Materials and Methods
Bacterial strains and growth conditions
All strains and plasmids used in this study are given in Supplementary Table S1 and details of their construction provided in Supplementary Materials and Methods. P. atrosepticum SCRI1043 (30) was grown at 25°C and E. coli at 37°C in Luria Broth (LB) at 180 rpm or on LB-agar (LBA) plates containing 1.5% (w/v) agar. When required, media were supplemented with the following: ampicillin (Ap; 100 μg/ml), chloramphenicol (Cm; 25 μg/ml), kanamycin (Km; 50 μg/ml), tetracycline (Tc; 10 μg/ ml) and D-glucose (0.2% w/v). Bacterial growth was measured in a Jenway 6300 spectrophotometer at 600 nm (OD600).
Molecular biology and DNA sequencing
Oligonucleotides were from Invitrogen or IDT and are listed in Supplementary Table S2. All strains and plasmids were confirmed by polymerase chain reaction (PCR) and DNA sequencing was performed at the Allan Wilson Centre, New Zealand. Plasmid DNA was prepared using Zyppy Plasmid Miniprep Kits (Zymo Research). DNA from PCR and agarose gels was purified using the GE Healthcare Illustra GFX PCR DNA and Gel Band Purification Kit. Restriction enzymes and T4 DNA ligase were from Roche or NEB.
Five millilitres cultures of P. atrosepticum ΔHAI2 with pTRB30 (vector control) or pPF189 were grown overnight without antibiotic selection (Figure (Figure1).1). Note that 10 μl were used to inoculate a fresh overnight culture and dilutions were plated onto LBA. This was repeated over 5 days and performed in triplicate. Colonies (100) from each replicate were patched onto LBA ± Km. Km sensitive (KmS) colonies were screened by PCR for new spacers as described later.
A pre-existing spacer:protospacer match accelerates Cas-dependent plasmid loss. (A) P. atrosepticum contains a Type I-F CRISPR-Cas system composed of three CRISPR arrays (1–3; gray arrows) and an operon of 6 cas genes (colored arrows). CRISPR2 consists of 10 spacers and spacer 6 (from leader proximal end; blue) perfectly matches a protospacer (red) in eca0560 in the chromosomal island HAI2, but has a TG PAM variant. (B) Schematic of the plasmid loss assays. P. atrosepticum ΔHAI2 carrying pTRB30 (control) or pPF189 (eca0560 primed; depicted) plasmids were grown without selection for 5 days and plasmid loss was scored by replica-plating on non-selective (NS) and selective (S) media. (C) Plasmid loss of a control plasmid (pTRB30) and the primed plasmid (pPF189) over 5 days when cultured in ΔHAI2 or ΔHAI2Δcas backgrounds. Data shown are the mean ± SD of three biological replicates.
P. atrosepticum containing plasmids pPF571 (vector control), pPF574 and pPF575 (priming vectors with protospacer 1 from the CRISPR1 array with a mutated PAM in F and R orientations) were grown overnight in 5 ml LB and passaged for 5 days by transfer of 10 μl to 5 ml fresh LB. Additionally, dilutions were plated on LBA + 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) for mCherry induction. White colonies were screened for CRISPR expansion and sequenced as outlined below.
CRISPR PCR and sequence analysis
Colonies displaying plasmid loss were screened by colony PCR using primers for CRISPR1 (PF174 and PF175), CRISPR2 (PF176 and PF177) or CRISPR3 (PF178 and PF179). The resulting products were separated on 2% agarose gels, purified and sequenced using PF175, PF177 or PF179 for CRISPR arrays 1, 2 or 3, respectively. Sequences were analyzed using CRISPRFinder (31), spacer sequences were extracted and assembled against target plasmids using Geneious™ and CRISPRTarget (23) to define the protospacer location, target strand and PAM.
Transformation and conjugation assays
Electrocompetent P. atrosepticum cells were prepared as described previously (32). For transformations, 50 ng of DNA was added to 50 μl of competent cells, incubated on ice for 10 min then electroporated (1 mm electro-cuvettes, 1800 V, capacitance 25 μF and resistance 200 ohms). Bacteria were recovered in 1 ml LB for 2 h at 25°C and then plated on LB containing the appropriate supplements and grown at 25°C. Transformation efficiency was calculated as transformants/ng of DNA and normalized to non-targeted plasmid controls.
For conjugation, the tested plasmids were transformed into E. coli S17–1 λpir. Donor (E. coli S17–1 λpir with tested plasmids) and recipient strains were grown overnight in LB with the appropriate antibiotics. The OD600 was adjusted to 1 and cells washed twice with LB. The donor and recipient strains were mixed (1:1 ratio), 5 μl of the mixture spotted on 0.2 μm filters (Millipore) on LBA and incubated overnight. Cells were resuspended in 2 ml phosphate buffered saline by vortexing the filters and dilution series were plated on LBA (total cells), LBA + Sp (donors) and glucose minimal medium + Km (transconjugants). The efficiency of conjugation was calculated as transconjugants per recipients.