Requirements for Pseudomonas aeruginosa Type I-F CRISPR-Cas Adaptation Determined Using a Biofilm Enrichment Assay

Abstract

CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated protein) systems are diverse and found in many archaea and bacteria. These systems have mainly been characterized as adaptive immune systems able to protect against invading mobile genetic elements, including viruses. The first step in this protection is acquisition of spacer sequences from the invader DNA and incorporation of those sequences into the CRISPR array, termed CRISPR adaptation. Progress in understanding the mechanisms and requirements of CRISPR adaptation has largely been accomplished using overexpression of cas genes or plasmid loss assays; little work has focused on endogenous CRISPR-acquired immunity from viral predation. Here, we developed a new biofilm-based assay system to enrich for Pseudomonas aeruginosa strains with new spacer acquisition. We used this assay to demonstrate that P. aeruginosa rapidly acquires spacers protective against DMS3vir, an engineered lytic variant of the Mu-like bacteriophage DMS3, through primed CRISPR adaptation from spacers present in the native CRISPR2 array. We found that for the P. aeruginosa type I-F system, the cas1 gene is required for CRISPR adaptation, recG contributes to (but is not required for) primed CRISPR adaptation, recD is dispensable for primed CRISPR adaptation, and finally, the ability of a putative priming spacer to prime can vary considerably depending on the specific sequences of the spacer.

Importance: Our understanding of CRISPR adaptation has expanded largely through experiments in type I CRISPR systems using plasmid loss assays, mutants of Escherichia coli, or cas1-cas2 overexpression systems, but there has been little focus on studying the adaptation of endogenous systems protecting against a lytic bacteriophage. Here we describe a biofilm system that allows P. aeruginosa to rapidly gain spacers protective against a lytic bacteriophage. This approach has allowed us to probe the requirements for CRISPR adaptation in the endogenous type I-F system of P. aeruginosa Our data suggest that CRISPR-acquired immunity in a biofilm may be one reason that many P. aeruginosa strains maintain a CRISPR-Cas system.

FIG 1

Cartoon of the type I-F CRISPR-Cas system in Pseudomonas aeruginosa strain UCBPP-PA14. Gene names and spacers numbers are indicated and described in the text.

FIG 2

Schematic of biofilm enrichment assay. Pseudomonas aeruginosa and DMS3vir were coinoculated at a multiplicity of infection of 0.01 using 2.5 × 10⁸ CFU and 2.5 × 10⁶ PFU of P. aeruginosa and DMS3vir, respectively, during each of the two biofilm incubations.

FIG 3

Number of viable P. aeruginosa cells in the planktonic and biofilm populations after the biofilm enrichment assay. After both the first (A) and second (B) 24-h challenge in biofilm-inducing medium, 1 ml of the planktonic culture was collected, serially diluted, and plated to measure the number of CFU. Additionally, after both the first (C) and second (D) 24-h challenge in biofilm-inducing medium, the total biofilm population at the air-liquid interface in each well was isolated using a cell scraper, washed, resuspended in PBS, serially diluted, and plated to measure the number of CFU. Error bars represent standard deviations from three replicates. *, significant difference (P < 0.05, Student's t test) of the specified condition from the equivalent condition without DMS3vir; ns, no significant difference.

FIG 4

Resistance to DMS3vir in the biofilm enrichment assay is gained through either type IV pilus loss of function or Cas1-dependent spacer acquisition. After the second biofilm incubation in the biofilm enrichment assay, both the biofilm population and the planktonic population of P. aeruginosa were plated for single colonies. At least 200 colonies under each specified condition from at least two replicates were repatched on LB agar. (A) Isolates that were resistant to DMS3vir and twitch negative were scored as a type IV pilus (T4P) mutant, while isolates that were resistant to DMS3vir, that were twitch positive, and that demonstrated spacer acquisition, as determined by PCR of the CRISPR arrays, were scored as spacer acquisition positive. Type IV pilus mutant and spacer acquisition-positive isolates are displayed under each condition as a percentage of the total DMS3vir resistant population under that condition. (B) After 24 h of coincubation of either WT or CRISPR-deficient (ΔCR) P. aeruginosa isolates with DMS3vir at a multiplicity of infection of 0.01 at 37°C in 5 ml of LB, 200 single colonies from two replicates under each condition were isolated, scored, and displayed as described in the legend to panel A. (C and D) One hundred randomly selected spacer acquisition-positive isolates from the WT and DMS3vir coincubation conditions in the biofilm enrichment assay were scored for insertion of either a single spacer or multiple spacers (C) and insertion of a new spacer into the CRISPR1, CRISPR2, or both CRISPR1 and CRISPR2 arrays (D), as determined by PCR.

FIG 5

The three putative priming spacers located in the native CRISPR2 locus of P. aeruginosa strain UCBPP-PA14 (top sequences) along with their cognate DMS3 target (bottom sequences). The PAM position is underlined, and the DMS3 location listed indicates the position of the 32-bp protospacer in the DMS3 genome.

FIG 6

DMS3 targets of newly acquired spacers demonstrate a bias. The 83 sequenced spacers acquired by WT P. aeruginosa, when incubated with DMS3vir in the biofilm enrichment assay, within 6,000 bp of the CRISPR2 spacer 1 (CR2_sp1) target are displayed as a function of both the distance from the CR2_sp1 target and which DMS3 strand (sense or antisense) that each targets. The dashed line represents the CR2_sp1 target location, with the x axis representing the distance from the CR2_sp1 target, divided into segments of 500 bp either upstream or downstream of the CR2_sp1 target. Each bar represents the number of spacers targeting within that particular 500-bp segment of DMS3, with blue bars representing targets on the positive-sense DMS3 strand and with the yellow bars representing targets on the negative-sense DMS3 strand.

FIG 7

Contributions to new spacer acquisition. RecD is dispensable for primed spacer acquisition, RecG and CRISPR2 spacer 1 are necessary for efficient primed spacer acquisition, and CRISPR2 spacer 20 is sufficient for primed spacer acquisition when the mismatches are in positions similar to those of CRISPR2 spacer 1 in the biofilm enrichment assay. After the second challenge in the biofilm enrichment assay, the biofilm population of P. aeruginosa (either the WT or the specified mutant) coincubated with either DMS3vir (first six columns) or DMS3vir-PC (last column on the right) was plated for single colonies. At least 200 colonies under each specified condition from at least two replicates were repatched on LB agar. Isolates that were resistant to DMS3vir and twitch negative were scored as type IV pilus (T4P) mutants, while isolates that were resistant to DMS3vir, twitch positive, and demonstrated spacer acquisition, as determined by PCR of the CRISPR arrays, were scored as spacer acquisition positive. The type IV pilus mutant and spacer acquisition-positive isolates from each condition are displayed as a percentage of the total DMS3vir-resistant population under that condition.