1. Microbiology and Infectious Disease
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A chimeric nuclease substitutes a phage CRISPR-Cas system to provide sequence-specific immunity against subviral parasites

  1. Zachary K Barth
  2. Maria HT Nguyen
  3. Kimberley D Seed  Is a corresponding author
  1. Department of Plant and Microbial Biology, University of California, Berkeley, United States
  2. Chan Zuckerberg Biohub, United States
Research Article
Cite this article as: eLife 2021;10:e68339 doi: 10.7554/eLife.68339
6 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Some ICP1 isolates encode a free-standing nuclease in place of CRISPR-Cas.

(A) A model of ICP1 interference of phage-inducible chromosomal island-like elements (PLEs) via CRISPR. When ICP1 infects a PLE(+) V. cholerae cell, ICP1 is able to overcome PLE restriction and reproduce if it possesses a CRISPR-Cas system with complementary spacers to the PLE. Cas and CR refer to the CRISPR associated genes and CRISPR array respectively. (B) Schematics of the region between gp87 and gp91 as it appears in ICP12001 (top) and ICP12006 (bottom). Genes represented by black arrows are conserved in all ICP1 isolates, while genes represented with gray arrows covary with gp88 or CRISPR-Cas. (C) An alignment between the T5orf172 domain of Gp88 and the GIY-YIG domains of several structurally resolved endonucleases. Secondary structure for Gp88 was predicted using HHPRED (Zimmermann et al., 2018). Alpha helices are shown in yellow shading, and beta strands are shown in blue shading. Key residues of the GIY-YIG motif are bolded. We included an atypical GIY-YIG endonuclease domain from a chloroplast-encoded glutoredoxin atGRXs16 to demonstrate the potential for alternative residues at core motif positions. A conserved glutamate that was previously found to be required for catalysis in I-TevI is denoted by an asterisk (and corresponds to the E180A mutation in Gp88 in subsequent experiments).

Figure 1—figure supplement 1
ICP12005 has an atypical CRISPR-Cas arrangement.

Genetic organization of the region between gp87 and gp91 in different ICP1 isolates (ICP12001[top], ICP12006[bottom], ICP12005[middle]). Other than ICP12005, all sequenced ICP1 isolates (Angermeyer et al., 2018) share their organization with ICP12001 or ICP12006.

Figure 2 with 2 supplements
The alternative nuclease Gp88 controls ICP1 host range in a natural isolate that lacks CRISPR-Cas.

(A) Tenfold dilutions of the phage isolate or mutant derivative indicated spotted on V. cholerae with the PLE indicated (bacterial lawns in gray, zones of killing are shown in black). Gp88* possess a single amino acid substitution (E180A) predicted to abolish nuclease activity. Spot assays were performed in biological triplicate, and a single representative image is shown. Replicate spot assays are shown in Figure 2—figure supplement 1 and Figure 2—figure supplement 2. (B) Replication of PLE1 and PLE2 in V. cholerae host strains calculated as the fold change in PLE DNA copy 20 minutes post infection with the ICP1 variant indicated.

Figure 2—figure supplement 1
Biological replicate of spot assays.

Tenfold dilutions of the phage isolate or mutant derivative indicated spotted on V. cholerae with the PLE indicated (bacterial lawns in gray, zones of killing are shown in black). This figure and Figure 2—figure supplement 2 each represent one biological replicate, while a third replicate is divided across Figure 2B, Figure 4B, and Figure 5B. Strain backgrounds appear in the same order that they first appear in the main text. For each biological replicate, spot assays were performed in parallel for the different strains.

Figure 2—figure supplement 2
Biological replicate of spot assays.

Tenfold dilutions of the phage isolate or mutant derivative indicated spotted on V. cholerae with the PLE indicated (bacterial lawns in gray, zones of killing are shown in black). This figure and Figure 2—figure supplement 1 each represent one biological replicate, while a third replicate is divided across Figure 2B, Figure 4B, and Figure 5B. Strain backgrounds appear in the same order that they first appear in the main text. For each biological replicate, spot assays were performed in parallel for the different strains.

Figure 3 with 2 supplements
Phage-inducible chromosomal island-like element (PLE) replicons are modular and are composed of a compatible RepA initiation factor and origin of replication (ori).

