Materials and Methods

Type I CRISPR-Cas provides robust immunity but incomplete attenuation of phage-induced cellular stress

Culture conditions and phage lysate preparation

Serratia sp. ATCC 39006 (Serratia) and Escherichia coli ST18 were grown overnight in lysogeny (LB) broth at 30 and 37°C, respectively in shaking conditions (160 rpm). When grown on plates, LB with agar (LBA, 1.5% w/v) was used and plates were incubated at an appropriate temperature until colony formation. When needed, antibiotics and supplements were added to the LB and LBA: Chloramphenicol (Cm; 25 μg/ml), Kanamycin (Km; 50 μg/ml), Tetracycline (Tc; 10 μg/ml), Gentamycin (Gm; 30 μg/ml) and 5-aminolevulinic acid (ALA; 50 μg/ml). New phage stocks were prepared as described elsewhere (23) using the double agar overlay method. Briefly, 100 μl of bacterial overnight culture and 100 μl of phage lysate were added to 4 ml of molten LBA overlay (0.35% w/v) and poured onto LBA plates. The phage concentration used was enough to produce almost confluent lysis. After an overnight incubation at 30°C, the overlays were pooled into a collection tube. Two drops of chloroform (NaHCO3-saturated) were added and the sample was vortexed to lyse the cells. Finally, cellular debris was removed by centrifugation (2000 g for 20 min) and the phage stock was transferred into a new tube and stored at 4°C. To calculate the phage titre, 10-fold serial dilutions were made in phage buffer (10 mM Tris Base (pH 7.4), 10 mM MgSO4, 0.01% w/v gelatine) and spotted (20 μl) onto LBA overlay previously seeded with 100 μl of Serratia. After an overnight incubation at 30°C, plaques were counted and phage titre was expressed as plaque forming units (pfu)/ml.

Primers, plasmids and strains

Primers, plasmids and strains used in this study are detailed in Supplementary Tables S1, S2 and S3, respectively.

Phage isolation

Phage JS26 was isolated from sewage samples collected from the Tahuna Waste Water Treatment Plant in Dunedin, New Zealand (45°54'16.1‘S; 170°31'16.8’E). Enrichment for Serratia phages was performed by inoculating an overnight Serratia culture with 100 μl of sewage sample and incubating overnight at 30°C under shaking conditions (160 rpm). A 10-fold dilution series was prepared and plated onto a top agar overlay seeded with Serratia overnight culture. Plaques showing different morphologies were picked and used to infect a new Serratia culture. This step was repeated until homogenous looking plaques were obtained to ensure phage purity.

Genome sequencing, annotation and comparative genomics

Phage genomic DNA (gDNA) was extracted from a high titre phage stock (∼109 pfu/ml) using the cetyltrimethylammonium bromide (CTAB) method described elsewhere (33). Samples were cleaned using the DNeasy Blood & Tissue Kit (QIAGEN) following the manufacturer's instructions and DNA was quantified using the Qubit dsDNA HS Assay Kit and the Qubit Fluorometer (2.0) (invitrogen) following the manufacturer's instructions. Isolated gDNA was sent to the Massey Genome Service (New Zealand) where libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina), QC was checked with the Quant-iT dsDNA HS Assay for quantification and analysed using SolexaQA++, fastQC and fastQsceen. Sequencing was performed using Illumina MiSeq (2 × 150 bp) and the resulting reads were processed and trimmed using SolexaQA++ (v3.1.7.1). The genome was assembled using SPAdes 3.9 (34), annotated with RASTtk (2.0) (35) and manually curated using BLASTp (2.10.0) (Supplementary Table S5). To identify putative tRNAs, tRNAscan-SE v. 2.0 (36) was used. The final genome sequence was deposited in GenBank under the accession number MN505213.1 and visualized using EasyFig (2.2.2) (37). Related phages were identified using PAirwise Sequence Comparison (PASC) (38), the phylogenetic tree was built through whole genome blast using VIrus Classification and Tree building Online Resource (VICTOR) (39) using distance formula _d_6 and branch support was inferred from 100 pseudo-bootstrap replicates each and modified with FigTree (v1.4.3).

