Materials and Methods
Bacterial strains, plasmids, and growth conditions.
Bacterial strains and plasmids used in this study are given in Table S1 in the supplemental material. P. atrosepticum SCRI1043 (49) was grown at 25°C and E. coli at 37°C in lysogeny broth (LB) at 180 rpm or on LB agar (LBA) plates containing 1.5% (wt vol−1) agar. When required, media were supplemented with chloramphenicol (Cm; 25 µg ml−1) or kanamycin (Km; 50 µg ml−1). Bacterial growth was measured in a Jenway 6300 spectrophotometer at 600-nm optical density (OD600).
Phage lysate preparation and titration.
The transducing phages, φTE (31) (genome size of ~142 kb) and φM1 (32, 40), were stored in phage buffer (10 mM Tris-HCl pH 7.4, 10 mM MgSO4, 0.01% wt vol−1 gelatin). Lysates were made by serially diluting phages in phage buffer, adding the mixture to 100 µl of a mixture consisting of P. atrosepticum culture (pregrown in 5-ml LB overnight) and 4 ml top LBA (0.35% φTE and 0.5% φM1 agar), and pouring the result onto LBA plates. Plates were incubated at 25°C overnight, plaques were counted, and the titer was determined. Top agar from plates with almost confluent lysis was harvested with 3 ml of phage buffer, subjected to vortex mixing with 500 µl chloroform (saturated with NaHCO3) for approximately 2 min, and centrifuged at 2,219 × g for 20 min at 4°C. The supernatant was collected, 100 µl of chloroform was added, and lysates were stored at 4°C. Titers of phages were determined as described above, typically resulting in high-titer stocks (5 × 1010 to 4 × 1011 PFU ml−1).
Duplicate 6-ml cultures of recipient strains were grown overnight and combined. The cultures were diluted to an OD600 of 2, and 10 ml (total, 1 × 1010 CFU) was pelleted and resuspended in 1 ml LB. Phage lysates were adjusted to 1 × 1011 PFU ml−1, and 100 µl (1 × 1010 PFU) was added at a multiplicity of infection (MOI) of 1. Transductions were incubated statically for 15 min at 25°C, and then 9 ml of LB (25°C) was added and the tubes were shaken for 45 min at 90 rpm on a slight angle at 25°C. Cells were pelleted at 2,219 × g for 9 min at room temperature, the supernatant was removed, and the pellet was resuspended in 10 ml LB. This step was repeated three times to remove excess phages, and the resulting material was finally resuspended in 1 ml LB. A 100-µl sample was plated onto LBA with the appropriate antibiotics, and the rest was pelleted and plated onto the same medium. Plates were incubated at 25°C for up to 5 days, and transductants were counted. Transduction efficiency was calculated as the number of transductants per PFU. Control samples of phage lysate and recipients were plated on the same antibiotics to check for contamination and spontaneous resistance, respectively, and no colonies were detected in any experiment. For infectivity controls, φTE titers were calculated for all lysates on their respective recipient strains to rule out differences in infection that might influence the determination of transduction efficiency (e.g., resulting from a receptor mutation).
P. atrosepticum Δcas (Cmr; strain PCF79 50) was made electrocompetent as described previously (36) and transformed with purified pTRB30 and pPF189 plasmids. φTE lysates were prepared on PCF79 transformed with pTRB30 and on PCF79 transformed with pPF189. The lysates were used to transduce the ΔHAI2, PIM06, and PIM17 strains. The CRISPR array spacer content of the PIM strains used in this study was determined by colony PCR and sequencing performed with primers for CRISPR1 (PF174 and PF175), CRISPR2 (PF176 and PF177), or CRISPR3 (PF178 and PF179) as described previously (30). All oligonucleotides used in this study are listed in Table S2. Plasmid transduction was verified by antibiotic resistance (gain of Kmr) and by PCR for the plasmid by using primers PF209 and PF210 (see Fig. S1A and B in the supplemental material). An infectivity control verified that all recipients had the same φTE sensitivity (Fig. S1C). Colonies were also checked for Cm sensitivity, and the results indicated that the colonies were not the original PCF79 strains used for lysate production. Mock lysates (containing no phages) were harvested from strain PCF79 containing either pTRB30 or pPF189 as described earlier but with phage buffer and no phage in the overlays. Mock lysates were used in transductions and did not facilitate plasmid transduction.
Oligonucleotides used in this study. Download TABLE S2, DOCX file, 0.01 MB.
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Transduction of chromosomal loci.
φTE lysates were prepared on six strains (PCF83 to PCF88) and used in transduction assays with strains ΔHAI2, PIM18, PIM28, and PIM86 as recipients with selection performed on Km (to detect Kmr transfer) or Cm (to detect Cmr control transfer). Phage lysate and recipient controls were also plated as described for the transduction assay. Representative transductants (where obtained) were patched onto LBA with Km and LBA with Cm. Putative transductants were verified by PCR for each marked locus with PF1212 (binds to marked insertion) in combination with the following primers: PF1575 (eca0128), PF1573 (eca0449), PF1457 (eca1388), PF1577 (eca1657), PF1459 (eca3296), and PF1460 (eca3672) (Fig. S2C). To verify the transduction of the cat gene, primers PF432 and PF433 were used (Fig. S2D). As a control transduction, φTE lysates were prepared using the wild-type P. atrosepticum strain (i.e., resulting in no marked transducing particles). When this was used in control transductions, no transductants (Kmr) were obtained.