(A) Genomic organization of PLE1 with insets corresponding to the PLE noncoding region and repA. Previously characterized PLE genes are labeled. Insets are Mauve alignments showing sequence conservation of the denoted loci across the different PLEs. Shared color denotes sequence conservation, with the height of the histogram representing nucleotide sequence identity. The susceptibility of each PLE(+) V. cholerae host to plaquing by phage encoding Gp88 is indicated. (B) Replication of hybrid PLEs in V. cholerae calculated as the fold change in PLE DNA copy 20 minutes post infection with ICP12006 ∆CRISPR. Strains with PLE1 ∆repA (possessing the native oriPLE1 or ∆ori::oriPLE2) or PLE2 ∆repA (possessing the native oriPLE2 or ∆ori::oriPLE1) were complemented with a vector expressing the repA gene from PLE1 or PLE2. The backbone, identity of the ori, and RepA variant are indicated as being from PLE1 or PLE2.

Figure 3—figure supplement 1
Alignment of repA from phaPLEs 1–5 encoding the conserved C-terminus.

The PLE1 sequence and identical nucleotides in the other PLE variants are shaded blue. Nucleotides that differ from PLE1 but co-occur in at least two PLE variants are shaded yellow. For positions where PLE1 is unique and both PLE2 and PLE3, and PLE4 and PLE5 have separate consensuses, the PLE2 and PLE3 consensus is bolded and unshaded, and the PLE4 and PLE5 consensus is underlined and unshaded. Nucleotides with no aligned matches are unshaded normal text.

Figure 3—figure supplement 2
PLE2 requires RepA and a cognate origin of replication to replicate.

Replication of PLE2 in V. cholerae calculated as the fold change in PLE DNA copy 20 minutes post infection with ICP12006 ∆CRISPR.

Figure 4 with 5 supplements
Gp88 is a PLE replication origin-directed nuclease.

(A) Sequence alignment of the N-terminal portion of Gp88 with the RepA_N domain from PLE1 RepA. Identical residues are denoted with a ‘*.’ Strong residue similarity is denoted by ‘:’, and weak similarity is denoted by ‘•.’ (B, C) Tenfold dilutions of the phage isolate or mutant derivative indicated spotted on V. cholerae with the PLE indicated (bacterial lawns in gray, zones of killing are shown in black). Spot assays were performed in parallel with those in Figure 2, and images labeled with the same PLE background are the same image. Spot assays were performed in biological triplicate, and a single representative image is shown. Biological replicates are shown in Figure 2—figure supplement 1. Gp88* possess a single amino acid substitution (E180A) predicted to abolish nuclease activity. (B) shows phage susceptibility of V. cholerae with PLE1, PLE4, and PLE5 ∆ori derivatives as compared to a strain without PLE. (C) shows phage susceptibility for V. cholerae with PLE2 ∆ori and PLE2 ∆ori::oriPLE1. (D) Nuclease assay showing the integrity of a PCR product amplified from the noncoding region containing the ori from the PLE variant indicated (numbers) treated with (+) and without (–) 500 nM of purified Gp88. Nuclease assays were performed in triplicate, replicates are presented in Figure 4—figure supplement 3. (E) Nuclease assay showing the integrity of a PCR product amplified from the noncoding region containing the ori from the PLE variant indicated (numbers) treated with (+) and without (–) 500 nM of purified Gp88*. Nuclease assays were performed in triplicate, replicates are presented in Figure 4—figure supplement 4.

Figure 4—source data 1

Original uncropped gels for Figure 4D (top) and Figure 4E (bottom).

The cropped images shown in the figure are indicated by the orange boxes.

https://cdn.elifesciences.org/articles/68339/elife-68339-fig4-data1-v1.pdf
Figure 4—figure supplement 1
The nuclease domain of Gp88 is similar to those of putative homing endonucleases in ICP1.

(A) A multiple sequence alignment between the C-terminal portion of Gp88 and several homologous sequences found in putative homing endonucleases in the ICP1 genome. Identical residues are denoted with a ‘*.’ Strong residue similarity is denoted by ‘:,’ and weak similarity is denoted by ‘•.’ (B) A phylogenetic tree constructed from the multiple sequence alignment in (A).

Figure 4—figure supplement 2
Protein preparations of Gp88 (Odn) and Gp88* (Odn*) used for in vitro assays.

Protein preparations were separated by SDS-PAGE and visualized by Coomassie staining. The marker (M) is indicated; the predicted molecular weight of untagged Gp88 is 26 kDa. (A) Gel showing wild-type Gp88 protein used for all cleavage assays except for those depicted in Figure 4—figure supplement 4 and Figure 6. (B) Gel showing wild-type Gp88 protein used for cleavage assays depicted in Figure 4—figure supplement 4 and Figure 6. (C) Gel showing Gp88* protein used for all in vitro assays that utilized Gp88*. Note that protein preparations (A) and (C) were performed in parallel; the preparation in (B) was completed separately.

