Arbitrium communication controls phage lysogeny through non-lethal modulation of a host toxin-antitoxin defence system.

Temperate Bacillus phages often utilize arbitrium communication to control lysis/lysogeny decisions, but the mechanisms by which this control is exerted remains largely unknown. Here we find that the arbitrium system of Bacillus subtilis phage ϕ3T modulates the host-encoded MazEF toxin-antitoxin system to this aim. Upon infection, the MazF ribonuclease is activated by three phage genes. At low arbitrium signal concentrations, MazF is inactivated by two phage-encoded MazE homologues: the arbitrium-controlled AimX and the later-expressed YosL proteins. At high signal, MazF remains active, promoting lysogeny without harming the bacterial host. MazF cleavage sites are enriched on transcripts of phage lytic genes but absent from the phage repressor in ϕ3T and other Spß-like phages. Combined with low activation levels of MazF during infections, this pattern explains the phage-specific effect. Our results show how a bacterial toxin-antitoxin system has been co-opted by a phage to control lysis/lysogeny decisions without compromising host viability.

Antagonistic interactions between phage and host factors control arbitrium lysis-lysogeny decision.

Phages can use a small-molecule communication arbitrium system to coordinate lysis-lysogeny decisions, but the underlying mechanism remains unknown. Here we determined that the arbitrium system in Bacillus subtilis phage phi3T modulates the bacterial toxin-antitoxin system MazE-MazF to regulate the phage life cycle. We show that phi3T expresses AimX and YosL, which bind to and inactivate MazF. AimX also inhibits the function of phi3T_93, a protein that promotes lysogeny by binding to MazE and releasing MazF. Overall, these mutually exclusive interactions promote the lytic cycle of the phage. After several rounds of infection, the phage-encoded AimP peptide accumulates intracellularly and inactivates the phage antiterminator AimR, a process that eliminates aimX expression from the aimP promoter. Therefore, when AimP increases, MazF activity promotes reversion back to lysogeny, since AimX is absent. Altogether, our study reveals the evolutionary strategy used by arbitrium to control lysis-lysogeny by domesticating and fine-tuning a phage-defence mechanism.

Getting Outside the Cell: Versatile Holin Strategies Used by Distinct Phages to Leave Their Bacillus thuringiensis Host.

Holins are small transmembrane proteins involved in the final stage of the lytic cycle of double-stranded DNA (dsDNA) phages. They cooperate with endolysins to achieve bacterial lysis, thereby releasing the phage progeny into the extracellular environment. Besides their role as membrane permeabilizers, allowing endolysin transfer and/or activation, holins also regulate the lysis timing. In this work, we provide functional characterization of the holins encoded by three phages targeting the Bacillus cereus group. The siphovirus Deep-Purple has a lysis cassette in which holP30 and holP33 encode two proteins displaying holin properties, including a transmembrane domain. The holin genes were expressed in Escherichia coli and induced bacterial lysis, with HolP30 being more toxic than HolP33. In Bacillus thuringiensis, the simultaneous expression of both holins was necessary to observe lysis, suggesting that they may interact to form functional pores. The myoviruses Deep-Blue and Vp4 both encode a single candidate holin (HolB and HolV, respectively) with two transmembrane domains, whose genes are not located near the endolysin genes. Their function as holin proteins was confirmed as their expression in E. coli impaired cell growth and viability. The HolV expression in B. thuringiensis also led to bacterial lysis, which was enhanced by coexpressing the holin with its cognate endolysin. Despite similar organizations and predicted topologies, truncated mutants of the HolB and HolV proteins showed different toxicity levels, suggesting that differences in amino acid composition influence their lysis properties. IMPORTANCE The phage life cycle ends with the host cell lysis, thereby releasing new virions into the environment for the next round of bacterial infection. Nowadays, there is renewed interest in phages as biocontrol agents, primarily due to their ability to cause bacterial death through lysis. While endolysins, which mediate peptidoglycan degradation, have been fairly well described, the pore-forming proteins, referred to as holins, have been extensively characterized in only a few model phages, mainly infecting Gram-negative bacteria. In this work, we characterized the holins encoded by a siphovirus and two myoviruses targeting members of the Gram-positive Bacillus cereus group, which comprises closely related species, including the well-known Bacillus anthracis, B. cereus sensu stricto, and Bacillus thuringiensis. Overall, this paper provides the first experimental characterization of holins encoded by B. cereus phages and reveals versatile lysis mechanisms used by these phages.

