Phage-based biocontrol strategies and their application in agriculture and aquaculture.

Meeting global food demands for a growing human population with finite natural resources is a major challenge. Aquaculture and agriculture are critical to satisfy food requirements, yet suffer significant losses from bacterial diseases. Therefore, there is an urgent need to develop novel antimicrobial strategies, which is heightened by increasing antibiotic resistance. Bacteriophages (phages) are viruses that specifically infect bacteria, and phage-derived therapies are promising treatments in the fight against bacterial diseases. Here, we describe multiple ways that phages and phage-based technologies can be used as antimicrobials. Antimicrobial activity can be achieved through lysis of targeted bacteria by virulent phages or lytic enzymes. Alternatively, phages can be engineered for the delivery of lethal genes and other cargoes to kill bacteria and to manipulate the bacterial response to conventional antibiotics. We also briefly highlight research exploring phages as potential biocontrol agents with examples from agriculture and aquaculture.

AbiEi Binds Cooperatively to the Type IV abiE Toxin-Antitoxin Operator Via a Positively-Charged Surface and Causes DNA Bending and Negative Autoregulation.

Bacteria resist phage infection using multiple strategies, including CRISPR-Cas and abortive infection (Abi) systems. Abi systems provide population-level protection from phage predation, via "altruistic" cell suicide. It has recently been shown that some Abi systems function via a toxin-antitoxin mechanism, such as the widespread AbiE family. The Streptococcus agalactiae AbiE system consists of a bicistronic operon encoding the AbiEi antitoxin and AbiEii toxin, which function as a Type IV toxin-antitoxin system. Here we examine the AbiEi antitoxin, which belongs to a large family of transcriptional regulators with a conserved N-terminal winged helix-turn-helix domain. This winged helix-turn-helix is essential for transcriptional repression of the abiE operon. The function of the AbiEi C-terminal domain is poorly characterized, but it contributes to transcriptional repression and is sufficient for toxin neutralization. We demonstrate that a conserved charged surface on one face of the C-terminal domain assists sequence-specific DNA binding and negative autoregulation, without influencing antitoxicity. Furthermore, AbiEi binds cooperatively to two inverted repeats within the abiE promoter and bends the DNA by 72°. These findings demonstrate that the mechanism of DNA binding by the widespread family of AbiEi antitoxins and transcriptional regulators can contribute to negative autoregulation.

Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer.

Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼ 350 new spacers acquired in priming events and identified a 5'-protospacer-GG-3' protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2-3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.

Gene regulation by engineered CRISPR-Cas systems.

The clustered regularly interspaced short palindromic repeat (CRISPR) arrays and their CRISPR associated (Cas) proteins constitute adaptive immune systems in bacteria and archaea that provide protection from bacteriophages, plasmids and other mobile genetic elements (MGEs). Recently, the ability to direct these systems to DNA in a sequence-specific manner has led to the emergence of new technologies for engineered gene regulation in bacteria and eukaryotes. These systems have the potential to enable facile high-throughput functional genomics studies aimed at identifying gene function and will be a crucial tool for synthetic biology. Here, we review the recent engineering of these systems for controlling gene expression.

A widespread bacteriophage abortive infection system functions through a Type IV toxin-antitoxin mechanism.

Bacterial abortive infection (Abi) systems are 'altruistic' cell death systems that are activated by phage infection and limit viral replication, thereby providing protection to the bacterial population. Here, we have used a novel approach of screening Abi systems as a tool to identify and characterize toxin-antitoxin (TA)-acting Abi systems. We show that AbiE systems are encoded by bicistronic operons and function via a non-interacting (Type IV) bacteriostatic TA mechanism. The abiE operon was negatively autoregulated by the antitoxin, AbiEi, a member of a widespread family of putative transcriptional regulators. AbiEi has an N-terminal winged-helix-turn-helix domain that is required for repression of abiE transcription, and an uncharacterized bi-functional C-terminal domain, which is necessary for transcriptional repression and sufficient for toxin neutralization. The cognate toxin, AbiEii, is a predicted nucleotidyltransferase (NTase) and member of the DNA polymerase ß family. AbiEii specifically bound GTP, and mutations in conserved NTase motifs (I-III) and a newly identified motif (IV), abolished GTP binding and subsequent toxicity. The AbiE systems can provide phage resistance and enable stabilization of mobile genetic elements, such as plasmids. Our study reveals molecular insights into the regulation and function of the widespread bi-functional AbiE Abi-TA systems and the biochemical properties of both toxin and antitoxin proteins.

Remarkable Mechanisms in Microbes to Resist Phage Infections.

