Materials and Methods

Multifactorial control of the expression of a CRISPR-Cas system by an extracytoplasmic function σ/anti-σ pair and a global regulatory complex

MATERIALS AND METHODSStrains, plasmids, growth conditions and strain constructionPlasmids and M. xanthus strains used in this study are listed, respectively, in Supplementary Tables S1 and 2. Myxococcus xanthus vegetative growth was carried out at 33°C in the rich casitone-Tris (CTT) medium (1% Casitone, 10 mM Tris–HCl pH 8.0, 1 mM KH2PO4-K2HPO4, 8 mM MgSO4; final pH 7.6), with antibiotic (kanamycin, Km, at 40 μg/ml or oxytetracycline, Tc, at 10 μg/ml) when necessary. CTT agar (1.5%) was used for growth on solid medium. For conditional gene expression, the medium was supplied with inducer (0.5 mM vanillate or 1 mM isopropyl-β-d-thiogalactopyranoside-IPTG) as required (50). Fruiting body development was induced on CF agar plates and examined using a Zeiss dissecting microscope. For this, cells grown in CTT to an OD550 of 0.6 (∼108 cells ml−1) were washed twice by centrifugation in TPM (10 mM Tris–HCl pH 8.0, 1 mM KH2PO4-K2HPO4, 8 mM MgSO4; final pH 7.6) buffer, concentrated 10-fold in TPM buffer, spotted (10 μl) on CF plates and incubated at 33°C. Escherichia coli DH5α, used for plasmid constructions, was grown at 37°C in Luria broth supplemented with the appropriate antibiotic. Plasmids were constructed using standard protocols, verified by DNA sequencing, and introduced into M. xanthus by electroporation, where they integrate by homologous recombination. Plasmids used for IPTG or vanillate-inducible gene expression and the integrative plasmids with a 1.38 or 1.31-kb M. xanthus DNA for integration at a chromosomal site with no promoter activity have been described elsewhere (50). Strains with in-frame gene deletions were constructed following the two-step allele exchange protocol using KmR negative selection/galK positive selection (galactose sensitivity, GalS), as described previously (50). The polymerase chain reaction (PCR) overlap extension method was employed for site-directed mutagenesis.Microarray analysisTotal RNA was isolated for microarray analysis as follows. The M. xanthus strain of interest was grown in 50 ml CTT to exponential phase (OD550 = 1), and 1 ml (for 5′ RACE) or 6 ml (for transcriptomic analysis) of the culture were then pelleted by centrifugation and stored at −70°C. Frozen cell pellets were resuspended in 300 μl of a 0.3 M sucrose/0.01 M sodium acetate pH 4.5 solution, transferred to an Eppendorf tube containing 300 μl of 2% sodium dodecyl sulfate, 0.01 M sodium acetate pH 4.5, incubated at 65°C for 1–2 min, mixed well with 400 μl of phenol and incubated at 65°C for 3 min after thorough mixing. After snap freezing for 1–2 min in liquid nitrogen, it was thawed, then centrifuged (16 000 g, 5 min) and the resulting aqueous layer was pipetted into a fresh tube with 600 μl of hot phenol. The mixing, 3 min-incubation at 65°C, snap-freezing in liquid nitrogen, and centrifugation were repeated as before. The aqueous phase obtained was mixed with 600 μl 1:1 phenol:chloroform in a new tube and centrifuged, and the process repeated using 600 μl pure chloroform. From this last step, the aqueous portion was mixed with 40 μl of 3 M sodium acetate (pH 4.5) and 900 μl of 96% ethanol, followed by 30-min incubation at −20°C and centrifuged (16 000 g, 20 min, 4°C). The supernatant was discarded, and the pellet was washed with 200 μl of ice-cold 70% ethanol, dried in a SpeedVac and suspended in 50 μl of 0.2% diethyl pyrocarbonate (DEPC)-treated water. It was then treated with 20 units of RNase-free recombinant DNase I in DNase I buffer and 20 units of Protector RNase Inhibitor (all from Roche) for 30 min and at 37°C. RNA was purified using QIAGEN RNeasy Mini Kit and its quality and quantity were assessed by gel electrophoresis and NanoDrop ND-1000 (Thermo Scientific) using an extinction coefficient at 260 nm of 40 