Phenotypic and genotypic characterization of the new Bacillus cereus phage SWEP1.

A new Bacillus cereus phage, SWEP1, was isolated from black soil. The host lysis activity of phage SWEP1 has a relatively short latent time (20 min) and a small burst size of 83 PFU. The genome of SWEP1 consists of 162,461 bp with 37.77% G+C content. The phage encodes 278 predicted proteins, 103 of which were assigned functionally. No tRNA genes were found. Comparative genomics analysis indicated that SWEP1 is related to Bacillus phage B4 (86.91% identity, 90% query coverage). Phenotypic and genotypic characterization suggested that SWEP1 is a new member of a new species in the genus Bequatrovirus, family Herelleviridae.

Enzyme-Linked Phage Receptor Binding Protein Assays (ELPRA) Enable Identification of Bacillus anthracis Colonies.

Bacteriophage receptor binding proteins (RBPs) are employed by viruses to recognize specific surface structures on bacterial host cells. Recombinant RBPs have been utilized for detection of several pathogens, typically as fusions with reporter enzymes or fluorescent proteins. Identification of Bacillus anthracis, the etiological agent of anthrax, can be difficult because of the bacterium's close relationship with other species of the Bacillus cereussensu lato group. Here, we facilitated the identification of B. anthracis using two implementations of enzyme-linked phage receptor binding protein assays (ELPRA). We developed a single-tube centrifugation assay simplifying the rapid analysis of suspect colonies. A second assay enables identification of suspect colonies from mixed overgrown solid (agar) media derived from the complex matrix soil. Thus, these tests identified vegetative cells of B. anthracis with little processing time and may support or confirm pathogen detection by molecular methods such as polymerase chain reaction.

Incidence and characterisation of Bacillus cereus bacteriophages from Thua Nao, a Thai fermented soybean product.

Bacillus cereus is considered to be an important food poisoning agent causing diarrhea and vomiting. In this study, the occurrence of B. cereus bacteriophages in Thai fermented soybean products (Thua Nao) was studied using five B. cereus sensu lato indicator strains (four B. cereus strains and one B. thuringiensis strain). In a total of 26 Thua Nao samples, there were only two bacteriophages namely BaceFT01 and BaceCM02 exhibiting lytic activity against B. cereus. Morphological analysis revealed that these two bacteriophages belonged to the Myoviridae. Both phages were specific to B. cereus and not able to lyse other tested bacteria including B. licheniformis and B. subtilis. The two phages were able to survive in a pH range between 5 and 12. However, both phages were inactive either by treatment of 50°C for 2 h or exposure of UV for 2 h. It should be noted that both phages were chloroform-insensitive, however. This is the first report describing the presence of bacteriophages in Thua Nao products. The characterization of these two phages is expected to be useful in the food industry for an alternative strategy including the potential use of the phages as a biocontrol candidate against foodborne pathogenic bacteria.

Strand Displacement and Unwinding Assays to Study the Concerted Action of the DNA Polymerase and SSB During Phi29 TP-DNA Replication.

The Bacillus subtilis phage Phi29 has a linear double-stranded DNA with a terminal protein (TP) covalently linked to each 5' end (TP-DNA). Phi29 single-stranded DNA-binding protein (SSB) is encoded by the viral gene 5 and binds the ssDNA generated during the Phi29 genome replication, stimulating the DNA elongation rate. Here, we describe some protocols to evaluate the effect of Phi29 SSB mutants on the DNA elongation rate and their unwinding activity during replication by Phi29 DNA polymerase using as substrate TP-DNA and also singly primed M13 DNA.