Bacteriophages (phages) specifically infect bacteria and are the most abundant biological entities on Earth. The constant exposure to phage infection imposes a strong selective pressure on bacteria to develop viral resistance strategies that promote prokaryotic survival. Thus, this parasite-host relationship results in an evolutionary arms race of adaptation and counteradaptation between the interacting partners. The evolutionary outcome is a spectrum of remarkable strategies used by the bacteria and phages as they attempt to coexist. These approaches include adsorption inhibition, injection blocking, abortive infection, toxin-antitoxin, and CRISPR-Cas systems. In this review, we highlight the diverse and complementary antiphage systems in bacteria, as well as the evasion mechanisms used by phages to escape these resistance strategies.

Chromosomal targeting by CRISPR-Cas systems can contribute to genome plasticity in bacteria.

The clustered regularly interspaced short palindromic repeats (CRISPR) and their associated (Cas) proteins form adaptive immune systems in bacteria to combat phage and other foreign genetic elements. Typically, short spacer sequences are acquired from the invader DNA and incorporated into CRISPR arrays in the bacterial genome. Small RNAs are generated that contain these spacer sequences and enable sequence-specific destruction of the foreign nucleic acids. Occasionally, spacers are acquired from the chromosome, which instead leads to targeting of the host genome. Chromosomal targeting is highly toxic to the bacterium, providing a strong selective pressure for a variety of evolutionary routes that enable host cell survival. Mutations that inactivate the CRISPR-Cas functionality, such as within the cas genes, CRISPR repeat, protospacer adjacent motifs (PAM), and target sequence, mediate escape from toxicity. This self-targeting might provide some explanation for the incomplete distribution of CRISPR-Cas systems in less than half of sequenced bacterial genomes. More importantly, self-genome targeting can cause large-scale genomic alterations, including remodeling or deletion of pathogenicity islands and other non-mobile chromosomal regions. While control of horizontal gene transfer is perceived as their main function, our recent work illuminates an alternative role of CRISPR-Cas systems in causing host genomic changes and influencing bacterial evolution.

Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands.

In prokaryotes, clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (Cas) proteins constitute a defence system against bacteriophages and plasmids. CRISPR/Cas systems acquire short spacer sequences from foreign genetic elements and incorporate these into their CRISPR arrays, generating a memory of past invaders. Defence is provided by short non-coding RNAs that guide Cas proteins to cleave complementary nucleic acids. While most spacers are acquired from phages and plasmids, there are examples of spacers that match genes elsewhere in the host bacterial chromosome. In Pectobacterium atrosepticum the type I-F CRISPR/Cas system has acquired a self-complementary spacer that perfectly matches a protospacer target in a horizontally acquired island (HAI2) involved in plant pathogenicity. Given the paucity of experimental data about CRISPR/Cas-mediated chromosomal targeting, we examined this process by developing a tightly controlled system. Chromosomal targeting was highly toxic via targeting of DNA and resulted in growth inhibition and cellular filamentation. The toxic phenotype was avoided by mutations in the cas operon, the CRISPR repeats, the protospacer target, and protospacer-adjacent motif (PAM) beside the target. Indeed, the natural self-targeting spacer was non-toxic due to a single nucleotide mutation adjacent to the target in the PAM sequence. Furthermore, we show that chromosomal targeting can result in large-scale genomic alterations, including the remodelling or deletion of entire pre-existing pathogenicity islands. These features can be engineered for the targeted deletion of large regions of bacterial chromosomes. In conclusion, in DNA-targeting CRISPR/Cas systems, chromosomal interference is deleterious by causing DNA damage and providing a strong selective pressure for genome alterations, which may have consequences for bacterial evolution and pathogenicity.

A widely conserved molecular switch controls quorum sensing and symbiosis island transfer in Mesorhizobium loti through expression of a novel antiactivator.

ICEMlSym(R7A) of Mesorhizobium loti is an integrative and conjugative element (ICE) that confers the ability to form a nitrogen-fixing symbiosis with Lotus species. Horizontal transfer is activated by TraR and N-acyl-homoserine lactone (AHL), which can stimulate ICE excision in 100% of cells. However, in wild-type cultures, the ICE is excised at low frequency. Here we show that QseM, a widely conserved ICE-encoded protein, is an antiactivator of TraR. Mutation of qseM resulted in TraR-dependent activation of AHL production and excision, but did not affect transcription of traR. QseM and TraR directly interacted in a bacterial two-hybrid assay in the presence of AHL. qseM expression was repressed by a DNA-binding protein QseC, which also activated qseC expression from a leaderless transcript. QseC differentially bound two adjacent operator sites, the lower affinity of which overlapped the -35 regions of the divergent qseC-qseM promoters. QseC homologues were identified on ICEs, TraR/TraM-regulated plasmids and restriction-modification cassettes, suggesting a conserved mode of regulation. Six QseC variants with distinct operators were identified that showed evidence of reassortment between mobile elements. We propose that QseC and QseM comprise a bimodal switch that restricts quorum sensing and ICEMlSym(R7A) transfer to a small proportion of cells in the population.