were used are: ybbD_apramycinR_R and ybbD_parS_site1_F (to attach parS site 1), ybbD_apramycinR_R and ybbD_parS_site2_F (to attach parS site 2), ybbD_apramycinR_R and ybbD_parS_site3_F (to attach parS site 3), ybbD_apramycinR_R and ybbD_parS_site4_F (to attach parS site 4), ybbD_apramycinR_R and ybbD_parS_site5_F (to attach parS site 5), ybbD_apramycinR_R and ybbD_parS_site6_F (to attach parS site 6), ybbD_apramycinR_R and ybbD_parS_site7_F (to attach parS site 7), and ybbD_apramycinR_R and ybbD_parS_scrambled_site3_F (to attach a scrambled sequence of parS site 3). These forward and reverse primers also carry 49 bp homology to the left or the right of the insertion point at the ybbD locus. The resulting PCR products were gel-extracted and electroporated into an arabinose-induced E. coli AB1157/pKD46 cells. Colonies that formed on LB + apramycin was restruck on LB + apramycin and incubated at 42 o C to cure of pKD46 plasmid. Finally, the correct insertion of the parS-apramycin R cassette was verified by PCR and Sanger sequencing.

Construction of Tn5-seq libraries for Illumina deep sequencing
Illumina deep sequencing libraries were constructed as follows: 1) End repair: the following components were mixed together in a DNA LoBind Eppi tube (Eppendorf): 50 µL of sheared genomic DNA, 10 µL of 10xT4 DNA ligase buffer, 2.5 µL 10mM dNTPs, 28.75 µL of autoclaved MiliQ water, 4 µL of T4 DNA polymerase (3,000 U/mL, NEB), 0.75 µL of Large Klenow Fragment (5,000 U/mL, NEB), and 4 µL of T4 polynucleotide kinase (10,000 U/mL, NEB). The reaction was incubated at room temperature for 30 minutes. End-repaired DNA was then purified by a MinElute Reaction Clean up kit (Qiagen) and eluted out using 30 µL of water.
2) A tailing: the following components were mixed together in a DNA LoBind Eppi tube (Eppendorf): 30 µL of purified DNA from step 1, 4 µL of 10xNEB buffer 2, 4 µL of 2mM dATP, and 3 µL of 3'-5' exo -Klenow fragment (5,000 U/mL, NEB). The reaction was incubated at 37 o C for 40 minutes. DNA was then purified by a MinElute Reaction Clean up kit (Qiagen) and eluted out using 15 µL of water.
3) Adaptor ligation: before this step an adaptor was prepared by annealing two singlestranded DNA oligos together. The sequences of the customised oligos are listed in the Supplementary Table S2. For ligating an A-tailed DNA to the adaptor, the following components was mixed in DNA LoBind Eppi tubes: 15 µL of A-tailed DNA from the previous step, 5 µL of adaptor, 2.5 µL of 10xT4 ligase buffer, 1 µL of water, and 1.5 µL of T4 DNA ligase enzyme. The reaction was incubated at room temperature for 30 minutes. DNA was subsequently purified using a Qiagen MinElute kit and eluted out using 50 µL of water. 5) DNA purification: DNA was loaded on a 2% agarose gel and a DNA band of desired fragment length was purified from primer dimer DNA and unincorporated oligos. Samples were submitted to the next-generation sequencing facility at the Tufts University. A custom read primer and a custom index read primer were used on a Hiseq2500 to read out the Tn5 insertion junctions and samples' indexes. The sequence of these custom primers are listed in the Supplementary Table S2.
For the list of Tn5-seq libraries in this study, see Supplementary Table S3.

