MATERIALS AND METHODSBacterial strains and growth conditionsStrains used in this study are listed in Supplementary Table S1. Unless otherwise noted, P. carotovorum and Escherichia coli strains were grown at 30°C and 37°C, respectively, either in Lysogeny Broth (LB) at 180 rpm or on LB-agar plates containing 1.5% (w/v) agar. When required, media were supplemented with ampicillin (100 μg/ml), chloramphenicol (25 μg/ml), l-arabinose (as noted) and/or isopropyl β-d-1-thiogalactopyranoside (IPTG) (as noted). Growth was monitored as the optical density at 600 nm (OD600) in a Jenway 6300 Spectrophotometer or in a Varioskan LUX Microplate Reader.DNA isolation and manipulationOligonucleotides used in this study are listed in Supplementary Table S2. Plasmid DNA was isolated using the Zyppy Plasmid Miniprep Kit (Zymo Research) and all plasmids were confirmed by DNA sequencing. Plasmids used in this study are listed in Supplementary Table S3 and their construction is outlined in the Supplementary Methods. Restriction digests, ligations, E. coli transformations and agarose gel electrophoresis were performed using standard techniques. Transformation of P. carotovorum strains was carried out by electroporation using a Bio-Rad GenePulser Xcell system (set to 1800 V, 25 μF, 200 Ω) in Bio-Rad electroporation cuvettes with a 0.1 cm electrode gap, followed by 2 h recovery in LB medium at 30°C at 180 rpm. DNA from PCR and agarose gels was purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare). Polymerases, restriction enzymes and T4 ligase were obtained from New England Biolabs or Thermo Scientific.Structural modelling and sequence analysisDNA sequence analyses were performed using Geneious 10.0.7 software (https://www.geneious.com) and ClustalΩ was used for sequence alignments (24). Protein BLAST (https://blast.ncbi.nlm.nih.gov) was used for identification of Aca2 homologs. The Aca2 structural model was generated by homology modelling using Phyre2 (25) with the MqsA structure bound to DNA (PDB: 3O9X) (26). The DNA-bound Aca2 dimer model was generated by aligning copies of the Phyre2 Aca2 model with each of the DNA-bound MqsA subunits using Pymol. The genome similarity of P. carotovorum strains RC5297 and ZM1 was compared using the ChunLab's online Average Nucleotide Identity (ANI) Calculator (27). Strengths of ribosome binding sites were calculated using the De Novo DNA RBS Calculator (Salis Lab, https://salislab.net/software/).Reporter assaysReporter assays for measuring acrIF8–aca2 promoter activity involved arabinose-inducible aca2 expression plasmids and/or reporter plasmids with eyfp under control of the acrIF8–aca2 promoter. To determine the effect of aca2 expression on acrIF8–aca2 expression, each eyfp reporter plasmid was tested with an aca2 expression plasmid (+Aca2; pPF1532) as well as the corresponding empty vector (–Aca2; pBAD30). Overnight starter cultures of P. carotovorum strains containing plasmids were grown in 96-well plates in an IncuMix incubator shaker (Select BioProducts) at 1200 rpm at 30°C. The OD600 for each was adjusted to 0.05 in LB medium containing the appropriate antibiotics, and if required, arabinose and IPTG were added to final concentrations of 0.05% (w/v) and 50 μM, respectively, unless otherwise noted. After 20 h of growth, fluorescence of plasmid-encoded mCherry and eYFP was measured by flow cytometry using a BD LSRFortessa cell analyzer. Cells were first gated based on forward and side scatter area, and mCherry-positive cells, as detected using a 610/20-nm bandpass filter with a detector gain of 606 V, were further analysed for eYFP levels using a 530/30-nm bandpass filter and detector at 600 V. Median fluorescence intensity of eYFP was measured for at least three replicates. Measurements for a control strain containing empty vectors were subtracted from the other samples to account for background fluorescence.Aca2 expression and purificationAn overnight culture of E. coli BL21(DE3) carrying the aca2 expression plasmids pPF1575 (wild-type Aca2) or pPF1857 (Aca2R30A) was diluted 1:100 in 500 mL LB supplemented with ampicillin. The culture was incubated at 25°C and 200 rpm until it reached an OD600 of ∼0.5, at which point IPTG was added to a final concentration of 1 mM. The culture was incubated at 18°C for 18 h and pelleted by centrifugation (10 000 g, 4°C, 10 min). For protein purification, 0.5 g of wet cell pellet was resuspended in 20 ml binding buffer (25 mM HEPES-NaOH, pH 7.5, 300 mM NaCl and 25 mM imidazole, 0.1 mM tris(2-carboxyethyl)phosphine (TCEP)) supplemented with one cOmplete Protease Inhibitor Cocktail tablet (Roche), 20 μg/ml DNase I and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by two cycles through a French Press at 10 000 psi. The lysate was cleared by centrifugation (10 000 g, 4°C, 10 min), followed by the addition of more PMSF (1 mM) and a second centrifugation step (10 000 g, 4°C, 10 min).The clarified lysate was loaded, using an FPLC system (ÄKTA pure, GE Healthcare), onto a 1 ml HisTrap FF column (GE Healthcare) that was pre-equilibrated with binding buffer. The column was washed with binding buffer and 5 column volumes (CV) of 10% elution buffer (0.1 mM TCEP, 25 mM HEPES-NaOH, pH 7.5, 300 mM