Proteomics

A Serratia culture (25 ml) was mixed with 500 μl of high titre phage stock (∼109 pfu/ml) and grown overnight at 30°C in shaking conditions (160 rpm). Cell debris were removed by centrifugation (2000 g for 20 min) and the supernatant containing phages was concentrated and purified with a sucrose cushion. Briefly, phage samples were pipetted onto a 20% (w/v) sucrose solution underlay. The sample was separated by centrifugation (120 000 g for 4 h at 20°C) and the pellet was resuspended in 250 μl of no-gelatin phage buffer and dialysed overnight against 2 l of milliQ water at 4°C. The phage solution was pooled together and cleaned from cell debris by centrifugation (10 000 g for 20 min). Next, a CsCl gradient was prepared by adding 2 ml of 1.2 g/ml and 1.6 g/ml CsCl solutions into a centrifuge tube. The sample was separated by centrifugation (120 000 g for 4 h at 20°C), and the white phage interface that formed in the CsCl gradient was harvested using a Pasteur pipette. The concentrated phage stock was washed by adding no-gelatin phage buffer and pelleted by an ultracentrifuge step (120 000 g for 2 h at 20°C). The pellet was resuspended in 100 μl milliQ water and stored at 4°C. For the proteomic analysis, 75 μl of phage sample was mixed with 20 μl of 1/10 (v/v) β-mercaptoethanol 4× SDS-buffer and incubated for 5 min at 95°C. The sample was separated by electrophoresis in a 12% (w/v) SDS-polyacrylamide gel for 1 h at 150 V. Page ladder (Thermo Fisher Scientific) was used as a reference. The gel was fixed for 2 h in 10% (v/v) acetic acid, 40% v/v ethanol. Finally, the gel was stained overnight in four parts of Colloidal Coomasie Blue stain (0.1% G250 w/v, 10% w/v ammonium sulphate and 2% v/v ortho-phospholic acid) and one part methanol.

Proteomic analysis of the phage structural proteins was performed as described elsewhere (40). Briefly, the gel lane was subjected to in-gel digestion with trypsin and analysed by protein identification by liquid chromatography–coupled tandem mass spectrometry (LC–MS/MS) in the Centre for Protein Research (University of Otago). Peptide reconstruction was performed with an Ultimate 3000 nano-flow uHPLC-System (Dionex Co., Thermo Fisher Scientific; Waltham, MA, USA) in-line coupled to the nanospray source of an LTQ-Orbitrap XL mass spectrometer (Thermo Scientific; Waltham, USA). Raw spectra were processed through the Proteome Discoverer software (Thermo Fisher Scientific, Waltham, MA, USA) using default settings to generate peak lists. Peak lists were then searched against a combined amino acid sequence database containing all JS26 sequence entries (GenBank accession number MN505213.1, 84 entries) integrated into the full SwissProt/UniProt sequence database using the Sequest HT (Thermo Fisher Scientific), Mascot (www.matrixscience.com) and MS Amanda search engines (Supplementary Table S6).

Electron microscopy

To examine phage by transmission electron microscopy (TEM), 10 μl of high titre phage stock (∼109 pfu/ml) was loaded onto plasma-glow discharged carbon coated 300 mesh copper grids. After 60 s, the excess specimen was removed by blotting and 10 μl of 1% (w/v) phosphotungstic acid (PTA) (pH 7.2) was applied to the grid to stain the samples and blotted off immediately. The grids were analyzed in the Otago Micro and Nano Imaging (OMNI) facility and viewed in a Philips CM100 BioTWIN transmission electron microscope (Philips/FEI Corporation, Eindhoven, Holland) and images captured using a MegaView lll digital camera (Soft Imaging System GmbH, Münster, Germany).

Efficiency of plaquing assay

To assess phage infectivity in different Serratia strains, an efficiency of plaquing (EOP) assay was performed. Serial 10-fold dilutions of high titre phage stocks (∼109 pfu/ml) were spotted onto LBA overlays seeded with Serratia (100 μl). After the spots were dry, the plates were incubated at 30°C overnight. The EOP was calculated as the ratio between the pfu/ml in Serratia strains and the wild-type control. All conditions were repeated in biological triplicates and the data was plotted as the mean ± SD.