ParB-(His)6 protein purification
Plasmid pET21b-ParB (HexaHistidine tag at the C-terminus of ParB, a gift from Christine Jacob-Wagner) was transformed into E. coli BL21/pRARE, and 10 mL overnight culture was used to inoculate 2 L LB medium + carbenicilin + chloramphenicol. Cells were grown at 37 o C to OD600 of ~0.4. The culture was then cooled to 30 o C before isopropyl-β-Dthiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM. The culture was left shaking for an additional 3 hrs at 30 o C before cells were harvested by centrifugation. Pelleted cells were resuspended in a buffer containing 100 mM Tris-HCl pH 8.0, 300 mM NaCl, 5% (v/v) glycerol, EDTA-free protease inhibitor tablet (Roche), and lysed by sonication (three cycles of 20 s with 40 s resting on ice in between each cycle). The cell debris was removed by centrifugation at 84,000 g for 30 min and the supernatant was filtered through a 0.45 µm membrane before being applied to a 1-ml Ni-loaded Hi-Trap Chelating HP column (GE Healthcare) that had been equilibrated with buffer A [100 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10 mM imidazole]. Protein was eluted from the column using an increasing (10 mM to 500 mM) imidazole gradient in the same buffer. ParB-(His)6 fractions were identified using SDS-PAGE, pooled together, and applied to a Heparin HP column (GE Healthcare) that had been equilibrated with buffer A [100mM Tris-HCl pH 8.0, 25mM NaCl, 5% (v/v) glycerol]. Protein was eluted from the column using an increasing (25 mM to 1 M NaCl) salt gradient in the same buffer A. ParB-(His)6 fractions were identified using SDS-PAGE, pooled together, and concentrated to approximately 2 mg/mL using a Vivaspin6 10 kDa cut-off protein concentrator (Vivascience). The concentrated protein was then exchanged into a storage buffer [50mM Tris-HCl, pH 8.0, 250 mM NaCl and 10% glycerol] using a Zeba desalting column (Thermo Scientific) before flash-frozen in liquid nitrogen. The concentration of ParB dimer was measured by Bradford method before Surface Plasmon Resonance (SPR) experiments. ParB-(His)6 is ~95% pure as judged by SDS-PAGE. ParB mutants: G101S and R104A were purified using the same procedure as ParB (WT) but from 8 L and 6 L of cultures, respectively. B. subtilis Spo0J/ParB, expressed from pET21b-Spo0J plasmid, was purified exactly as for Caulobacter ParB-(His)6 but from 4 L of cultures.

Bi-parental E. coli-Caulobacter conjugation and parS + plasmid toxicity assay
E. coli S17-1 cells were transformed with low-copy number plasmids containing individual parS site by heat shock and plated out on LB + kanamycin. Resulting colonies were grown to mid-exponential phase in preparation for a bi-parental conjugation with the wild-type Caulobacter CB15N. Briefly, E. coli cells were pelleted and resuspended in fresh PYE to wash off residual antibiotics from the growing culture. 100 µL of E. coli cells at OD600 of 0.4 was mixed with 500 µL of exponentially growing Caulobacter at OD600 of 0.4. The mixture of E. coli and Caulobacter cells were centrifuged at 13,000 rpm for 1 minute and the pellet was resuspended in 50 µL of fresh PYE before being spotted on a nitrocellulose membrane. The membrane was then laid on top of a fresh PYE plate and incubated at 30 o C for 5 hrs. After the incubation, cells were released from the nitrocellulose membrane by vortexing in an Eppendorf tube containing 500 µL of fresh PYE. A ten-fold serial dilution was performed for each conjugation and 5 µL of each dilution was spotted on PYE plates supplemented with just nalidixic acid or with both nalidixic acid and kanamycin. Plates were incubated at 30 o C for 3 days to allow Caulobacter colonies to form.

Immunoblot analysis
For Western blot analysis, Caulobacter or E. coli cells were pelleted and resuspended directly in 1xSDS sample buffer, then heated to 95°C for 5 min before loading. Total protein was run on 10% Tris-HCl gels (Bio-Rad) at 150 V for separation. Resolved proteins were transferred to polyvinylidene fluoride membranes using the Trans-Blot Turbo Transfer System (BioRad) and probed with 1:10,000 dilution of primary α-FLAG antibodies (Sigma-Aldrich), or 1:5,000 dilution of α-T18 antibody (Abcam) and subsequently by a secondary HRP-conjugated antibody (1:5,000). Blots were imaged using an Amersham Imager 600 (GE Healthcare), and quantified using ImageStudio Lite (Licor).

Bacterial-two hybrid assay
Bacterial-two hybrid assays were performed exactly as described in the Euromedex manual.