NaCl and 500 mM imidazole). Aca2 was eluted using a linear imidazole gradient to 100% elution buffer over 10 CV. Fractions containing Aca2, as determined using NuPAGE 4-12% Bis-Tris Protein Gels (Thermo Scientific), were pooled into dialysis tubing (SnakeSkin 3.5 kDa MWCO, Thermo Scientific) and TEV protease was added. The sample was dialyzed overnight at 4°C in size-exclusion chromatography (SEC) buffer (0.1 mM TCEP, 25 mM HEPES-NaOH, pH 7.5 and 300 mM NaCl). Reverse-IMAC was performed to separate the cleaved Aca2 from the rest of the sample. The dialyzed sample was centrifuged (15 000 g, 4°C, 5 min) and soluble protein loaded onto an IMAC column. The column was washed with 10 CV binding buffer and 10 CV elution buffer. Cleaved Aca2 was collected in the unbound fraction and peak fractions were pooled and further purified by size-exclusion chromatography using a Superose 12 10/300 GL column (GE Healthcare) pre-equilibrated in SEC buffer. Fractions containing Aca2 were pooled and glycerol was added to a final concentration of 5% (v/v). Protein concentrations were determined using the Qubit Protein Assay Kit (Thermo Scientific). Aliquots were snap-frozen using dry ice and ethanol and stored at -80°C.Electrophoretic mobility shift assays (EMSAs)DNA probes for EMSAs were PCR-amplified using the primers listed in Supplementary Table S2 from the plasmids listed in Supplementary Table S3. Probes were fluorescently labelled with IRDye-700 on their 5′ ends, whereas for binding specificity controls, unlabelled primers of the same sequence were used.EMSAs involved 20 μl reactions containing 20 mM HEPES-NaOH, pH 7.5, 100 mM NaCl, 0.1 mM TCEP, 5 mM MgCl2, 0.1 μg/μl BSA, 0.01 μg/μl poly(dI•dC), 0.05 μg/ml poly-l-lysine, labelled DNA probe (final concentration 0.25 nM) and purified Aca2 to final concentrations as indicated. For competition assays, excess unlabelled probe (final concentration 50 nM) was incubated with Aca2 for 15 min prior to addition of the labelled probe. All binding reactions were incubated for 15 min at room temperature in the dark. Next, 5 μl loading dye (0.5× TBE (45 mM Tris, pH 8.3, ∼45 mM boric acid and 1 mM EDTA), 34% glycerol (v/v), 0.2% bromophenol blue (w/v)) was added and samples were loaded on 8% polyacrylamide gels (19:1 acrylamide/bis-acrylamide (Bio-Rad), 0.5× TBE, 2.5% (v/v) glycerol, 0.6 mg/ml ammonium persulfate and 0.05% (v/v) tetramethylethylenediamine) which had been pre-run for at least 30 min at 4°C. Gel electrophoresis was performed at 100 V and 4°C in the dark for ∼1 h. DNA was imaged at 700 nm using the LI-COR Odyssey Fc imaging system and Image Studio software. Image Studio was used to quantitate band shifts from at least three independent assays, and dissociation constants were determined using GraphPad Prism (Version 7.00).DNA bending assaysDNA bending assays were based on (28). Probes for bending assays were generated by PCR using the IRDye700-labelled oligonucleotides and the templates indicated in Supplementary Table S2. Probes were characterized by varying flexure displacement, defined as the ratio of (a) the distance from the centre of the putative protein binding site to the 5′ end of the probe, and (b) the total length of the probe. The DNA-binding reactions were assembled according to EMSA conditions with 20 nM Aca2 and probe concentrations of 1 nM. After imaging, mobility of shifted and unshifted DNA probes (Rbound and Rfree, respectively) was determined as the distance between the probe and the gel well. The ratio Rbound/Rfree was plotted as a function of flexure displacement, and a quadratic equation of the form y = ax2 + bx + c describing the best-fit curve was used to determine the bending angle according to the formula αbend = cos–1(2c – a)/2c = cos–1(2c + b)/2c, as described in (29). The bending angle was calculated as the mean ± standard deviation of at least three independent DNA bending assays.
CRISPR-Cas systems are widespread bacterial adaptive defence mechanisms that provide protection against bacteriophages. In response, phages have evolved anti-CRISPR proteins that inactivate CRISPR-Cas systems of their hosts, enabling successful infection. Anti-CRISPR genes are frequently found in operons with genes encoding putative transcriptional regulators. The role, if any, of these anti-CRISPR-associated (aca) genes in anti-CRISPR regulation is unclear. Here, we show that Aca2, encoded by thePectobacterium carotovorumtemperate phage ZF40, is an autoregulator that represses the anti-CRISPR–aca2operon. Aca2 is a helix-turn-helix domain protein that forms a homodimer and interacts with two inverted repeats in the anti-CRISPR promoter. The inverted repeats are similar in sequence but differ in their Aca2 affinity, and we propose that they have evolved to fine-tune, and downregulate, anti-CRISPR production at different stages of the phage life cycle. Specific, high-affinity binding of Aca2 to the first inverted repeat blocks the promoter and induces DNA bending. The second inverted repeat only contributes to repression at high Aca2 concentrations in vivo, and no DNA binding was detectable in vitro. Our investigation reveals the mechanism by which an Aca protein regulates expression of its associated anti-CRISPR.