ng-cm/μl.An 8 × 15k microarray platform format (8 microarrays per slide each with 15 000 probes) was designed using eArray (https://earray.chem.agilent.com/earray/; Agilent Technologies, Santa Clara, USA), and in each microarray spot 7316 out of the 7441 annotated genes in the M. xanthus genome (excluding 79 RNA genes, 43 pseudogenes and genes with duplicate annotations) were represented by 60-bp amplicon probes (in duplicate and distributed randomly per microarray, with empty array features filled with randomly chosen amplicon probes). Total RNA isolated as described above was checked for quality and quantity using the Agilent Bioanalyzer 2100 and the Prokaryote Total RNA Nano assay, with those having an RNA integrity number ≥ 7 chosen for further analysis. This RNA was reverse-transcribed to yield cDNA that, after labeling with the cyanine Cy3 (green) or Cy5 (red) fluorescent dyes (GE Healthcare), was hybridized to the microarray spot. Relative mRNA levels were determined using the Agilent two-color microarray-based prokaryote analysis for gene expression protocols, version 1.3. For each test and reference condition two or more independent microarray experiments were performed and analyzed separately. Microarray hybridization and data analyses were performed at Bioarray SL (Spain). Hybridized microarrays were scanned and the image analyzed to transform color intensity to numeric data using the Agilent Feature Extractor Software v.10.7. Raw intensity data were read and checked for quality using the Limma package for R (51), corrected for background using the normexp+offset method (52), and normalized with loess (intra-array) and quantile (inter-array) procedures (53). A simple linear model was fit to the data and variances were corrected using an empirical Bayes approach implemented in the Bioconductor limma package (54). Genes were considered as significantly affected if they displayed a mean log2 ratio < −1 or > +1 (corresponding to 2-fold change up or down) relative to the control and with adjusted P-values ≤ 0.05 (95% confidence limit) from two independent array runs. Microarray data have been deposited at NCBI Gene Expression Omnibus (GEO) with accession number {"type":"entrez-geo","attrs":{"text":"GSE112385","term_id":"112385"}}GSE112385.Real-time quantitative reverse transcription PCR (qRT-PCR)Total RNA (2 μg) isolated as above was reverse transcribed to cDNA using random hexamer primers (Promega) and Transcriptor Reverse Transcriptase (Roche) in a 20 μl reaction mix as per instructions. The cDNA sample (2 μl) was added to a PCR mixture prepared using Power SYBR® Green PCR Master Mix (Applied Biosystems) that contained the respective primers at 100 nM. Reactions were performed in triplicate for cDNA obtained from each biological replicate; a control with an equivalent volume of RNA, but not reverse transcribed, was tested to rule out the presence of contaminant DNA. Reported values are the average and the standard error for three biological replicates. Primers (Supplementary Table S3) to amplify ∼50–150 bp region within each transcript were designed (16S rRNA was the internal standard) using Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). RNA quantitation was carried out in an Applied Biosystems StepOne equipment using the 1-Step RT-PCR program cycle without the reverse transcription step. Melting and dissociation curves were determined from 60–95°C, 30 s and 95°C, 15 s. Each primer pair was tested for RT-PCR analysis on a standard curve generated from five 10-fold serial dilutions of cDNA. Only primer pairs with efficiency close to 100% were used and data were analyzed using the Applied Biosystems system software.5′ RACE and RT-PCR analysisTotal RNA was isolated from the ΔddvA or wild-type (WT) strain using High Pure RNA kit (Roche) or RNeasy Mini kit (Qiagen) as described above, and RNA recovered after two rounds of treatment with recombinant DNase I (Roche) to fully