Extraordinary Multi-Organismal Interactions Involving Bacteriophages, Bacteria, Fungi, and Rotifers: Quadruple Microbial Trophic Network in Water Droplets.

Our observations of predatory fungi trapping rotifers in activated sludge and laboratory culture allowed us to discover a complicated trophic network that includes predatory fungi armed with bacteria and bacteriophages and the rotifers they prey on. Such a network seems to be common in various habitats, although it remains mostly unknown due to its microscopic size. In this study, we isolated and identified fungi and bacteria from activated sludge. We also noticed abundant, virus-like particles in the environment. The fungus developed absorptive hyphae within the prey. The bacteria showed the ability to enter and exit from the hyphae (e.g., from the traps into the caught prey). Our observations indicate that the bacteria and the fungus share nutrients obtained from the rotifer. To narrow the range of bacterial strains isolated from the mycelium, the effects of bacteria supernatants and lysed bacteria were studied. Bacteria isolated from the fungus were capable of immobilizing the rotifer. The strongest negative effect on rotifer mobility was shown by a mixture of Bacillus sp. and Stenotrophomonas maltophilia. The involvement of bacteriophages in rotifer hunting was demonstrated based on molecular analyses and was discussed. The described case seems to be an extraordinary quadruple microbiological puzzle that has not been described and is still far from being understood.

Microbial single-cell RNA sequencing by split-pool barcoding.

Single-cell RNA sequencing (scRNA-seq) has become an essential tool for characterizing gene expression in eukaryotes, but current methods are incompatible with bacteria. Here, we introduce microSPLiT (microbial split-pool ligation transcriptomics), a high-throughput scRNA-seq method for Gram-negative and Gram-positive bacteria that can resolve heterogeneous transcriptional states. We applied microSPLiT to >25,000 Bacillus subtilis cells sampled at different growth stages, creating an atlas of changes in metabolism and lifestyle. We retrieved detailed gene expression profiles associated with known, but rare, states such as competence and prophage induction and also identified unexpected gene expression states, including the heterogeneous activation of a niche metabolic pathway in a subpopulation of cells. MicroSPLiT paves the way to high-throughput analysis of gene expression in bacterial communities that are otherwise not amenable to single-cell analysis, such as natural microbiota.

A CRISPR-Cas9 tool to explore the genetics of Bacillus subtilis phages.

Here, we present pRH030, a new CRISPR-Cas9 tool for the genetic engineering of Bacillus phages and beyond. It is based on the Streptococcus pyogenes cas9 with its native constitutive promoter, tracrRNA, and a gRNA precursor. The constitutive expression of Cas9 was conducive to the inactivation of viral attackers and enhanced phage mutagenesis efficiency up to 100%. The gRNA precursor can be built up to an artificial CRISPR array with up to 5 spacers (target sequences) assembled from ordinary oligonucleotides and directly cloned into pRH030. Required time and resources remain comparable to a single gRNA cloning. These properties make pRH030 an attractive new system for the modification of Bacillus phages and qualify it for research beyond genetic construction.

The PlyB Endolysin of Bacteriophage vB_BanS_Bcp1 Exhibits Broad-Spectrum Bactericidal Activity against Bacillus cereus Sensu Lato Isolates.