Phage infection time courses

Serratia strains were grown to exponential phase (OD600 = 0.3) and diluted to an OD600 = 0.05. Bacterial cultures (180 μl) were pipetted into 96-well plates and infected with 20 μl phage lysate to produce a multiplicity of infection (moi) = 0, 1 and 10. The plates were incubated in a Varioskan Flash plate reader (Thermo Fisher Scientific) for 20 h at 30°C and shaking (240 rpm) and OD600 measurements were taken every 12 min. The experiment was repeated in biological triplicates and the data was plotted as the mean ± SD.

Phage receptor characterization

Receptor identification was performed by transposon mutagenesis. Overnight cultures of donor (E. coli ST18 pKRCPN2 carrying transposon Tn-DS1028uidA_Km) and recipient (_Serratia) strains were adjusted to an OD600 = 1, washed free from antibiotics and mixed in equal ratios. The samples were spotted (20 μl) onto LBA + ALA and incubated overnight at 30°C to allow conjugation. Next, the spots were scraped and resuspended in LB + Km to select for the transposon insertion events. The mutant pool was grown overnight and seeded onto an LBA overlay. High titre JS26 stocks (∼109 pfu/ml) were spotted (20 μl) onto the lawn and plates were incubated at 30°C until colonies appeared within the phage spot. Phage resistant mutants were restreaked onto a new plate to ensure purity.

Transposon insertion sites were identified by arbitrary PCR (41). A first round of random colony PCR was performed on phage resistant clones using a random primers PF106, PF107 and PF108 and transposon nested primers PF226 and PF1212. PCR products were cleaned using the GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare) and used as DNA template for a second round of PCR with adapter primer PF109 (that binds to the 5′ ends of PF106-PF108) and Tn-DS1028uidA_Km nested primers (either PF226 or PF1212). Bands were extracted, sequenced and mapped against _Serratia genome to detect the transposon insertion site. EOP assay were performed on transposon mutants (PCF619, PCF620, PCF621, PCF623) to test phage resistance with an ΔflhDC mutant (PCF879) used as a control. Moreover, the swimming ability of the mutants was evaluated in a motility assay. The mutants were stabbed onto LBA (0.35% w/v) and grown overnight at 30°C. Motility was assessed by measuring the halo of swimming from the inoculated spot and compared with a Serratia WT as the control.

One step growth curve

A Serratia culture (25 ml) was grown up to exponential phase (OD600 = 0.3) and infected with JS26 at an moi = 0.1. The culture was incubated at 30°C in shaking conditions (160 rpm) and samples were taken at different time point (5, 20, 40, 60, 80, 100, 120 140, 160 min post infection (mpi)). At each time point, a 100 μl aliquot was taken and mixed with 900 μl of LB. The samples were immediately diluted in a 10-fold series and spotted (20 μl) onto LBA overlays previously seeded with Serratia. The experiment was repeated in three biological replicates and the data was plotted as mean of pfu/ml ± SD.

Cell survival assay

To determine the moi at which synchronicity of infection was achieved, a cell survival assay was performed. Serratia cultures (5 ml) were grown up to an early exponential phase (OD600 = 0.3) and infected with a range of moi (moi = 0, 0.1, 1, 25, 50). The infected cultures were incubated at 30°C, shaking (160 rpm) and samples were taken (500 μl) at time points 0, 5 and 25 mpi. The aliquots were mixed with 500 μl LB and centrifuged (17 000 g for 2 min) to remove free phages. The bacterial pellet was washed with 1 ml of LB, resuspended in 500 μl LB and used to prepare in 10-fold serial dilutions. The bacterial dilutions were spotted (20 μl) onto LB plates. The percentage of cell survival was calculated as cfu/ml in infected samples/cfu/ml in uninfected control × 100%. The experiment was repeated in biological triplicates and the data was plotted as mean ± SD.