eliminate genomic DNA was suspended in 50 μl RNase-free water (pre-treated with 0.2% DEPC). For 5′ RACE (rapid amplification of cDNA ends), 2 μg of this purified RNA was reverse-transcribed using an oligonucleotide primer that hybridizes to the 3′ end of the gene of interest and the 5′/3′ RACE 2nd Generation kit in the presence of RNase inhibitor Protector (Roche). The cDNA product was purified, tagged with a 5′ polyA adaptor and used as template in a PCR reaction with, as primers, dT-Anchor and a cDNA-specific oligonucleotide that hybridizes to the 3′ end of the gene of interest, followed by a second PCR reaction with PCR Anchor instead of the dT-Anchor. The product was purified using High Pure PCR Product Purification Kit (Roche) and sequenced. For RT-PCR, 500 ng total RNA was reverse-transcribed using random hexamers primers (Promega) and Transcriptor Reverse Transcriptase (Roche). A total of 2 μl of the cDNA product was used as template for subsequent PCR using primer pairs that generate fragments of ≤500 bp spanning across the junctions of the annotated cas genes or across the leader to CRISPR4 spacers. As the negative (−RT) control, RNA that was not subjected to reverse transcription, but otherwise treated identically, was used as template in the PCR reaction.β-galactosidase activitySpecific β-galactosidase activity (in nanomoles of o-nitrophenyl β-D-galactoside hydrolyzed/min/mg protein and at least three independent measurements) was performed on cells grown in CTT to early exponential phase or on cells undergoing development using a SpectraMax 340 microtitre plate reader (Molecular Devices), as described elsewhere (45). X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside)-agarose overlay assay for qualitative analysis of reporter lacZ expression was carried out as reported previously (50).Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR)Cells grown in 50 ml CTT (with or without vanillate as required) at 33°C to mid-late exponential phase (OD550 = 0.70) were treated with rifampicin (25 μg/ml) for 30 min and then cross-linked with formaldehyde (1% v/v; Sigma-Aldrich) in the presence of 10 mM sodium phosphate (pH 7.6) for 10 min at room temperature with shaking (100 rpm). After cooling in ice for 30 min, cells were pelleted, washed twice with phosphate-buffered saline solution and stored at −80°C until further use. For chromatin immunoprecipitation (ChIP), the frozen pellet was thawed, resuspended in lysis buffer (470 μl, 10 mM Tris pH 7.5, 1 mM ethylenediaminetetraacetic acid (EDTA), 100 mM NaCl, 2.2 mg/ml lysozyme) and incubated at room temperature with shaking (100 rpm) for 30 min. It was mixed with ChIP buffer (550 μl, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris pH 8.1, 167 mM NaCl, 1X Roche complete protease inhibitor cocktail), incubated for 10 min at 37°C and sonicated (sixty 30 s on/30 s off cycles) in a Bioruptor (Diagenode) to generate fragments of ∼0.5 kb. The sample was clarified by centrifugation and an aliquot of 20 μl supernatant was kept aside as the input sample. The rest was mixed with 550 μl ChIP buffer with 0.01% sodium dodecyl sulphate (SDS) and 5 μl of monoclonal anti-FLAG (Sigma-Aldrich) or anti-CarD antibodies (46), incubated at 4°C overnight with rotation and immunoprecipitated (5–6 h, 4°C) with rotation with 30 μl of protein A magnetic Dynabeads (Life Technologies) previously washed with PBS plus 1 mg/ml bovine serum albumin. The beads were then washed once each with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.1, 150 mM NaCl), the same buffer at 0.5 M NaCl, and with LiCl buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris pH 8.1), and twice with TE buffer (10 mM Tris pH 8.0, 1 mM EDTA). The protein–DNA complex was recovered in two 100 μl fractions of elution buffer (1% SDS, 0.1 M NaHCO3) and incubated at 65°C for 10 min to disrupt the cross-links, treated with 5 μl proteinase K (20 μg/μl) at 42°C for 2 h and