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1. Temperature, by Controlling Growth Rate, Regulates CRISPR-Cas Activity in Pseudomonas aeruginosa

   https://doi.org/10.1128/mBio.02184-18  

   Høyland-Kroghsbo, Nina Molin; Muñoz, Katrina Arcelia; Bassler, Bonnie L.; Vogel, Joerg (December 2018, mBio)

   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. 

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2. Bacteriophage T4 Escapes CRISPR Attack by Minihomology Recombination and Repair

   https://doi.org/10.1128/mBio.01361-21  

   Wu, Xiaorong; Zhu, Jingen; Tao, Pan; Rao, Venigalla B. (June 2021, mBio)

   Hatfull, Graham F. (Ed.)

   ABSTRACT Bacteria and bacteriophages (phages) have evolved potent defense and counterdefense mechanisms that allowed their survival and greatest abundance on Earth. CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated) is a bacterial defense system that inactivates the invading phage genome by introducing double-strand breaks at targeted sequences. While the mechanisms of CRISPR defense have been extensively investigated, the counterdefense mechanisms employed by phages are poorly understood. Here, we report a novel counterdefense mechanism by which phage T4 restores the genomes broken by CRISPR cleavages. Catalyzed by the phage-encoded recombinase UvsX, this mechanism pairs very short stretches of sequence identity (minihomology sites), as few as 3 or 4 nucleotides in the flanking regions of the cleaved site, allowing replication, repair, and stitching of genomic fragments. Consequently, a series of deletions are created at the targeted site, making the progeny genomes completely resistant to CRISPR attack. Our results demonstrate that this is a general mechanism operating against both type II (Cas9) and type V (Cas12a) CRISPR-Cas systems. These studies uncovered a new type of counterdefense mechanism evolved by T4 phage where subtle functional tuning of preexisting DNA metabolism leads to profound impact on phage survival. IMPORTANCE Bacteriophages (phages) are viruses that infect bacteria and use them as replication factories to assemble progeny phages. Bacteria have evolved powerful defense mechanisms to destroy the invading phages by severing their genomes soon after entry into cells. We discovered a counterdefense mechanism evolved by phage T4 to stitch back the broken genomes and restore viral infection. In this process, a small amount of genetic material is deleted or another mutation is introduced, making the phage resistant to future bacterial attack. The mutant virus might also gain survival advantages against other restriction conditions or DNA damaging events. Thus, bacterial attack not only triggers counterdefenses but also provides opportunities to generate more fit phages. Such defense and counterdefense mechanisms over the millennia led to the extraordinary diversity and the greatest abundance of bacteriophages on Earth. Understanding these mechanisms will open new avenues for engineering recombinant phages for biomedical applications. 

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3. The impact of phage and phage resistance on microbial community dynamics

   https://doi.org/10.1371/journal.pbio.3002346  

   Alseth, Ellinor O; Custodio, Rafael; Sundius, Sarah A; Kuske, Rachel A; Brown, Sam P; Westra, Edze R (April 2024, PLOS Biology)

   Barr, Jeremy J (Ed.)

   Where there are bacteria, there will be bacteriophages. These viruses are known to be important players in shaping the wider microbial community in which they are embedded, with potential implications for human health. On the other hand, bacteria possess a range of distinct immune mechanisms that provide protection against bacteriophages, including the mutation or complete loss of the phage receptor, and CRISPR-Cas adaptive immunity. While our previous work showed how a microbial community may impact phage resistance evolution, little is known about the inverse, namely how interactions between phages and these different phage resistance mechanisms affect the wider microbial community in which they are embedded. Here, we conducted a 10-day, fully factorial evolution experiment to examine how phage impact the structure and dynamics of an artificial four-species bacterial community that includes eitherPseudomonas aeruginosawild-type or an isogenic mutant unable to evolve phage resistance through CRISPR-Cas. Additionally, we used mathematical modelling to explore the ecological interactions underlying full community behaviour, as well as to identify general principles governing the impacts of phage on community dynamics. Our results show that the microbial community structure is drastically altered by the addition of phage, withAcinetobacter baumanniibecoming the dominant species andP.aeruginosabeing driven nearly extinct, whereasP.aeruginosaoutcompetes the other species in the absence of phage. Moreover, we find that aP.aeruginosastrain with the ability to evolve CRISPR-based resistance generally does better when in the presence ofA.baumannii, but that this benefit is largely lost over time as phage is driven extinct. Finally, we show that pairwise data alone is insufficient when modelling our microbial community, both with and without phage, highlighting the importance of higher order interactions in governing multispecies dynamics in complex communities. Combined, our data clearly illustrate how phage targeting a dominant species allows for the competitive release of the strongest competitor while also contributing to community diversity maintenance and potentially preventing the reinvasion of the target species, and underline the importance of mapping community composition before therapeutically applying phage. 

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4. A synthetic biology approach to assemble and reboot clinically relevant Pseudomonas aeruginosa tailed phages

   https://doi.org/10.1128/spectrum.02897-23  

   Ipoutcha, Thomas; Racharaks, Ratanachat; Huttelmaier, Stefanie; Wilson, Cole J; Ozer, Egon A; Hartmann, Erica M (March 2024, Microbiology Spectrum)

   Andam, Cheryl P (Ed.)

   The rise in the frequency of antibiotic resistance has made bacterial infections, specifically Pseudomonas aeruginosa, a cause for greater concern. Phage therapy is a promising solution that uses naturally isolated phages to treat bacterial infections. Ecological limitations, which stipulate a discrete host range and the inevitable evolution of resistance, may be overcome through a better understanding of phage biology and the utilization of engineered phages. In this study, we developed a synthetic biology approach to construct tailed phages that naturally target clinically relevant strains of Pseudomonas aeruginosa. As proof of concept, we successfully cloned and assembled the JG024 and DMS3 phage genomes in yeast using transformation-associated recombination cloning and rebooted these two phage genomes in two different strains ofP. aeruginosa. We identified factors that affected phage reboot efficiency like the phage species or the presence of antiviral defense systems in the bacterial strain. We have successfully extended this method to two other phage species and observed that the method enables the reboot of phages that are naturally unable to infect the strain used for reboot. This research represents a critical step toward the construction of clinically relevant, engineered P. aeruginosa phages. IMPORTANCE Pseudomonas aeruginosais a bacterium responsible for severe infections and a common major complication in cystic fibrosis. The use of antibiotics to treat bacterial infections has become increasingly difficult as antibiotic resistance has become more prevalent. Phage therapy is an alternative solution that is already being used in some European countries, but its use is limited by the narrow host range due to the phage receptor specificity, the presence of antiviral defense systems in the bacterial strain, and the possible emergence of phage resistance. In this study, we demonstrate the use of a synthetic biology approach to construct and reboot clinically relevant P. aeruginosa tailed phages. This method enables a significant expansion of possibilities through the construction of engineered phages for therapy applications. 

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5. Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

   https://doi.org/10.3390/biology10060530  

   Thompson, Marlo K.; Sobol, Robert W.; Prakash, Aishwarya (June 2021, Biology)

   The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples. 

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