Lytic bacteriophages (or phages) drive bacterial mortality by elaborating exquisite abilities to bind, breach, and destroy bacterial cell membranes and subjugate critical bacterial cell functions. These antimicrobial activities make phages ideal candidates to serve as, or provide sources of, biological control measures for bacterial pathogens. In this study, we isolated the Myoviridae phage vB_BanS_Bcp1 (here referred to as Bcp1) from landfill soil, using a Bacillus anthracis host. The antimicrobial activities of both Bcp1 and its encoded endolysin, PlyB, were examined across different B. cereussensu lato group species, including B. cereussensu stricto, Bacillus thuringiensis, and Bacillus anthracis, with pathogenic potential in humans and multiple different uses in biotechnological applications. The Bcp1 phage infected only a subset (11 to 66%) of each B. cereussensu lato species group tested. In contrast, functional analysis of purified PlyB revealed a potent bacteriolytic activity against all B. cereussensu lato isolates tested (n = 79). PlyB was, furthermore, active across broad temperature, pH, and salt ranges, refractory to the development of resistance, bactericidal as a single agent, and synergistic with a second endolysin, PlyG. To confirm the potential for PlyB as an antimicrobial agent, we demonstrated the efficacy of a single intravenous treatment with PlyB alone or combination with PlyG in a murine model of lethal B. anthracis infection. Overall, our findings show exciting potential for the Bcp1 bacteriophage and the PlyB endolysin as potential new additions to the antimicrobial armamentarium.IMPORTANCE Organisms of the Bacillus cereussensu lato lineage are ubiquitous in the environment and are responsible for toxin-mediated infections ranging from severe food poisoning (B. cereussensu stricto) to anthrax (Bacillus anthracis). The increasing incidence of many of these infections, combined with the specter of antibiotic resistance, has created a need for novel antimicrobials with potent activity, including bacteriophages (or phages) and phage-encoded products (i.e., endolysins). In this study, we describe a broadly infective phage, Bcp1, and its encoded endolysin, PlyB, which exhibited a rapidly bacteriolytic effect against all B. cereussensu lato isolates tested with no evidence of evolving resistance. Importantly, PlyB was highly efficacious in a mouse model of lethal bacteremia with B. anthracis Both the Bcp1 phage and the PlyB endolysin represent novel mechanisms of action compared to antibiotics, with potential applications to address the evolving problem of antimicrobial resistance.

Isolation of A Novel Bacillus thuringiensis Phage Representing A New Phage Lineage and Characterization of Its Endolysin.

Phages, the parasites of bacteria, are considered as a new kind of antimicrobial agent due to their ability to lyse pathogenic bacteria. Due to the increase of available phage isolates, the newly isolated phage showed increasing genomic similarities with previously isolated phages. In this study, the novel phage vB_BthS_BMBphi, infecting the Bacillus thuringiensis strain BMB171, is isolated and characterized together with its endolysin. This phage is the first tadpole-like phage infecting the Bacillus strains. Genomic analysis shows that the phage genome is dissimilar to all those of previously characterized phages, only exhibiting low similarities with partial regions of the B. thuringiensis prophages. Phylogenetic analysis revealed that the phage was distant from the other Bacillus phages in terms of evolution. The novel genome sequence, the distant evolutionary relationship, and the special virion morphology together suggest that the phage vB_BthS_BMBphi could be classified as a new phage lineage. The genome of the phage is found to contain a restriction modification system, which might endow the phage with immunity to the restriction modification system of the host bacterium. The function of the endolysin PlyBMB encoded by the phage vB_BthS_BMBphi was analyzed, and the endolysin could lyse all the tested Bacillus cereus group strains, suggesting that the endolysin might be used in controlling pathogenic B. cereus group strains. The findings of this study enrich the understanding of phage diversity and provide a resource for controlling the B. cereus group pathogenic bacteria.

Structural and functional insights into the regulation of the lysis-lysogeny decision in viral communities.

Communication is vital for all organisms including microorganisms, which is clearly demonstrated by the bacterial quorum-sensing system. However, the molecular mechanisms underlying communication among viruses (phages) via the quorum-sensing-like 'arbitrium' system remain unclear. Viral or host densities are known to be related to an increased prevalence of lysogeny; however, how the switch from the lytic to the lysogenic pathway occurs is unknown. Thus, we sought to reveal mechanisms of communication among viruses and determine the lysogenic dynamics involved. Structural and functional analyses of the phage-derived SAIRGA and GMPRGA peptides and their corresponding receptors, phAimR and spAimR, indicated that SAIRGA directs the lysis-lysogeny decision of phi3T by modulating conformational changes in phAimR, whereas GMPRGA regulates the lysis-lysogeny pathway by stabilizing spAimR in the dimeric state. Although temperate viruses are thought to share a similar lytic-lysogenic cycle switch model, our study suggests the existence of alternative strain-specific mechanisms that regulate the lysis-lysogeny decision. Collectively, these findings provide insights into the molecular mechanisms underlying communication among viruses, offering theoretical applications for the treatment of infectious viral diseases.