Generation of native type I-E and I-F anti-phage strains

Serratia strains harbouring anti-JS26 spacers (Supplementary Tables S3 and S4) in their type I-E and I-F chromosomal arrays (CRISPR1 and CRISPR2 respectively) were generated by primed spacer acquisition (42). Plasmids pPF1257 (type I-E priming vector) and pPF1258 (type I-F priming vector), carrying a phage fragment and a protospacer primed by a spacer in CRISPR1 and CRISPR2, respectively, were constructed as follows. A fragment (∼1 kb) of the tape measure gene (gp61) was amplified by PCR (using primers PF2296 and PF2297) from phage gDNA and digested with SpeI and KpnI. The insert was cloned into the two priming vectors (pPF1125 and pPF1126) previously digested by the same enzymes. Plasmids were transformed into E. coli ST18, plated onto LBA + ALA + Cm and cloning was checked by PCR using primers PF1403 and PF1372, and confirmed by sequencing. The resulting plasmid was conjugated into Serratia by mixing equal ratios of donor and recipient strains and spotting mating spots onto LBA + ALA and incubated at 30°C overnight. The mating spots were streaked onto LBA + Cm to select for transconjugants.

Colonies were grown overnight in LB in the absence of antibiotic selection to allow for plasmid targeting and cultures passaged overnight for 2–3 days. Aliquots were taken each day, and dilutions were plated onto LBA and LBA + Cm to check for plasmid loss. Array expansion was checked by PCR screening with PF1989/PF1887 (CRISPR1) and PF1990/PF1888 (CRISPR2), and acquisition of anti-JS26 spacers was confirmed by sequencing.

RNA extraction and sample preparation and RNA-seq analysis

Serratia WT or anti-JS26 strains (PCF524 and PCF525) were grown to exponential phase (OD600 = 0.3) in 25 ml LB at 30°C and in shaking conditions (160 rpm). Cultures were infected at an moi = 50 and 2 ml samples were taken at 0 (prior to phage infection), 5, 20 and 40 mpi. Immediately after extraction, the samples were centrifuged (17 000 g for 1 min at 4°C) to remove the free phage and the bacterial pellet was resuspended in 2 ml of RNAlater (Invitrogen) and stored at –20°C. The experiment was repeated in biological triplicates.

Total RNA was extracted using the RNeasy kit (QIAGEN). Briefly, samples resuspended in RNAlater were harvested by centrifugation (17 000 g for 1 min at 4°C) and resuspended in 250 μl of 10 mM Tris–HCl (pH 8.0). The resuspended pellet was transferred to a pre-chilled bead beater tube containing 350 μl of RLT buffer (lysis buffer) and β-mercaptoethanol. Cell lysis was performed with a bead beater, using three 10 s pulses interspaced by a 1 min incubation in ice. The samples were chilled for 3 min on ice and cellular debris was removed by centrifugation (17 000 g for 1 min at 4°C). The homogenate was transferred into a clean Eppendorf and 1.5× volume of 100% ethanol was added and mixed by vortexing. The sample was transferred into the RNeasy mini spin column, centrifuged (>8000 g for 15 s) and the flow through discarded. Next, the column was washed twice with 500 μl of Buffer RPE (washing buffer) followed by centrifugation (>8000 g for 15 s). RNA was eluted by adding 50 μl of RNase free water followed by centrifugation (>8000 g for 1 min). The sample was treated with TURBO DNase (Invitrogen) following the manufacturer's instructions. Finally, impurities were removed by centrifugation (>8000 g for 1 min) and the RNA samples were transferred to a new tube and stored at –20°C.

The absence of DNA contamination was confirmed by PCR using primers PF796 and PF797 that amplify a fragment encoding the flhDC genes in Serratia. RNA concentration was estimated using a nanodrop and the quality assessed by an RNA nano chip on the Bioanalyzer (Agilent). The total RNA samples were ribo-depleted using Ribo-zero rRNA removal kit (epicentre) and directional cDNA libraries were prepared with the TruSeq stranded mRNA library preparation kit (Illumina) following manufacturer's instructions.

