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Antagonistic coevolution of parasite infectivity and host resistance may alter the biological functionality of species, yet these dynamics in nature are still poorly understood. Here we show the molecular details of a long-term phage–bacterium arms race in the environment. Bacteria ( Flavobacterium columnare ) are generally resistant to phages from the past and susceptible to phages isolated in years after bacterial isolation. Bacterial resistance selects for increased phage infectivity and host range, which is also associated with expansion of phage genome size. We identified two CRISPR loci in the bacterial host: a type II-C locus and a type VI-B locus. While maintaining a core set of conserved spacers, phage-matching spacers appear in the variable ends of both loci over time. The spacers mostly target the terminal end of the phage genomes, which also exhibit the most variation across time, resulting in arms-race-like changes in the protospacers of the coevolving phage population. Arms races between phage and bacteria are well known from lab experiments, but insight from field systems is limited. Here, the authors show changes in the resistance and CRISPR loci of bacteria and the infectivity, host range and genome size of phage over multiple years in an aquaculture environment.

Parasitism by bacteriophages has led to the evolution of a variety of defense mechanisms in their host bacteria. However, it is unclear what factors lead to specific defenses being deployed upon phage infection. To explore this question, we co-evolved the bacterial fish pathogen Flavobacterium columnare and its virulent phage V156 in presence and absence of a eukaryotic host signal (mucin) for sixteen weeks. The presence of mucin leads to a dramatic increase in CRISPR spacer acquisition, especially in low nutrient conditions where over 60% of colonies obtain at least one new spacer. Additionally, we show that the presence of a competitor bacterium further increases CRISPR spacer acquisition in F. columnare. These results suggest that ecological factors are important in determining defense strategies against phages, and that the phage-bacterium interactions on mucosal surfaces may select for the diversification of bacterial immune systems.

CRISPR-Cas systems are immune systems that protect bacteria and archaea against their viruses, bacteriophages. Immunity is achieved through the acquisition of short DNA fragments from the viral invader’s genome. CRISPR-Cas immune systems adapt to new threats by acquiring new spacers from invading nucleic acids such as phage genomes. However, some CRISPR-Cas loci lack genes necessary for spacer acquisition despite variation in spacer content between microbial strains. It has been suggested that such loci may use acquisition machinery from cooccurring CRISPR-Cas systems within the same strain. Here, following infection by a virulent phage with a double-stranded DNA (dsDNA) genome, we observed spacer acquisition in the native host Flavobacterium columnare that carries an acquisition-deficient CRISPR-Cas subtype VI-B system and a complete subtype II-C system. We show that the VI-B locus acquires spacers from both the bacterial and phage genomes, while the newly acquired II-C spacers mainly target the viral genome. Both loci preferably target the terminal end of the phage genome, with priming-like patterns around a preexisting II-C protospacer. Through gene deletion, we show that the RNA-cleaving VI-B system acquires spacers in trans using acquisition machinery from the DNA-cleaving II-C system. Our observations support the concept of cross talk between CRISPR-Cas systems and raise further questions regarding the plasticity of adaptation modules. IMPORTANCE CRISPR-Cas systems are immune systems that protect bacteria and archaea against their viruses, bacteriophages. Immunity is achieved through the acquisition of short DNA fragments from the viral invader’s genome. These fragments, called spacers, are integrated into a memory bank on the bacterial genome called the CRISPR array. The spacers allow for the recognition of the same invader upon subsequent infection. Most CRISPR-Cas systems target DNA, but recently, systems that exclusively target RNA have been discovered. RNA-targeting CRISPR-Cas systems often lack genes necessary for spacer acquisition, and it is thus unknown how new spacers are acquired and if they can be acquired from DNA phages. Here, we show that an RNA-targeting system “borrows” acquisition machinery from another CRISPR-Cas locus in the genome. Most new spacers in this locus are unable to target phage mRNA and are therefore likely redundant. Our results reveal collaboration between distinct CRISPR-Cas types and raise further questions on how other CRISPR-Cas loci may cooperate.