at 65°C for 6 h, followed by DNA isolation using High Pure PCR product purification kit (Roche). The input sample was also subjected to this cross-link reversal and DNA extraction protocol. qRT-PCR was then carried out using the SYBR Green reaction mix (BioRad) and specific primers (Supplementary Table S3) in 0.1 ml MicroAMP FAST optical 48-well reaction plates in a StepOne qPCR apparatus (Applied Biosystems). Standard curves were obtained for each DNA region of interest and its corresponding primer pair with serial dilutions of the input DNA sample. Signal enrichment at each promoter was estimated as: (i) percent of input and normalized relative to the sample obtained with cells lacking the immunoprecipitated protein; or (ii) ratio of the promoter-specific to intragenic signals of ChIP reactions relative to the values for the input sample; the intragenic region used as the non-promoter control spans nucleotides 402–460 of the fruA gene (55). Reported values are the mean and standard error from three independent experiments.Electrophoretic mobility shift assays (EMSA)Purification of CarD, CarG and M. xanthus RNAP-σA and electrophoretic mobility shift assays (EMSA) were carried out as described previously (45,56). The 32P-5′-end radiolabeled double-stranded DNA probes used for EMSA were obtained by PCR-amplification from plasmid constructs bearing the required regions as DNA template. The DNA probes thus obtained were: (i) 304-bp segments corresponding to the entire CRISPR4 leader with the WT or mutant (Mut) putative −35 promoter element at the leader, to test for RNAP-σA binding; (ii) 170-bp segments covering the DdvS-dependent promoter regions, to test for CarD and CarD–CarG binding. Samples (20 μl) in EMSA buffer (80 mM KCl, 25 mM Tris pH 8.0, 5 mM MgCl2, 1 mM dithiothreitol, 10% glycerol, 200 ng/μl bovine serum albumin) with 1 μg non-specific competitor polydG-dC and 1 nM of a given radiolabeled DNA probe (∼13 000 cpm) were incubated for 30 min with RNAP-σA (100 nM) at 37°C, or CarD (440 or 520 nM) with or without CarG (12 μM) at 4°C. Then, to samples containing RNAP-σA, heparin (1 μg) was added and incubated for an additional 5 min to allow formation of only heparin-resistant open promoter complexes, followed by electrophoresis in 4% non-denaturing polyacrylamide gels at 200 V for 1.5 h in TBE buffer (45 mM Tris and boric acid, 1 mM EDTA) at 10°C. Gels were vacuum-dried and analyzed by autoradiography.Northern blot analysis of pre-crRNA and mature crRNAsTotal RNA from 5 ml cell cultures in CTT (OD550 = 0.6) was isolated with 1 ml of Trizol reagent (Invitrogen) and 0.2 ml of chloroform (Sigma-Aldrich) and precipitated with 0.5 ml isopropanol (100%). The pellet was washed with 1 ml of pre-cooled 75% ethanol, resuspended in 50 μl of DEPC-treated water and RNA concentration determined for pre-crRNA analysis. To isolate RNA from fruiting bodies at each time point analyzed, cells from 10 ml CTT cultures (OD550 = 0.6) were washed and suspended in 2 ml TPM, then distributed on four CF plates as 20 μl spots and incubated at 33°C. At given times, developing cells were collected in tubes containing 1 ml TPM and a 0.5 ml equivalent of glass beads (0.1 mm diameter) for lysis using a Mini-beadbeater (BioSpec), and RNA was recovered from the supernatant as before. For mature crRNA detection, the total RNA was resuspended in 300 μl DEPC-treated water and large RNA molecules were selectively precipitated out by incubating with 5% polyethylene glycol 8000 and 0.5 M NaCl for 30 min in ice. The supernatant, recovered by centrifugation, was mixed with 100% ethanol (three volumes) and 3 M sodium acetate (pH 5; 0.1 volume), incubated overnight at −20°C to precipitate small RNAs, washed twice with 80% ethanol, dried and resuspended in DEPC-treated water. The RNA concentration was determined and northern blot analysis of mature crRNA was carried out as described below.To detect pre-crRNA, ∼20 μg of total