Structural basis of the arbitrium peptide-AimR communication system in the phage lysis-lysogeny decision.

A bacteriophage can replicate and release virions from a host cell in the lytic cycle or switch to a lysogenic process in which the phage integrates itself into the host genome as a prophage. In Bacillus cells, some types of phages employ the arbitrium communication system, which contains an arbitrium hexapeptide, the cellular receptor AimR and the lysogenic negative regulator AimX. This system controls the decision between the lytic and lysogenic cycles. However, both the mechanism of molecular recognition between the arbitrium peptide and AimR and how downstream gene expression is regulated remain unknown. Here, we report crystal structures for AimR from the SPbeta phage in the apo form and the arbitrium peptide-bound form at 2.20 Å and 1.92 Å, respectively. With or without the peptide, AimR dimerizes through the C-terminal capping helix. AimR assembles a superhelical fold and accommodates the peptide encircled by its tetratricopeptide repeats, which is reminiscent of RRNPP family members from the quorum-sensing system. In the absence of the arbitrium peptide, AimR targets the upstream sequence of the aimX gene; its DNA binding activity is prevented following peptide binding. In summary, our findings provide a structural basis for peptide recognition in the phage lysis-lysogeny decision communication system.

Studies on Lytic, Tailed Bacillus cereus-specific Phage for Use in a Ferromagnetoelastic Biosensor as a Novel Recognition Element.

This study investigated the feasibility of the lytic, tailed Bacillus cereus-specific phage for use in a ferromagnetoelastic (FME) biosensor as a novel recognition element. The phage was immobilized at various concentrations through either direct adsorption or a combination of 11-mercapto-1-undecanoic acid (11-MUA) and [N-(3-dimethylaminopropyl)-N'-carbodiimide hydrochloride and N-hydroxysuccinimide (EDC/NHS)]. The effects of time and temperature on its lytic properties were investigated through the exposure of B. cereus (4 and 8 logCFU/ml) to the phage (8 logPFU/ml) for various incubation periods at 22°C and at various temperatures for 30 and 60 min. As the phage concentration increased, both immobilization methods also significantly increased the phage density (p < 0.05). SEM images confirmed that the phage density on the FME platform corresponded to the increased phage concentration. As the combination of 11-MUA and EDC/NHS enhanced the phage density and orientation by up to 4.3-fold, it was selected for use. When various incubation was conducted, no significant differences were observed in the survival rate of B. cereus within 30 min, which was in contrast to the significant decreases observed at 45 and 60 min (p < 0.05). In addition, temperature exerted no significant effects on the survival rate across the entire temperature range. This study demonstrated the feasibility of the lytic, tailed B. cereus-specific phage as a novel recognition element for use in an FME biosensor. Thus, the phage could be placed on the surface of foods for at least 30 min without any significant loss of B. cereus, as a result of the inherent lytic activity of the B. cereus-specific phage as a novel recognition element.

Directional mechanical stability of Bacteriophage φ29 motor's 3WJ-pRNA: Extraordinary robustness along portal axis.

The molecular motor exploited by bacteriophage φ29 to pack DNA into its capsid is regarded as one of the most powerful mechanical devices present in viral, bacterial, and eukaryotic systems alike. Acting as a linker element, a prohead RNA (pRNA) effectively joins the connector and ATPase (adenosine triphosphatase) components of the φ29 motor. During DNA packing, this pRNA needs to withstand enormous strain along the capsid's portal axis-how this remarkable stability is achieved remains to be elucidated. We investigate the mechanical properties of the φ29 motor's three-way junction (3WJ)-pRNA using a combined steered molecular dynamics and atomic force spectroscopy approach. The 3WJ exhibits strong resistance to stretching along its coaxial helices, demonstrating its super structural robustness. This resistance disappears, however, when external forces are applied to the transverse directions. From a molecular standpoint, we demonstrate that this direction-dependent stability can be attributed to two Mg clamps that cooperate and generate mechanical resistance in the pRNA's coaxial direction. Our results suggest that the asymmetric nature of the 3WJ's mechanical stability is entwined with its biological function: Enhanced rigidity along the portal axis is likely essential to withstand the strain caused by DNA condensation, and flexibility in other directions should aid in the assembly of the pRNA and its association with other motor components.

