Materials and Methods

A CRISPR-associated factor Csa3a regulates DNA damage repair in CrenarchaeonSulfolobus islandicus

MATERIALS AND METHODSCell growth and DNA damage treatment Sulfolobus islandicus strains, including the wild-type (E233S) and the derived strains, are listed in Supplementary Table S1. These strains were cultured at 78°C in the SCV medium (basal medium supplemented with 0.2% sucrose, 0.2% casamino acids and 1% vitamin solution), or SCVU medium (SCV medium with addition of 20 μg/ml uracil at a final concentration) (22). Sulfolobus genomic DNA damage treatment using 4-nitroquinoline-1-oxide (NQO), which mimics UV radiation, was performed as previously described (23). Briefly, NQO dissolved in DMSO solution was added to Sulfolobus cultures at the early exponential growth phase (OD600 = 0.2) to a final concentration specified in each experiment. Cell concentration was determined at OD600 during incubation, and cell samples were taken for aggregation assay, cell viability assay, RNA extraction, and flow cytometry analysis.Protein expression and purificationExpression and purification of the Csa3a protein were conducted as described previously (7).Transcriptome analysisStrains (two biological repeats for each strain) for transcriptome analysis were cultured to log phase (OD600 = 0.2). Thereafter, a 1 ml culture of each strain was transferred to 100 ml fresh SCVU medium in 250-ml flasks. Exponentially growing cultures of S. islandicus E233S (the wild-type strain, WT,) and Δcsa3a were diluted to OD600 = 0.2 and cultured in the presence or absence of 2 μM NQO for 6 h. Then, total RNA was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). Genomic DNA in the total RNA sample was removed using DNase I (Roche, Basel, Switzerland). The quality and quantity of purified total RNA were determined by measuring the absorbance at 260 and 280 nm using a NanoDrop ND-1000 spectrophotometer (Labtech, Wilmington, MA, USA). Total RNA integrity was verified by electrophoresis on a 1.5% agarose gel. A total of 3 μg RNA per sample was used as input material for cDNA library preparations. Sequencing libraries were generated using the NEBNext Ultra™ RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer's recommendations, and index codes were added to assign sequences to each sample. First-strand cDNA synthesis was performed using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H−). Subsequently, second-strand cDNA synthesis was performed using DNA polymerase I and RNase H, followed by 15 cycles of PCR enrichment. Sequencing was performed with an Illumina HiSeq2000 instrument. Raw data were initially processed to obtain clean reads by eliminating adapter sequences and low-quality bases. Clean reads were aligned to the reference genome sequence of S. islandicus REY15A (GenBank Accession No. {"type":"entrez-nucleotide","attrs":{"text":"NC_017276","term_id":"385774741","term_text":"NC_017276"}}NC_017276). An index of the reference genome was built using Bowtie software v2.0.6, and paired-end clean reads were aligned to the reference genome using TopHat software v2.0.9. To count the number of reads mapped to each gene, HTSeq software v0.6.1 was used, following which the reads per kilobase per million mapped reads (RPKM) for each gene was calculated based on the length of the gene and the number of reads mapped to the gene. Each strain was sequenced in duplicate. To determine the expression level of each gene in different groups, transcript expression levels were expressed as the RPKM. Next, P-values were used to identify differentially expressed genes (DEGs) between the 2 groups using the chi-squared test (2 × 2), and the significance threshold of the P-value in multiple tests was set based on the false discovery rate (FDR). Furthermore, Fold-changes (log2RPKM1/RPKM2) were estimated according to normalized gene-expression levels. Threshold for DEGs was set at P-values < 0.01 and log2 fold-change >1 (FDR < 0.05). Transcriptome data were deposited in the SRA database under Accession PRJNA608153. The DEGs are listed in Supplementary Table S2.Electrophoretic mobility shift assayPCR was used to generate probes for electrophoretic mobility shift assay (EMSA) using one of the primers with 5′-end HEX-label (Supplementary Table S3). PCR products of the probes were first cloned into the T-vector as the template for inverse PCR to introduce mutations at the desired sites. Mutated probes were amplified from above plasmids introduced with mutations using one of the primers with 5′-end HEX-label. Then, the PCR products were purified in 6% native polyacrylamide gel electrophoresis (PAGE) for EMSA. EMSA binding reactions (15 μl) containing 5 ng/μl of HEX-labeled probes and different concentrations of Csa3a protein (as described in the figure legends) were incubated for 20 min at 40°C in the binding buffer 20 mM Tris–HCl, pH8.0, 50 mM KCl, 5% glycerol, 1 mM ethylenediaminetetraacetic acid, 1 mM dithiothreitol, 5 ng/μl poly(dI-dC). For the specific competition, increasing amounts of unlabeled specific probes were added to the reaction mixture. Thereafter, samples were loaded onto a 4% low melting agarose gel buffered with 1× TAE solution. DNA–protein complexes were separated at 200 V for 40 min and the resulting bands was detected using a FUJIFILM scanner (FLA-5100).Localized surface plasmon resonance analysisPCR was used to generate the probes for the localized surface plasmon resonance (LSPR) analysis using the primers listed in Supplementary Table S3. PCR products were purified through ethanol precipitation. The Csa3a protein was immobilized on the chip with