MATERIALS AND METHODSBacterial strains, plasmids, and phage. The strains and plasmids used in this study are listed in Table S1. To construct the chromosomal 3×flag-tagged csy4 in PA14, DNA fragments flanking the 3′ terminus of csy4, including a 3×flag tag, were amplified, sewed together by overlap extension PCR, and cloned into pEXG2 (a generous gift from Joseph Mougous, University of Washington, Seattle, WA) using HindIII and XbaI restriction sites (47). The plasmid to make the ΔCRISPR Δcas PA14 mutant was engineered using the identical strategy and DNA fragments surrounding the CRISPR cas locus, flanked by EcoRI and XbaI restriction sites. The resulting plasmids were used to transform E. coli SM10λpir and were subsequently mobilized into PA14 via mating. Exconjugants were selected on Luria-Bertani (LB) containing gentamicin (30 μg/ml) and irgasan (100 μg/ml), followed by recovery of mutants on M9 medium containing 5% (wt/vol) sucrose. Candidate mutants were confirmed by PCR and sequencing. A PA14 strain harboring a CRISPR spacer matching phage JBD44a was generated by cloning 1,581 bp of JBD44a gene gp33 using native HindIII sites into the adaptation-promoting pCR2SP1 seed plasmid (18). The plasmid was propagated in PA14 on LB agar with 50 µg/ml gentamicin, and single colonies were streaked repeatedly on LB agar and tested for plasmid loss. CRISPR-adapted clones were identified using PCR with primers that enabled an assessment of incorporation of spacers into either CRISPR1 or CRISPR2. Newly integrated spacers were identified and mapped using sequencing. A strain with the new spacer AGCCACAACANAGGCCAGAGAAGCTGCTGCGA in CRISPR2 that targeted gene gp33 was selected and tested for resistance to JBD44a by a cross-streak assay. The primers used are listed in Table S2.TABLE S1Bacterial strains, phage, and plasmids. Download Table S1, PDF file, 0.05 MB.Copyright © 2018 Høyland-Kroghsbo et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.TABLE S2Primers used in this study. Download Table S2, PDF file, 0.04 MB.Copyright © 2018 Høyland-Kroghsbo et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.Growth conditions. PA14 and mutants were grown at the indicated temperatures in LB broth or on LB solidified with 15 g agar/liter. LB was supplemented with 50 μg/ml gentamicin where appropriate. For AI supplementation assays, 3OC12-HSL and C4-HSL (Sigma) or the solvent dimethyl sulfoxide (DMSO) was used. Growth of bacterial cultures was measured by OD600, where 1 unit equals 109 CFU/ml.Adaptation assay. WT PA14 and the ΔlasI ΔrhlI mutant were transformed with pCR2SP1 seed, as described previously (18), and plated on LB medium containing 50 μg/ml gentamicin; in the case of the ΔlasI ΔrhlI mutant, either DMSO (control) or 2 μM 3OC12-HSL plus 10 μM C4-HSL (designated AI) was added. The plates were incubated at 37, 30, 23, and 15°C until the colonies were 1 mm in diameter. Single colonies were tested for population-wide integration of new CRISPR spacers by PCR using DreamTaq Green PCR master mix (Thermo Fisher); primers used were designed to anneal upstream of the CRISPR2 array and inside the second spacer, which enabled the detection of expansion of this array. The PCR products were subjected to agarose gel electrophoresis, and band intensities were analyzed using the ImageQuant TL software (GE Healthcare).Relative plasmid copy number. PA14 harboring pHERD30T, the empty vector backbone for the pCR2SP1 seed plasmid, was grown at 37, 30, 23, and 15°C on LB agar supplemented with gentamicin (50 μg/ml). Total DNA was extracted from individual colonies that were 1 mm in size using a DNeasy blood and tissue kit (Qiagen). qPCR was performed using PerfeCTa SYBR Green FastMix, Low ROX (Quantabio) with primers specific for pHERD30T and chromosomal rpoB.Western blot analysis. The PA14 csy4-3×flag and ΔlasI ΔrhlI csy4-3×flag mutant strains were streaked onto LB medium and grown at 37, 30, 23, or 15°C until individual colonies reached 1 mm in diameter. Single colonies were harvested and lysed with BugBuster protein extraction reagent (Millipore), following the manufacturer’s instructions. Fifty micrograms of protein was separated by SDS-PAGE on a 4 to 20% Mini-PROTEAN TGX polyacrylamide gel (Bio-Rad) and blotted onto a polyvinylidene difluoride (PVDF) membrane (catalog no. 1620174; Bio-Rad). The membrane was incubated for 1 h with monoclonal Anti-FLAG M2-peroxidase (HRP) antibody (catalog no. A8592; Sigma), monoclonal anti-RpoB antibody (catalog no. ab191598; Abcam), both at a 1:3,000 dilution, or polyclonal anti-GroEL antibody (catalog no. G6532; Sigma) at a 1:15,000 dilution in Tris-buffered saline with Tween 20 (TBST) and 5% skim milk. Anti-rabbit antibody (catalog no. W4011; Promega) was used as the secondary antibody for detection of the anti-RpoB and anti-GroEL antibodies. The membrane was washed in TBST and was developed using SuperSignal West Femto maximum sensitivity substrate (catalog no. 34095; Thermo Scientific).
Article TitleTemperature, by Controlling Growth Rate, Regulates CRISPR-Cas Activity inPseudomonas aeruginosa
Clustered regularly interspaced short palindromic repeat (CRISPR)-associated (CRISPR-Cas) systems are adaptive defense systems that protect bacteria and archaea from invading genetic elements. InPseudomonas aeruginosa, quorum sensing (QS) induces the CRISPR-Cas defense system at high cell density when the risk of bacteriophage infection is high. Here, we show that another cue, temperature, modulatesP. aeruginosaCRISPR-Cas. Increased CRISPR adaptation occurs at environmental (i.e., low) temperatures compared to that at body (i.e., high) temperature. This increase is a consequence of the accumulation of CRISPR-Cas complexes, coupled with reducedP. aeruginosagrowth rate at the lower temperature, the latter of which provides additional time prior to cell division for CRISPR-Cas to patrol the cell and successfully eliminate and/or acquire immunity to foreign DNA. Analyses of a QS mutant and synthetic QS compounds show that the QS and temperature cues act synergistically. The diversity and level of phage encountered byP. aeruginosain the environment exceed that in the human body, presumably warranting increased reliance on CRISPR-Cas at environmental temperatures.