Figure 4—figure supplement 2—source data 1

Original uncropped gels for Figure 4—figure supplement 2.

The cropped images shown in the figure are indicated by the orange boxes.

https://cdn.elifesciences.org/articles/68339/elife-68339-fig4-figsupp2-data1-v1.pdf
Figure 4—figure supplement 3
Replicates of Gp88 nuclease assay.

Two replicates of a nuclease assay showing the integrity of a PCR product amplified from the noncoding region containing the ori from the PLE variant indicated (numbers) treated with (+) and without (–) 500 nM of purified Gp88. A third replicate is depicted in Figure 4D.

Figure 4—figure supplement 3—source data 1

Original uncropped gels for Figure 4—figure supplement 3.

The cropped images shown in the figure are indicated by the orange boxes.

https://cdn.elifesciences.org/articles/68339/elife-68339-fig4-figsupp3-data1-v1.pdf
Figure 4—figure supplement 4
Replicates of Gp88* nuclease assay.

Two replicates of a nuclease assay showing the integrity of a PCR product amplified from the noncoding region containing the ori from the PLE variant indicated (numbers) treated with (+) and without (–) 500 nM of purified Gp88*. A third replicate is depicted in Figure 4E.

Figure 4—figure supplement 4—source data 1

Original uncropped gels for Figure 4—figure supplement 4.

The cropped images shown in the figure are indicated by the orange boxes.

https://cdn.elifesciences.org/articles/68339/elife-68339-fig4-figsupp4-data1-v1.pdf
Figure 4—figure supplement 5
Cleavage of an altered PLE4 probe.

Two replicates of a nuclease assay showing the integrity of a PCR product amplified from the noncoding region containing the ori from the PLE1 or PLE4 treated with (+) and without (–) 500 nM of purified Gp88. The PLE4 probe used in this experiment is amplified with a different set of primers compared to the probe used in Figure 4D such that cleavage of the probe at or near the iterons would produce products that could be differentiated on the basis of size.

Figure 4—figure supplement 5—source data 1

Original uncropped gels for Figure 4—figure supplement 5.

The cropped images shown in the figure are indicated by the orange boxes.

https://cdn.elifesciences.org/articles/68339/elife-68339-fig4-figsupp5-data1-v1.pdf
Figure 5 with 1 supplement
ICP1-encoded Odn (Gp88) requires the PLE iterons for cleavage.

(A) Nuclease assay showing the integrity of a PCR product amplified from the noncoding region containing the ori from wild-type (WT) PLE1 and the ∆iteron mutant, with purified Odn (31.25–500 nM) titrated in. 500 nM catalytically inactive Odn (Odn*) with a single amino acid substitution (E180A) with the WT PLE1 sequence was also included (far left). Nuclease assays were performed in triplicate and replicates are presented in Figure 5—figure supplement 1. (B) Tenfold dilutions of the phage isolate or mutant derivative indicated spotted on V. cholerae with the PLE indicated (bacterial lawns in gray, zones of killing are shown in black). Spot assays were performed in parallel with those in Figures 2 and 4, and images labeled with the same PLE background are the same image. Spot assays were performed in biological triplicate, and a single representative image is shown. Replicate assays are shown in Figure 2—figure supplement 1.

Figure 5—source data 1

Original uncropped gel for Figure 5A.

The cropped image shown in the figure is indicated by the orange box.

https://cdn.elifesciences.org/articles/68339/elife-68339-fig5-data1-v1.pdf
Figure 5—figure supplement 1
Replicates of nuclease assays.

Nuclease assays showing the integrity of a PCR product amplified from the noncoding region containing the ori from wild-type (WT) PLE1 and the ∆iteron mutant, with purified Odn (31.25–500 nM) titrated in. 500 nM catalytically inactive Odn (Odn*) with a single amino acid substitution (E180A) with the WT PLE1 sequence was also included. This assay was performed in triplicate, and two replicates are shown here. The third replicate is shown in Figure 5A.

Figure 5—figure supplement 1—source data 1

Original uncropped gels for Figure 5—figure supplement 1.

The cropped images shown in the figure are indicated by the orange boxes.

https://cdn.elifesciences.org/articles/68339/elife-68339-fig5-figsupp1-data1-v1.pdf
Figure 6 with 2 supplements
Mutations in PLE1 present in V. cholerae isolates from Pakistan render the PLE1 ori resistant to Odn-mediated cleavage.