different titers of probes in the mobile phase, as indicated in the figure legends. The kinetic parameters of the binding reactions were calculated and analyzed by Trace Drawer software (Ridgeview Instruments AB, Sweden) and One to One fitting model.Flow cytometry analysisThe flow cytometry analysis was conducted as described previously (23). Briefly, 300 μl of Sulfolobus cells were fixed with 700 μl of absolute ethanol and stored at 4°C for 12 h. Then, fixed cells were collected through centrifugation at 2, 800 rpm for 20 min and washed with 1 ml of 10 mM Tris–NaCl buffer, pH 7.5, with 10 mM MgCl2. Cells were collected again and stained with 40 μg/ml ethidium bromide (Sigma-Aldrich, St. Louis, MO, USA) and 100 μg/ml mithramycin A (Apollo chemical, Tamworth, UK). We analyzed the stained cell samples in an Apogee A40 cytometer (Apogeeflow, Hertfordshire, UK) equipped with a 405 nm laser. A dataset of at least 60 000 cells were collected for each sample.Cell aggregation assayCell aggregation in S. islandicus cultures was estimated through direct microscopy of cell aggregates in fresh cultures, as previously described (19). Briefly, each strain was cultured in the absence or presence of 2 μM NQO for 12 h, after which aliquots were placed on glass slides, covered with coverslips and directly observed under a Nikon Eclipse 80i microscope (Nikon, Kobe, Japan). Data were collected from at least 24 fields of view images and more than 1000 single cells for each sample. For each analysis, three independent growth experiments were conducted.Report gene assayReporter plasmids were constructed using the Sulfolobus–E. coli shuttle vector pSeSD with the S. solfataricus galactosidase gene (lacS) as the reporter gene (24). For this experiment, we selected the promoters of the single DDR genes or the first genes of the DDR operons (SiRe_1878: upsX, SiRe_1879: upsE, SiRe_1881: upsA, SiRe_1316: cedA1, SiRe_1857: cedB, SiRe_1717: tfb3 and SiRe_1231: cdc6–2) that were up-regulated in the csa3a overexpression cells. Promoter fragments of these genes were amplified from S. islandicus REY15A genomic DNA using Phanta DNA Polymerase (Vazyme, Nanjing, China), and the primers listed in Supplementary Table S3. The PCR products (ca. 200 bp) were purified using the Cycle-Pure kit (Omega Bio-Tek, USA). Purified DNAs were digested and inserted into the pSe-lacS vector (25) to yield the reporter plasmids: pSe-upsX-lacS, pSe-upsE-lacS, pSe-upsA-lacS, pSe-cedA1-lacS, pSe-cedB-lacS, pSe-tfb3-lacS and pSe-cdc6–2-lacS (Supplementary Table S1).These reporter gene plasmids were electroporated into S. islandicus wild-type (E233S) and Δcsa3a strains, respectively. Three single colonies of each transformant were selected and cultured for 6 h in SCV either in the presence or absence of 2 μM NQO. Cell mass was collected for each strain and used to prepare the cellular extracts. The protein content of the cellular extracts was determined by microBCA Protein Assay Reagent (Thermo Scientific), whereas the β-galactosidase activity was determined as previously described (24).Cell viability analysisWe estimated the cell viability of Sulfolobus cultures by determining their colony formation units (CFU)/ml culture. Exponentially growing cultures of S. islandicus (OD600 = 0.2) were treated with NQO to a final concentration of 2 μM and incubated for 6 h. Then, using 1 ml of culture, cells were pelleted by centrifugation and re-suspended in 1 ml fresh SCVU medium. The resulting cell suspensions were serially diluted and plated using the two-layer plating method. Exactly 100 μl of the diluted samples were plated onto gelrite plates in triplicate. Colonies appearing on plates after 7 days of incubation were counted, to determine CFUs/ml culture.Plate titration experimentThe plate titration experiment of Sulfolobus was estimated by determining the density of the lawns on the plate. Exponentially growing cultures of S. islandicus (OD600 = 0.2) were treated with NQO with a final concentration of 2 μM and incubated for 48 h. Then, using 1 ml of culture, cells were serially diluted, and 10 μl of the diluted sample was dripped on the SCVU plate and incubated at 78°C for 2 days.DNA transfer and repair assaysDNA transfer assay was conducted as described previously (16), with modification. Targeting plasmid pTcas5 was constructed by cloning an oligonucleotide matching the protospacer on the cas5 gene in a mini-CRISPR cassette (Repeat-Spacer-Repeat) on the pSeSD plasmid. Then, 1 μg of pTcas5 plasmid was electroporated into 50 μl competent cells of S. islandicus wt (ΔpyrEFΔlacS) or Δcsa3a (ΔpyrEFΔlacSΔcsa3a) cells. After electroporation, 1 ml preheated medium was added into the culture containing the transformed cells. The mixed cultures were incubated with or without 2 ml of preheated Δcas5 (ΔpyrEFΔlacSΔcas5) mating partner cells (OD600 = 0.5) for 2 h at 78°C, then, plated on the SCV medium without uracil at 78°C for 6 days. We calculated increased folds of transformation efficiencies of wt or Δcsa3a cells incubated with Δcas5 partner cells compared with that of wt or Δcsa3a cells (Ewt::pTcas5+Δcas5/Ewt::pTcas5 or EΔcsa3a::pTcas5+Δcas5/EΔcsa3a::pTcas5; E: transformation efficiency), respectively. Deletion of the cas5 gene locus on the chromosomes of the single colonies of wt::pTcas5 × Δcas5 or Δcsa3a::pTcas5 × Δcas5 conjugants were determined by PCR analysis.

Article TitleA CRISPR-associated factor Csa3a regulates DNA damage repair in CrenarchaeonSulfolobus islandicus

Abstract

Transcriptome data were deposited in the SRA database under Accession PRJNA608153.


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