Phototope star detection kit

Mock- and p150CAF-1-depleted Xenopus high-speed egg extract (HSE) were prepared as previoulsy (Ray-Gallet and Almouzni, 2004 (link)). Nucleosome assembly was performed on pBS plasmid damaged by UV (500 J/m2) to promote DNA synthesis as previously described (Ray-Gallet and Almouzni, 2004 (link)) except that the reaction mixed contained 3.2 µM of biotin-14-dCTP (Invitrogen, Ref 19518–0189) instead of [α³²P]-dCTP. The p150CAF-1-depleted extracts were complemented with 50 ng of isolated/reconstituted SpCAF-1 complex composed of WT or mutated Pcf1. After DNA purification, samples were by processed for gel electrophoresis (1% agarose) to resolve topoisomers as previously described (Ray-Gallet and Almouzni, 2004 (link)). After staining with Ethidium bromide to visualize total DNA and gel transfer on a Nylon N+membrane (GE Healthcare Ref RPN203B) (Qbiogen) for 45 min at 40 mbar in 10 x SSC, the membrane was rinsed in PBS, air dried and DNA was crosslinked to the membrane using Stratalinker (Bio-Rad). DNA synthesis was visualized by detecting biotin with the Phototope-Star detection kit (New England Biolabs Ref N7020S) and images acquired on a Chemidoc system (Bio-Rad).

DNA shift assays of LdtR, as well as its mutated versions, was carried out as described previously [9 (link)]. Fragments of the selected promoters were generated by PCR using biotinylated primers (Table 2). The reaction mix for EMSA contained 1 ng of 5’biotin-labelled DNA probe, 50 nM Tris-HCl pH 7.2, 150 mM KCl, 10 mM MgCl₂, 0.01% Triton X100, 12.5 ng/μl both Poly(dI-dC) and Poly(dA-dT) nonspecific competitor DNAs, and purified LdtR (0–1000 nM) as indicated. The mix was incubated for 20 min at 37°C and the electrophoresis was conducted at 4°C on 6% acrylamide/bisacrylamide non-denaturing gels in 0.5X Tris-borate EDTA buffer (TBE) pH 8.3. The DNA was transferred to a Hybond-N⁺ membrane (GE Healthcare, Pittsburgh, PA, USA) by electroblotting at 250 mA for 45 min in a Semi Dry Electroblotting System (Fisher Scientific). The detection of the DNA bands was carried out with the Phototope-Star Detection Kit (New England Biolabs, Ipswich, MA, USA). The membranes were exposed to Autoradiography Film (Mid-Sci, St. Louis, MI, USA).

DNA gel shift assays were performed according to the protocol described in Pagliai et al. (2014). A fragment of the CLIBASIA_03135 promoter was generated by PCR using biotin‐labelled primers (Table 4) and then purified using QIAquick spin columns (Qiagen). Briefly, the reaction for EMSA contained 1 ng of 5′‐labelled DNA probe, 50 mM Tris–HCl pH 7.2, 150 mM KCl, 10 mM MgCl₂, 0.01% Triton X‐100, 12.5 ng μl⁻¹ of an equimolar mix of Poly(dI‐dC) and Poly(dA‐dT) non‐specific DNA competitor and purified LdtR protein (0 – 500 nM). The whole reaction was incubated at 37°C for 20 min and then separated on 6% acrylamide–bisacrylamide non‐denaturing gels using 0.5× Tris–borate–EDTA buffer, pH 8.3 (TBE). Samples were subjected to electrophoresis for 2 h at 100 V using ice‐cold 0.5× TBE buffer, and then, the DNA was transferred from the polyacrylamide gel to a Hybond‐N⁺ membrane (GE Healthcare) by blotting at 250 mA for 45 min using a semidry transfer unit, followed by a UV cross‐link of the DNA to the membrane. The biotin‐labelled DNA was visualized using the Phototope‐Star detection kit (New England Biolabs, Ispwich, Massachusetts, USA).

The Southern blot procedure was modified from Wu et al.^{51 (link)}. Tomato genomic DNA was extracted from leaf tissue using the Qiagen Plant DNA extraction kit. DNA (10 µg) from the CHI lines and the PAP1 lines were digested with XbaI and BamHI, respectively. The digested DNA was separated by electrophoresis and blotted onto a nylon membrane (Zeta-probe GT membrane, Bio-Rad Laboratories, Hercules, CA), following the manufacturer’s instructions. The probe for the CHI and PAP1 genes was isolated from vectors harboring each gene^{51 (link)}. The membranes were prehybridized overnight at 65 °C in 7% SDS and 0.25 M Na₂HPO₄ and then hybridized overnight at 65 °C in the same solution containing the probe labeled by the NEBlot Phototope Kit (New England Biolabs, Ipswich, MA). Membranes were washed twice for 40 min each with 20 mM Na₂HPO₄ and 5% SDS at 65 °C and then washed twice again for 30 min each with 20 mM Na₂HPO₄ and 1% SDS at 65 °C. The signal was detected by the Phototope-Star Detection Kit (New England Biolabs, Ipswich, MA, U.S.A).

Chromosomal DNA (3 μg) from R6, R6 P_Zn-hlp, and R6 P_Zn-hlpΔhlp was digested with HindIII and separated by 0.8% agarose gel electrophoresis, transferred to Nylon membranes and hybridized to a biotinylated 141-bp hlp probe obtained by PCR amplification with 5′ biotinylated oligonucleotides HU20BIOT and HU61RBIOT (Table 1). Blots were developed with the Phototope-Star Detection Kit (New England Biolabs) following the manufacturer’s instructions.

Cultures were grown exponentially until OD₆₂₀ = 0.1. At this time (time 0) antibiotics were added at concentrations equivalent to 1×MIC for each strain and samples were taken during 4 h. Phage and chromosomal DNAs were obtained as follows. First, 2.92 g of NaCl were added to 50 mL of culture, and the solution kept at 4°C for 1 h. The suspension was centrifuged at 2,200×g for 20 min at 4°C. The supernatant, containing phages, was precipitated overnight at 4°C with polyethylene glycol 800 (1 g/10 mL solution). The suspension was centrifuged at 11,200×g during 15 min at 4°C and the pellet suspended in 0.5 mL of buffer (100 mM Tris-HCl pH 7.5, 0.1 M NaCl, 10 mM MgCl₂). To eliminate cellular nucleic acids, the phage suspension was treated with RNase and DNase (100 μg/mL each) during 1 h at 37°C. Phage particles were digested 90 min with proteinase K (150 μg/mL) in the presence of 0.5% SDS and 50 mM EDTA at 50°C. Phenol extraction and ethanol precipitation allowed the isolation of phage DNA. A 191-bp phage probe containing hol1 was obtained with biotinylated P9 and P9-R oligonucleotides [24] (link) using strain 949 as template. The Phototope-Star Detection Kit (New England Biolabs) was used following the manufacturer's instructions.