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. 2018 Nov 13;9(6):e02184-18.
doi: 10.1128/mBio.02184-18.

Temperature, by Controlling Growth Rate, Regulates CRISPR-Cas Activity in Pseudomonas Aeruginosa

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Free PMC article

Temperature, by Controlling Growth Rate, Regulates CRISPR-Cas Activity in Pseudomonas Aeruginosa

Nina Molin Høyland-Kroghsbo et al. mBio. .
Free PMC article

Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR)-associated (CRISPR-Cas) systems are adaptive defense systems that protect bacteria and archaea from invading genetic elements. In Pseudomonas 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, modulates P. aeruginosa CRISPR-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 reduced P. aeruginosa growth 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 by P. aeruginosa in the environment exceed that in the human body, presumably warranting increased reliance on CRISPR-Cas at environmental temperatures.IMPORTANCE P. aeruginosa is a soil dwelling bacterium and a plant pathogen, and it also causes life-threatening infections in humans. Thus, P. aeruginosa thrives in diverse environments and over a broad range of temperatures. Some P. aeruginosa strains rely on the CRISPR-Cas adaptive immune system as a phage defense mechanism. Our discovery that low temperatures increase CRISPR adaptation suggests that the rarely occurring but crucial naive adaptation events may take place predominantly under conditions of slow growth, e.g., during the bacterium's soil dwelling existence and during slow growth in biofilms.

Keywords: CRISPR; Pseudomonas; growth rate; phage; quorum sensing.

Figures

FIG 1
FIG 1
CRISPR adaptation is temperature dependent. Integration of new CRISPR spacers into the CRISPR2 locus was measured by PCR amplification of the CRISPR2 array region from single colonies of PA14 and the Δcas3 mutant. Both strains harbored the CRISPR-targeted plasmid, pCR2SP1 seed, containing a seed mutation that promotes adaptation. Each adaptation event results in acquisition of a new spacer (32 bp) and repeat (28 bp), which is exhibited by a 60-bp increase in size of the CRISPR locus and can be visualized by gel electrophoresis. (A) Adaptation of PA14 cells carrying pCR2SP1 seed at 37, 30, 23, or 15°C. The Δcas3 mutant is incapable of cleaving DNA bound by the Csy1–4 complex and serves as a negative control for adaptation. Data are shown for representative colonies. (B) Quantitation of the spacer population in panel A, n = 6.
FIG 2
FIG 2
Temperature does not affect the relative copy number of pHERD30T in PA14. 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). The copy number of plasmid DNA relative to chromosomal DNA was measured by qPCR of total DNA using primers for pHERD30T and rpoB. Error bars represent standard deviation (SD) from n = 3 replicates (P = 0.1752, one-way analysis of variance [ANOVA]). ns, not significant.
FIG 3
FIG 3
Csy4 levels are modestly upregulated at low temperatures. Western blot of PA14 Csy4-3×FLAG grown at 37, 30, 23, and 15°C. Top, abundance of RpoB, which was used as the endogenous control. Bottom, abundance of Csy4-3×FLAG. Quantitation of the relative abundance of Csy4-3×FLAG normalized to RpoB is shown below the blot. The data are representative of >3 independent experiments.
FIG 4
FIG 4
Growth rate affects CRISPR-Cas activity. (A) Retention of the control parent plasmid pHERD30T (black) and the CRISPR-targeted plasmid pCR2SP1 (gray) in PA14 grown at 37°C to an OD600 of 1 with aeration at 250 rpm (denoted rapid growth) or 150 rpm (denoted slow growth). One hundred percent denotes no plasmid loss. SD represents 3 replicates. (P < 0.0001, Student’s t test). (B) Adaptation of PA14 cells carrying pCR2SP1 grown as in panel A at T = 0 and at the end of the experiment, at OD600 of 1. (C) Western blot of PA14 Csy4-3×FLAG grown as in panel A to an OD600 of 1. Top, abundance of RpoB, which was used as the endogenous control. Bottom, abundance of Csy4-3×FLAG. Quantitation of the relative abundance of Csy4-3×FLAG normalized to RpoB is shown below the blot.
FIG 5
FIG 5
QS and low temperature act synergistically to enhance CRISPR-Cas-mediated adaptation. PCR amplification of the CRISPR2 array and visualization by gel electrophoresis, as in Fig. 1. The PA14 ΔlasI ΔrhlI mutant does not produce 3OC12-HSL or C4-HSL. 3OC12-HSL and C4-HSL (designated AI) were supplied at saturating concentrations (2 μM and 10 μM, respectively), as denoted. The data are representative of >3 independent experiments.

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