Bioinformatic analysis of type III CRISPR systems reveals key properties and new effector families.

Recognition of RNA from invading mobile genetic elements (MGE) prompts type III CRISPR systems to activate an HD nuclease domain and/or a nucleotide cyclase domain in the Cas10 subunit, eliciting an immune response. The cyclase domain can generate a range of nucleotide second messengers, which in turn activate a diverse family of ancillary effector proteins. These provide immunity by non-specific degradation of host and MGE nucleic acids or proteins, perturbation of membrane potentials, transcriptional responses, or the arrest of translation. The wide range of nucleotide activators and downstream effectors generates a complex picture that is gradually being resolved. Here, we carry out a global bioinformatic analysis of type III CRISPR loci in prokaryotic genomes, defining the relationships of Cas10 proteins and their ancillary effectors. Our study reveals that cyclic tetra-adenylate is by far the most common signalling molecule used and that many loci have multiple effectors. These typically share the same activator and may work synergistically to combat MGE. We propose four new candidate effector protein families and confirm experimentally that the Csm6-2 protein, a highly diverged, fused Csm6 effector, is a ribonuclease activated by cyclic hexa-adenylate.

Chromosomal Inversions and the Demography of Speciation in Drosophila montana and Drosophila flavomontana.

Chromosomal inversions may play a central role in speciation given their ability to locally reduce recombination and therefore genetic exchange between diverging populations. We analyzed long- and short-read whole-genome data from sympatric and allopatric populations of 2 Drosophila virilis group species, Drosophila montana and Drosophila flavomontana, to understand if inversions have contributed to their divergence. We identified 3 large alternatively fixed inversions on the X chromosome and one on each of the autosomes 4 and 5. A comparison of demographic models estimated for inverted and noninverted (colinear) chromosomal regions suggests that these inversions arose before the time of the species split. We detected a low rate of interspecific gene flow (introgression) from D. montana to D. flavomontana, which was further reduced inside inversions and was lower in allopatric than in sympatric populations. Together, these results suggest that the inversions were already present in the common ancestral population and that gene exchange between the sister taxa was reduced within inversions both before and after the onset of species divergence. Such ancestrally polymorphic inversions may foster speciation by allowing the accumulation of genetic divergence in loci involved in adaptation and reproductive isolation inside inversions early in the speciation process, while gene exchange at colinear regions continues until the evolving reproductive barriers complete speciation. The overlapping X inversions are particularly good candidates for driving the speciation process of D. montana and D. flavomontana, since they harbor strong genetic incompatibilities that were detected in a recent study of experimental introgression.

Antiviral type III CRISPR signalling via conjugation of ATP and SAM.

CRISPR systems are widespread in the prokaryotic world, providing adaptive immunity against mobile genetic elements1,2. 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 domain3-5 or indirectly, via synthesis of cyclic oligoadenylate second messengers to activate diverse ancillary effectors6-9. 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 nucleases10. SAM-AMP thus represents a new class of second messenger for antiviral signalling, which may function in different roles in diverse cellular contexts.

Multispecies coinfections and presence of antibiotics shape resistance and fitness costs in a pathogenic bacterium.

Increasing antimicrobial resistance (AMR) poses a challenge for treatment of bacterial diseases. In real life, bacterial infections are typically embedded within complex multispecies communities and influenced by the environment, which can shape costs and benefits of AMR. However, knowledge of such interactions and their implications for AMR in vivo is limited. To address this knowledge gap, we investigated fitness-related traits of a pathogenic bacterium (Flavobacterium columnare) in its fish host, capturing the effects of bacterial antibiotic resistance, coinfections between bacterial strains and metazoan parasites (fluke Diplostomum pseudospathaceum) and antibiotic exposure. We quantified real-time replication and virulence of sensitive and resistant bacteria and demonstrate that both bacteria can benefit from coinfection in terms of persistence and replication, depending on the coinfecting partner and antibiotic presence. We also show that antibiotics can benefit resistant bacteria by increasing bacterial replication under coinfection with flukes. These results emphasize the importance of diverse, inter-kingdom coinfection interactions and antibiotic exposure in shaping costs and benefits of AMR, supporting their role as significant contributors to spread and long-term persistence of resistance.

Mucin induces CRISPR-Cas defense in an opportunistic pathogen.

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.

Bacteriophage Resistance Affects Flavobacterium columnare Virulence Partly via Mutations in Genes Related to Gliding Motility and the Type IX Secretion System.