RNA obtained above was electrophoresed in a 1.2% agarose gel with 20 mM MOPS/5 mM sodium acetate/1 mM EDTA/7% formaldehyde buffer. The gel was washed with water (six changes, 90 min), followed by capillary transfer to a Hybond N+ membrane (GE Lifesciences) overnight and cross-linked by UV-irradiation (1.2 × 105 μJ/cm2; Hoefer UVC 500 apparatus). Pre-crRNA was detected using a radiolabeled double-stranded 1044-bp DNA probe (spanning the segment from CRISPR4 spacer 37 to spacer 52) generated using DNA polymerase I Klenow (Takara) and α-32P-dCTP (specific activity 3000 Ci/mmol) following standard protocols. 23S RNA, probed using a specific 1078-bp double-stranded DNA probe, was used as the loading control. Probes were incubated at 95°C for 5 min and rapidly cooled in ice prior to use. The pre-crRNA blot was incubated for 2 h at 65°C in 0.9 M NaCl, 1% SDS, 100 μg/ml sheared, denatured salmon sperm DNA and hybridized overnight at 65°C with probe in fresh buffer containing 10% dextran sulfate sodium salt and 50 μg/ml of the non-specific DNA. After hybridization, the blots were washed (3–5 times over 20 min) with 0.3 M NaCl, 0.03 M sodium citrate (SSC) buffer containing 0.1% SDS at 65°C, and analyzed by autoradiography (Kodak X-OMAT film; intensifying screen at −70°C). For northern blots of mature crRNAs, RNA (10 μg) free of large RNAs obtained above was electrophoresed alongside 5′-end 32P-radiolabeled RNA markers (Decade, Ambion) in a 7 M urea-15% polyacrylamide gel (19:1 acrylamide:bisacrylamide) with TBE buffer, electrotransferred to a Hybond N+ membrane (250 mA for 45 min in TBE) and UV cross-linked as before. Single-stranded synthetic DNA probes (20 pmol) to detect mature crRNA corresponding to the CRISPR4 leader-proximal spacer 52 (5′-TGCATGAGTCGAAAGAGATTTTGAAGCGCCGGAC-3′) and leader-distal spacer 1 (5′-GTCTGGCAACACCAGAATGGCGGAACCGACTA-3′) were 5′-end-labeled with γ-32P-ATP (6000 Ci/mmol specific activity; Perkin Elmer) and T4 polynucleotide kinase (Takara) using standard protocols. The loading control was 5S RNA probed with 5′-CTTAACTTCCGTGTTCGGGATGGGAACGGGTGGGAC-3′. The mature crRNA blot was treated for 2 h at 30°C with 40% formamide, 7% SDS, 0.3 M NaCl, 0.05 M sodium phosphate pH 7 and Denhardt’s solution (57) with 100 μg/ml sheared, denatured salmon sperm DNA (58) and hybridized with specific probes in the same solution for 16 h at 30°C. The blots were washed with SSC buffer containing 0.2% SDS solution at 50°C, and analyzed by autoradiography as before.Bioinformatic analysisDatabase searches and analysis were carried out with BLASTN (for nucleotide sequences) and DELTA-BLAST (for proteins) at http://blast.ncbi.nlm.nih.gov/Blast.cgi. CRISPR-Cas data were analyzed using CRISPI (http://crispi.genouest.org/; (59)) and CRISPRTarget (http://bioanalysis.otago.ac.nz/CRISPRTarget/crispr_analysis.html (60)). Genomic context and putative operons in the M. xanthus genome were analyzed using Artemis software (http://www.sanger.ac.uk/resources/software/artemis (61)). Putative DdvS promoters within a maximum distance to gene of 200 bp were identified in the M. xanthus genomic region from positions 8909344 to 8883115 (∼26 kb at the ddvS–ddvA CRISPR4-Cas locus) with as query the sequence GTAAn16CGT (n is any deoxyribonucleotide) in Virtual Footprint Regulon Prediction (http://prodoric.tu-bs.de/vfp; (62)). RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi; (63)) and mfold (http://unafold.rna.albany.edu/?q=mfold; (64) servers were used for secondary structure predictions.

Article TitleMultifactorial control of the expression of a CRISPR-Cas system by an extracytoplasmic function σ/anti-σ pair and a global regulatory complex

Abstract

Microarray data are accessible through GEO accession number{"type":"entrez-geo","attrs":{"text":"GSE112385","term_id":"112385"}}GSE112385, deposited at the NCBI′s GEO (https://www.ncbi.nlm.nih.gov/geo/).


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