Experimental comparison of forces resisting viral DNA packaging and driving DNA ejection.

We compare forces resisting DNA packaging and forces driving DNA ejection in bacteriophage phi29 with theoretical predictions. Ejection of DNA from prohead-motor complexes is triggered by heating complexes after in vitro packaging and force is inferred from the suppression of ejection by applied osmotic pressure. Ejection force from 0% to 80% filling is found to be in quantitative agreement with predictions of a continuum mechanics model that assumes a repulsive DNA-DNA interaction potential based on DNA condensation studies and predicts an inverse-spool conformation. Force resisting DNA packaging from ∼80% to 100% filling inferred from optical tweezers studies is also consistent with the predictions of this model. The striking agreement with these two different measurements suggests that the overall energetics of DNA packaging is well described by the model. However, since electron microscopy studies of phi29 do not reveal a spool conformation, our findings suggest that the spool model overestimates the role of bending rigidity and underestimates the role of intrastrand repulsion. Below ∼80% filling the inferred forces resisting packaging are unexpectedly lower than the inferred ejection forces, suggesting that in this filling range the forces are less accurately determined or strongly temperature dependent.

Membrane Penetration by Bacterial Viruses.

The bacteriophage ϕ29 infects Gram-positive Bacillus subtilis with a short noncontractile tail. Recent studies showed that the ϕ29 tail protein gp9 forms a hexameric tube with six long loops of membrane-active peptides blocking in the tube at the distal end of the tail. The long loops exit on genome release and form a membrane pore for passage of the genome. The membrane penetration mechanism of the ϕ29 tail might be common among tailed bacteriophages.

CRISPR-Cas systems exploit viral DNA injection to establish and maintain adaptive immunity.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems provide protection against viral and plasmid infection by capturing short DNA sequences from these invaders and integrating them into the CRISPR locus of the prokaryotic host. These sequences, known as spacers, are transcribed into short CRISPR RNA guides that specify the cleavage site of Cas nucleases in the genome of the invader. It is not known when spacer sequences are acquired during viral infection. Here, to investigate this, we tracked spacer acquisition in Staphylococcus aureus cells harbouring a type II CRISPR-Cas9 system after infection with the staphylococcal bacteriophage ϕ12. We found that new spacers were acquired immediately after infection preferentially from the cos site, the viral free DNA end that is first injected into the cell. Analysis of spacer acquisition after infection with mutant phages demonstrated that most spacers are acquired during DNA injection, but not during other stages of the viral cycle that produce free DNA ends, such as DNA replication or packaging. Finally, we showed that spacers acquired from early-injected genomic regions, which direct Cas9 cleavage of the viral DNA immediately after infection, provide better immunity than spacers acquired from late-injected regions. Our results reveal that CRISPR-Cas systems exploit the phage life cycle to generate a pattern of spacer acquisition that ensures a successful CRISPR immune response.

Isolation and Characterization of Bacillus cereus Bacteriophages from Foods and Soil.

The aim of this study was to isolate and characterize Bacillus cereus bacteriophages of various origins. Twenty-seven bacteriophages against B. cereus were isolated from various Korean traditional fermented foods and soils. Plaque size, transmission electron microscopy, virulence profile, and in vitro lytic activity of bacteriophage isolates were examined. Transmission electron microscopy confirmed B. cereus bacteriophages belonging to the family Siphoviridae. Among B. cereus bacteriophages with broad host range, 18 isolates (66.7%) did not harbor any B. cereus virulence factors. Among them, bacteriophage strain CAU150036, CAU150038, CAU150058, CAU150064, CAU150065, and CAU150066 effectively inhibited B. cereus in vitro within 1 h. Therefore, they are considered potential candidates for controlling the contamination of B. cereus in food or other applications.