Phage therapy is becoming a widely recognized alternative for fighting pathogenic bacteria due to increasing antibiotic resistance problems. However, one of the common concerns related to the use of phages is the evolution of bacterial resistance against the phages, putatively disabling the treatment. Experimental adaptation of the phage (phage training) to infect a resistant host has been used to combat this problem. Yet, there is very little information on the trade-offs of phage infectivity and host range. Here we co-cultured a myophage FCV-1 with its host, the fish pathogen Flavobacterium columnare, in lake water and monitored the interaction for a one-month period. Phage resistance was detected within one day of co-culture in the majority of the bacterial isolates (16 out of the 18 co-evolved clones). The primary phage resistance mechanism suggests defense via surface modifications, as the phage numbers rose in the first two days of the experiment and remained stable thereafter. However, one bacterial isolate had acquired a spacer in its CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas locus, indicating that also CRISPR-Cas defense was employed in the phage-host interactions. After a week of co-culture, a phage isolate was obtained that was able to infect 18 out of the 32 otherwise resistant clones isolated during the experiment. Phage genome sequencing revealed several mutations in two open reading frames (ORFs) likely to be involved in the regained infectivity of the evolved phage. Their location in the genome suggests that they encode tail genes. Characterization of this evolved phage, however, showed a direct cost for the ability to infect several otherwise resistant clones—adsorption was significantly lower than in the ancestral phage. This work describes a method for adapting the phage to overcome phage resistance in a fish pathogenic system.

Virus capsids mediate the transfer of viral genetic information from one cell to another, thus the origin of the first viruses arguably coincides with the origin of the viral capsid. Capsid genes are evolutionarily ancient and their emergence potentially predated even the origin of first free-living cells. But does the origin of the capsid coincide with the origin of viruses, or is it possible that capsid-like functionalities emerged before the appearance of true viral entities? We set to investigate this question by using a computational simulator comprising primitive replicators and replication parasites within a compartment matrix. We observe that systems with no horizontal gene transfer between compartments collapse due to the rapidly emerging replication parasites. However, introduction of capsid-like genes that induce the movement of randomly selected genes from one compartment to another rescues life by providing the non-parasitic replicators a mean to escape their current compartments before the emergence of replication parasites. Capsid-forming genes can mediate the establishment of a stable meta-population where parasites cause only local tragedies but cannot overtake the whole community. The long-term survival of replicators is dependent on the frequency of horizontal transfer events, as systems with either too much or too little genetic exchange are doomed to succumb to replication-parasites. This study provides a possible scenario for explaining the origin of viral capsids before the emergence of genuine viruses: in the absence of other means of horizontal gene transfer between compartments, evolution of capsid-like functionalities may have been necessary for early life to prevail.

CRISPR systems are widespread in the prokaryotic world, providing adaptive immunity against mobile genetic elements 1 , 2 . Type III CRISPR systems, with the signature gene cas10 , use CRISPR RNA to detect non-self RNA, activating the enzymatic Cas10 subunit to defend the cell against mobile genetic elements either directly, via the integral histidine–aspartate (HD) nuclease domain 3 – 5 or indirectly, via synthesis of cyclic oligoadenylate second messengers to activate diverse ancillary effectors 6 – 9 . A subset of type III CRISPR systems encode an uncharacterized CorA-family membrane protein and an associated NrN family phosphodiesterase that are predicted to function in antiviral defence. Here we demonstrate that the CorA-associated type III-B (Cmr) CRISPR system from Bacteroides fragilis provides immunity against mobile genetic elements when expressed in Escherichia coli . However, B. fragilis Cmr does not synthesize cyclic oligoadenylate species on activation, instead generating S -adenosyl methionine (SAM)-AMP (SAM is also known as AdoMet) by conjugating ATP to SAM via a phosphodiester bond. Once synthesized, SAM-AMP binds to the CorA effector, presumably leading to cell dormancy or death by disruption of the membrane integrity. SAM-AMP is degraded by CRISPR-associated phosphodiesterases or a SAM-AMP lyase, potentially providing an ‘off switch’ analogous to cyclic oligoadenylate-specific ring nucleases 10 . SAM-AMP thus represents a new class of second messenger for antiviral signalling, which may function in different roles in diverse cellular contexts. The Bacteroides fragilis type III CRISPR protein Cmr conjugates ATP to S -adenosyl methionine, generating S -adenosyl methionine (SAM)-AMP, a novel second messenger with a role in antiviral signalling.