Increasing problems with antibiotic resistance have directed interest toward phage therapy in the aquaculture industry. However, phage resistance evolving in target bacteria is considered a challenge. To investigate how phage resistance influences the fish pathogen Flavobacterium columnare, two wild-type bacterial isolates, FCO-F2 and FCO-F9, were exposed to phages (FCO-F2 to FCOV-F2, FCOV-F5, and FCOV-F25, and FCO-F9 to FCL-2, FCOV-F13, and FCOV-F45), and resulting phenotypic and genetic changes in bacteria were analyzed. Bacterial viability first decreased in the exposure cultures but started to increase after 1 to 2 days, along with a change in colony morphology from original rhizoid to rough, leading to 98% prevalence of the rough morphotype. Twenty-four isolates (including four isolates from no-phage treatments) were further characterized for phage resistance, antibiotic susceptibility, motility, adhesion, and biofilm formation, protease activity, whole-genome sequencing, and virulence in rainbow trout fry. The rough isolates arising in phage exposure were phage resistant with low virulence, whereas rhizoid isolates maintained phage susceptibility and high virulence. Gliding motility and protease activity were also related to the phage susceptibility. Observed mutations in phage-resistant isolates were mostly located in genes encoding the type IX secretion system, a component of the Bacteroidetes gliding motility machinery. However, not all phage-resistant isolates had mutations, indicating that phage resistance in F. columnare is a multifactorial process, including both genetic mutations and changes in gene expression. Phage resistance may not, however, be a challenge for development of phage therapy against F. columnare infections since phage resistance is associated with decreases in bacterial virulence. IMPORTANCE Phage resistance of infectious bacteria is a common phenomenon posing challenges for the development of phage therapy. Along with a growing world population and the need for increased food production, constantly intensifying animal farming has to face increasing problems of infectious diseases. Columnaris disease, caused by Flavobacterium columnare, is a worldwide threat for salmonid fry and juvenile farming. Without antibiotic treatments, infections can lead to 100% mortality in a fish stock. Phage therapy of columnaris disease would reduce the development of antibiotic-resistant bacteria and antibiotic loads by the aquaculture industry, but phage-resistant bacterial isolates may become a risk. However, phenotypic and genetic characterization of phage-resistant F. columnare isolates in this study revealed that they are less virulent than phage-susceptible isolates and thus not a challenge for phage therapy against columnaris disease. This is valuable information for the fish farming industry globally when considering phage-based prevention and curing methods for F. columnare infections.

Cooperation between Different CRISPR-Cas Types Enables Adaptation in an RNA-Targeting System.

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.

Adapting a Phage to Combat Phage Resistance.

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.

Aquaculture as a source of empirical evidence for coevolution between CRISPR-Cas and phage.

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'.

Gliding Motility and Expression of Motility-Related Genes in Spreading and Non-spreading Colonies of Flavobacterium columnare.

Gliding motility facilitates the movement of bacteria along surfaces in many Bacteroidetes species and results in spreading colonies. The adhesins required for the gliding are secreted through a gliding motility-associated protein secretion system, known as the type IX secretion system (T9SS). The fish pathogen Flavobacterium columnare produces spreading (rhizoid [Rz], soft [S]) and non-spreading (rough [R]) colony types, of which only the spreading Rz type is virulent. In this study, we explored the spreading behavior of these colony types by microscopic imaging and measured the expression of genes associated with gliding motility and T9SS (gldG, gldH, gldL, sprA, sprB, sprE, sprF, sprT, and porV) under high and low resource levels by using RT-qPCR (reverse transcription quantitative PCR). The spreading colony types responded to the low resource level with increased colony size. The non-spreading colony type, as well as the cells growing under high nutrient level expressed only moderate cell movements. Yet, a low nutrient level provoked more active gliding motility in individual cells and increased spreading by cooperative gliding. The gene expression survey demonstrated an increased expression level of sprA (a core component of T9SS) and sprF (needed for adhesin secretion) under low nutrient conditions. Surprisingly, the expression of gliding motility genes was not consistently associated with more active spreading behavior. Furthermore, no genetic differences were found between spreading and non-spreading colony types in the studied genes associated with gliding motility. Our study demonstrates that environmental nutrient level is an important regulator of both gliding motility and the expression of some of the associated genes. These results may help to understand the connections between nutrient concentration, gliding motility, and virulence of F. columnare.

Long-term genomic coevolution of host-parasite interaction in the natural environment.

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.

Scoping the effectiveness and evolutionary obstacles in using plasmid-dependent phages to fight antibiotic resistance.

AIM: 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. MATERIALS & METHODS: The lytic plasmid-dependent phage PRD1 and the multiresistance conferring plasmid RP4 in an Escherichia coli host were utilized to assess the genetic and phenotypic changes induced by combined phage and antibiotic selection. RESULTS & CONCLUSIONS: 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.

Chasing the Origin of Viruses: Capsid-Forming Genes as a Life-Saving Preadaptation within a Community of Early Replicators.

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.

Evolutionary rescue of bacteria via horizontal gene transfer under a lethal ß-lactam concentration.

ß-Lactams are a commonly used class of bactericidal antibiotics. The number of ß-lactam-resistant pathogens is constantly increasing in hospitals around the world. Interestingly, most of the ß-lactam-resistant bacteria carry mobile genetic elements, such as conjugative plasmids, that render the pathogen resistant. These elements mediate their own transfer from one bacterium to another, producing new resistant strains via horizontal gene transfer. Here we investigated whether it is possible that transfer of the resistance element from another bacterium may evolutionarily rescue a susceptible bacterium exposed to a lethal concentration of the ß-lactam ampicillin. Indeed, the rescuing occurs even at very high, clinically significant antibiotic levels, suggesting that pathogens may acquire the resistance 'on the fly' from commensal bacteria during treatment.