The samples were sequenced in the Otago Genomic Facility (OGF, New Zealand) using Illumina HiSeq 2500 v4 Rapid single-read sequencing, with ∼10 million reads (101 bp) generated per sample. The reads were trimmed using Trimmomatic (43) and their quality evaluated through FASTQC analysis. Finally, raw sequence reads were aligned to Serratia (accession number CP025085) and JS26 (accession number MN505213.1) genomes using Bowtie2 with default parameters (44). The alignment was converted to BAM files format using SAMtools (45). The differential expression between the uninfected controls (timepoint 0) and the infected samples (5, 20 and 40 mpi) was evaluated using the DESeq2 (46) R/Bioconductor package (3.5.2) with a Wald test, followed by a Benjamini and Hochberg procedure. All sets were defined by a false discovery rate of 10%. Genes differentially expressed in Serratia WT and anti-JS26 I-E and I-F strains are listed in Supplementary Tables S7, S8 and S9, respectively. Classification of differentially expressed genes was performed using the gene set enrichment analysis platform (GSEA-pro V3.0) using operon, Gene Ontology (GO) (47) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) (48) databases.

Adaptation assay

To determine if the phage-encoded cas4 homologue affected spacer acquisition in Serratia, cas4 was cloned into an overexpression vector under an arabinose inducible promoter (with cas4, pPF2291; empty vector, pPF783) and overexpressed during adaptation assays. The cas4 gene was amplified from JS26 gDNA through PCR using primers PF4692 and PF4693. Gibson assembly (NEBuilder HiFi DNA Assembly) was used to clone the insert into the expression vector pPF783 previously digested with restriction enzymes EcoRI and SphI. The construct was transformed into E. coli ST18 and conjugated into Serratia. Adaptation assays during cas4 overexpression were performed as previously described (22). Briefly, plasmids pPF719 and pPF953 (non-targeted ‘naïve’ control), pPF1233 and pPF1048 (high and medium ‘primed’ type I-E) and pPF1242 and pPF1243 (high and medium ‘primed’ type I-F) were conjugated from E. coli ST18 into Serratia carrying pPF783 or pPF2291. Transconjugants were streaked onto LBA with Tc and Gm to ensure maintenance of the two plasmids. Single colonies were used to inoculate 5 ml LB cultures supplemented with arabinose (0.05% w/v) and Gm. These were incubated overnight at 30°C under shaking condition (160 rpm) and passaged for 3 days by daily transfer of 10 μl of culture into 5 ml of fresh LB supplemented with arabinose (0.05% w/v) and Gm. CRISPR array expansion was determined by PCR directly on cells from passaged cultures using primers PF633 and PF2177 for the type I-E array (CRISPR1) and PF1888 and PF1990 for the type I-F array (CRISPR2). PCR products were run on 3% agarose gels with a 1 kb + DNA ladder (invitrogen), stained with ethidium bromide and visualized under UV light. The experiment was performed in biological triplicates.

CRISPRi of the phage cas4 homologue

To determine if the JS26 cas4 homologue was required for phage replication, EOP and phage infection time courses were performed on Serratia carrying a dCas9 expression vector with (pPF2786) or without (pPF1755) a single guide RNA (sgRNA) targeting the cas4 homologue in JS26. EOP and phage infection time courses experiments were performed as previously described with the addition of arabinose (0.1% w/v) to induce expression of dCas9 and Km for plasmid maintenance.

Article TitleType I CRISPR-Cas provides robust immunity but incomplete attenuation of phage-induced cellular stress

Abstract

The DNA sequencing reads from JS26 and the raw reads from the RNA-sequencing experiment have been deposited in the NCBI SRA with the BioProject number PRJNA746619. Processed data files have been deposited in GEO under accession number{"type":"entrez-geo","attrs":{"text":"GSE186673","term_id":"186673"}}GSE186673. The JS26 genome has been deposited in the NCBI GenBank database with the accession number{"type":"entrez-nucleotide","attrs":{"text":"NC_053012.1","term_id":"1985467647","term_text":"NC_053012.1"}}NC_053012.1. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (90) partner repository with the dataset identifier PXD029878.


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