Type III CRISPR systems detect the presence of RNA from mobile genetic elements (MGE) in prokaryotes, providing antiviral immunity. On activation, the catalytic Cas10 subunit conjugates ATP to form cyclic oligoadenylate (cOA) signalling molecules that activate ancillary effectors, providing an immune response. Cellular ring nucleases degrade cOA to reset the system. Here, we describe the structure and mechanism of a new family of ring nucleases, Crn4, associated with type III-D CRISPR systems. The crystal structure of Crn4 reveals a small homodimeric protein with a fold unrelated to any known ring nuclease or, indeed, any known protein structure. Crn4 degrades a wide range of cOA species to linear oligoadenylates in vitro and ameliorates type III CRISPR immunity in vivo. Phage and plasmids also encode Crn4 orthologues that may function as anti-CRISPRs. These observations expand our understanding of ring nucleases and reveal a new protein fold for cyclic nucleotide recognition.

So far, studies on the bacterial immune system CRISPR-Cas and its ecological and evolutionary effects have been largely limited to laboratory conditions. While providing crucial information on the constituents of CRISPR-Cas, such studies may overlook fundamental components that affect bacterial immunity in natural habitats. Translating laboratory-derived predictions to nature is not a trivial task, owing partly to the instability of natural communities and difficulties in repeated sampling. To this end, we review how aquaculture, the farming of fishes and other aquatic species, may provide suitable semi-natural laboratories for examining the role of CRISPR-Cas in phage/bacterium coevolution. Existing data from disease surveillance conducted in aquaculture, coupled with growing interest towards phage therapy, may have already resulted in large collections of bacterium and phage isolates. These data, combined with premeditated efforts, can provide empirical evidence on phage–bacterium dynamics such as the bacteriophage adherence to mucus hypothesis, phage life cycles and their relationship with CRISPR-Cas and other immune defences. Typing of CRISPR spacer content in pathogenic bacteria can also provide practical information on diversity and origin of isolates during outbreaks. In addition to providing information of CRISPR functionality and phage–bacterium dynamics, aquaculture systems can significantly impact perspectives on design of phage-based disease treatment at the current era of increasing antibiotic resistance. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

So far, studies on the bacterial immune system CRISPR-Cas and its ecological and evolutionary effects have been largely limited to laboratory conditions. While providing crucial information on the constituents of CRISPR-Cas, such studies may overlook fundamental components that affect bacterial immunity in natural habitats. Translating laboratory-derived predictions to nature is not a trivial task, owing partly to the instability of natural communities and difficulties in repeated sampling. To this end, we review how aquaculture, the farming of fishes and other aquatic species, may provide suitable semi-natural laboratories for examining the role of CRISPR-Cas in phage/bacterium coevolution. Existing data from disease surveillance conducted in aquaculture, coupled with growing interest towards phage therapy, may have already resulted in large collections of bacterium and phage isolates. These data, combined with premeditated efforts, can provide empirical evidence on phage–bacterium dynamics such as the bacteriophage adherence to mucus hypothesis, phage life cycles and their relationship with CRISPR-Cas and other immune defences. Typing of CRISPR spacer content in pathogenic bacteria can also provide practical information on diversity and origin of isolates during outbreaks. In addition to providing information of CRISPR functionality and phage–bacterium dynamics, aquaculture systems can significantly impact perspectives on design of phage-based disease treatment at the current era of increasing antibiotic resistance. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.

To investigate the potential evolutionary obstacles in the sustainable therapeutic use of plasmid-dependent phages to control the clinically important conjugative plasmid-mediated dissemination of antibiotic resistance genes to pathogenic bacteria. The lytic plasmid-dependent phage PRD1 and the multiresistance conferring plasmid RP4 in an host were utilized to assess the genetic and phenotypic changes induced by combined phage and antibiotic selection. Resistance to PRD1 was always coupled with either completely lost or greatly reduced conjugation ability. Reversion to full conjugation efficiency was found to be rare, and it also restored the susceptibility to plasmid-dependent phages. Consequently, plasmid-dependent phages constitute an interesting candidate for development of sustainable anticonjugation/antiresistance therapeutic applications.