(A) The PLE1Mut iteron region. Iterons are bolded and sub-repeats are denoted with arrows. The underlined sequence is identical to the sequence in red. An asterisk (*) denotes the location of an A to T substitution. (B) An alignment of the iterons from PLE5, PLE4, PLE1, and PLE1Mut. Iterons are in bold with sub-repeats indicated with arrows. Sequence deviating from a consensus is shown in red. Regions with 100% conservation are denoted with an asterisk. (C) Nuclease assay showing the integrity of a PCR product amplified from the noncoding region containing the ori from wild-type (WT) PLE1 or PLE1Mut with purified Odn (31.25–500 nM) titrated in. Nuclease assays were performed in triplicate, and replicates are presented in Figure 6—figure supplement 1. (D) Replication of PLE1 WT and PLE1Mut in V. cholerae calculated as the fold change in PLE DNA copy 20 minutes post infection with the ICP1 variant indicated. (E) Tenfold dilutions of the phage isolate or mutant derivative indicated spotted on V. cholerae with the PLE indicated (bacterial lawns in gray, zones of killing are shown in black). Biological replicates of the spot assays are presented in Figure 6—figure supplement 2.

Figure 6—source data 1

Original uncropped gel for Figure 6C.

The cropped image shown in the figure is indicated by the orange box.

https://cdn.elifesciences.org/articles/68339/elife-68339-fig6-data1-v1.pdf
Figure 6—source data 2

Values for the graph in Figure 6D.

https://cdn.elifesciences.org/articles/68339/elife-68339-fig6-data2-v1.xlsx
Figure 6—figure supplement 1
Replicates of nuclease assays.

Nuclease assays showing the integrity of a PCR product amplified from the noncoding region containing the ori from wild-type PLE1 (WT) or PLE1Mut with (+) and without (–) the addition of 500 nM oOdn. Nuclease assays were performed in triplicate. Two replicates are shown here, and the third is depicted in Figure 6C.

Figure 6—figure supplement 1—source data 1

Original uncropped gels for Figure 6—figure supplement 1.

The cropped images shown in the figure are indicated by the orange boxes.

https://cdn.elifesciences.org/articles/68339/elife-68339-fig6-figsupp1-data1-v1.pdf
Figure 6—figure supplement 2
Biological replicates of PLE1Mut spot assays.

Tenfold dilutions of the phage isolate or mutant derivative indicated spotted on V. cholerae with the PLE indicated (bacterial lawns in gray, zones of killing are shown in black). A representative replicate is also shown in Figure 6E.