Transduction of HAI2.
To check HAI2 transfer, lysates of φTE were prepared on three strains (PCF89 to PCF91) and used in transduction assays performed with the ΔHAI2 strain or the wild-type strain as the recipient. Since homologous recombination positively affects transduction efficiency, transduction to islandless recipients occurred at a 10-fold-lower frequency (~0.5 × 10−9 transductants/PFU) (Fig. 2B) than was seen with wild-type recipients (~0.5 × 10−8 transductants/PFU). Putative HAI2 transductants were verified by plating on LBA with Km or Cm and by using PCR to amplify features of the various strains. PCR was performed with primer pairs as follows: cas1 with PF390 and PF391, cat (Cmr gene) with PF432 and PF433, attP (circularized pHAI2) with PF1225 and PF1226, attB (the HAI2 insertion site) with PF1227 and PF1228, attL (island border regions) with PF1227 and PF1226, attR with PF1225 and PF1228, and, finally, a gene in the island, eca0560, with PF1446 and PF1447. To check CRISPR-Cas inhibition of transfer, φTE prepared on PCF90 was used in transduction assays with strains ΔHAI2, PIM06, and PIM31 as recipients.
Transduction with antiphage strains.
Transduction assays with antiphage strains were carried out using overnight cultures at an OD600 adjusted to 2 (1 × 109 CFU), and the cultures were resuspended in 1 ml LB in 50-ml tubes. Phages were added at an MOI of 1 (1 × 109 PFU). Assays with φTE were incubated for 15 min statically, followed by 45 min of shaking, before a 100-µl sample and then the remaining amount were plated. Reaction mixtures used for assays with φM1 were incubated for a total of 20 min with shaking, prior to plating onto media with antibiotics.
Efficiency of plating and efficiency of transduction assays.
Strains that had acquired φTE- and φM1-targeting spacers were isolated as previously described (51). The value representing the efficiency of plating was defined as the titer of the phage-resistant test strains (PCF188, PCF190, PCF193, PCF254, and PCF256)/the titer of the control strain (SCRI1043). The transducing lysates were prepared on PCF88 and PCF79 with pTRB30 plasmid, and the chromosomal marker and plasmid were transduced into recipients. Efficiency of transduction was determined by calculation of the test transduction efficiency/control (SCRI1043) efficiency.
Mixed-culture transduction assays.
Monocultures of the phage-sensitive WT (PCF326) strain and phage-resistant CRISPR (PCF332 anti-φTE and PCF400 anti-φM1) strains were grown overnight and combined in WT/anti-φ ratios of 100:0, 90:10, 50:50, 10:90, and 0:100, and 1 × 109 cells were used for each assay. Assays were performed with six replicates for both φTE and φM1. Phage lysate was added at an MOI of 1, and cells were shaken for 1 h, in 1 ml LB, before being plated onto LBA with selection. Transductants were patched onto LBA containing Nal or Sm to identify the host strain. Colony forming units (CFUs) were determined by taking initial and final samples (10 µl), plating onto LBA, and patching 100 colonies onto LBA containing Nal or Sm. Total PFU counts were determined by adding an aliquot (10 µl) of culture to LB containing chloroform. PFU fold change was calculated as final PFU/initial PFU. Transduction fold enhancement for WT strains was calculated as follows: (number of transductants/average number of transductants in the 100:0 assay)/proportion of WT cells in the assay.
Transduction of CRISPR by escape phages.
Transduction assays were performed as described above (see "Transduction with antiphage strains"). The transduction of CRISPR-Cas into the Δcas strain was identified by plating transductants onto LBA carrying Km to select for transfer of the chromosomal marker. Transductants were then patched onto LBA with or without Cm to screen for loss of Cmr, indicating that the cas genes had been transferred. PCR screening of CRISPR arrays to detect the transduction of spacers was performed using primers PF174 and PF175 for CRISPR1 and primers PF176 and PF177 for CRISPR2 (Table S2).
Nested PCR to screen for spacer acquisition.
Nested PCR was performed using primers PF1730 and PF1732 (round 1) followed by PF1730 and PF1733 (round 2). PCR products from round 1 were purified using an Illustra GFX PCR DNA and gel band purification kit (GE Healthcare) and were used as the round 2 template. To test the level of detection provided by PCR, the strains (PIM20 and ΔHAI2) were grown overnight. The PIM20 strain was serially diluted and combined with the ΔHAI2 strain at appropriate proportions based on OD600 values to create samples with 100 to 1010 PIM20 cells in a pool of 1010 total cells. An aliquot of each sample was used for PCR. Island transduction was carried out as described above (see "Transduction of HAI2"), using a donor with a Kmr marker in eca0573 (PCF89). Following transduction, pooled culture was used as the template for round 1 PCR.