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Gene (Vibrio cholerae)RepAPLE1(PLE1 ORF11)Barth et al., 2020bWP_002040284.1
Gene (Vibrio cholerae)RepAPLE2(PLE2 ORF14)This paperAGG36643.1
Gene (Bacteriophage ICP1)Odn (ICP1_2001_Dha_0 gp88)This paperYP_004251029
Gene (Bacteriophage ICP1)Odn* (ICP1_2001_Dha_0 gp88E180A)This paperThe E180A mutation is predicted to abolish catalytic activity
Recombinant DNA reagentPtac-repAPLE1(plasmid)Barth et al., 2020bpZKB129Inducible RepA from PLE1
Recombinant DNA reagentPtac-repAPLE2(plasmid)This paperpKS2159Inducible RepA from PLE2
Recombinant DNA reagentpE-SUMO-Odn (plasmid)This paperpKS2187Vector to express 6xHisSumo-fusion protein, fused to N-terminus of Odn (Gp88)
Recombinant DNA reagentpE-SUMO-Odn* (plasmid)This paperpKS2189Vector to express 6xHisSumo-fusion protein, fused to N-terminus of Odn* (Gp88E180A)
Strain, strain background (Vibrio cholerae)PLE V. cholerae
(E7946)
Levine et al., 1982KDS6
Strain, strain background (Vibrio cholerae)PLE1 V. cholerae
(PLE1 E7946)
O'Hara et al., 2017KDS36
Strain, strain background (Vibrio cholerae)PLE2 V. cholerae
(PLE2 E7946)
O'Hara et al., 2017KDS37
Strain, strain background (Vibrio cholerae)PLE3 V. cholerae
(PLE3 E7946)
O'Hara et al., 2017KDS38
Strain, strain background (Vibrio cholerae)PLE4 V. cholerae
(PLE4 E7946)
O'Hara et al., 2017KDS39
Strain, strain background (Vibrio cholerae)PLE5 V. cholerae
(PLE5 E7946)
O'Hara et al., 2017KDS40
Strain, strain background (Vibrio cholerae)PLE1 ∆ori V. cholerae
(PLE1 E7946)
This paperKDS297Used for all spot assays
Strain, strain background (Vibrio cholerae)PLE2 ∆ori V. cholerae
(PLE2 E7946)
This paperKDS298Figure 3—figure supplement 2
Strain, strain background (Vibrio cholerae)PLE2 ∆repA V. cholerae
(PLE2 E7946)
This paperKDS299Figure 3—figure supplement 2
Strain, strain background (Vibrio cholerae)PLE1 ∆repA ∆ori::oriPLE2; Ptac-repAPLE1 V. cholerae E7946This paperKDS300Figure 3B
Strain, strain background (Vibrio cholerae)PLE1 ∆repA ∆ori::oriPLE2; Ptac-repAPLE2 V. cholerae E7946This paperKDS301Figure 3B
Strain, strain background (Vibrio cholerae)PLE2 ∆repA ∆ori::oriPLE1; Ptac-repAPLE1 V. cholerae E7946This paperKDS302Figure 3B
Strain, strain background (Vibrio cholerae)PLE2 ∆repA ∆ori::oriPLE1; Ptac-repAPLE2 V. cholerae E7946This paperKDS303Figure 3B
Strain, strain background (Vibrio cholerae)PLE4 ∆ori V. cholerae
(PLE4 E7946)
This paperKDS304Used for all spot assays
Strain, strain background (Vibrio cholerae)PLE5 ∆ori V. cholerae
(PLE5 E7946)
This paperKDS305Used for all spot assays
Strain, strain background (Vibrio cholerae)PLE1 ∆iterons V. cholerae
(PLE1 E7946)
Barth et al., 2020bKDS263Used for all spot assays
Strain, strain background (Vibrio cholerae)PLE2 ∆ori::ori PLE1V. cholerae
(PLE2 E7946)
This paperKDS306Used for all spot assays
Strain, strain background (Vibrio cholerae)PLE1∆ori::oriMut∆lacZ::KanR V. cholerae E7946 (referred to as PLE1Mut)This paperKDS319Ori engineered to match what is observed in PLE1(+) strains from Pakistan: biosample accession numbers SAMN08979118, SAMN08979175, SAMN08979185, SAMN08979188, and SAMN08979253
Strain, strain background (Escherichia coli)pE-SUMO-Odn E. coli BL21This paperKDS307Expression strain for Gp88/Odn
Strain, strain background (Escherichia coli)pE-SUMO-Odn* E. coli BL21This paperKDS308Expression strain for Gp88*/Odn*
Strain, strain background (Bacteriophage ICP1)2006 WT (ICP1_2006_Dha_E)O'Hara et al., 2017MH310934
Strain, strain background (Bacteriophage ICP1)2006 ∆CR; ∆Cas2_3 (ICP1_2006_Dha_E)McKitterick and Seed, 2018
Strain, strain background (Bacteriophage ICP1)2001 WT (ICP1_2001_Dha_0)Seed et al., 2011HQ641347
Strain, strain background (Bacteriophage ICP1)2001 ∆odn (ICP1_2001_Dha_0)This paperKSϕ93odn is gp88
Strain, strain background (Bacteriophage ICP1)2001 odn* (ICP1_2001_Dha_0)This paperKSϕ134odn* isgp88E180A
Sequence-based reagent5'-AGGGTTTGAGTGCGATTACG-3'O'Hara et al., 2017zac14qPCR primer targeting a conserved portion of the PLE noncoding region
Sequence-based reagent5'-TGAGGTTTTACCACCTTTTGC-3'O'Hara et al., 2017zac15qPCR primer targeting a conserved portion of the PLE noncoding region
Sequence-based reagent5'-GTCATTTAACGCATCTTATCACC-3'This paperKS459F-primer used to amplify noncoding region probes for PLE1 and PLE5
Sequence-based reagent5'-GGCTTAGCAACTGTCTACGG-3'This paperzac267F-primer used to amplify noncoding region probes for PLE2, PLE3, and PLE4
Sequence-based reagent5'-GTTACGTCTGATTGCTGACG-3'This paperKS321R-primer used to amplify noncoding region probes for PLE1
Sequence-based reagent5'-CCGCTTATATCAATTTCACTAATATCT-3'This paperzac269R-primer used to amplify noncoding region probes for PLE2 and PLE3
Sequence-based reagent5'-GGACGGCTAAACCATTCTCG-3'This paperKS323R-primer used to amplify
noncoding region probes for PLE4 and PLE5
Sequence-based reagent5’-CATAAGGTTGGCTCCTCAATG-3’This paperKS458R-primer used to amplify noncoding region probe for PLE4 in Figure 4—figure supplement 5

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