The life cycle of SPß and related phages.

Phages are viruses of bacteria and are the smallest and most common biological entities in the environment. They can reproduce immediately after infection or integrate as a prophage into their host genome. SPß is a prophage of the Gram-positive model organism Bacillus subtilis 168, and it has been known for more than 50 years. It is sensitive to dsDNA damage and is induced through exposure to mitomycin C or UV radiation. When induced from the prophage, SPß requires 90 min to produce and release about 30 virions. Genomes of sequenced related strains range between 128 and 140 kb, and particle-packed dsDNA exhibits terminal redundancy. Formed particles are of the Siphoviridae morphotype. Related isolates are known to infect other B. subtilis clade members. When infecting a new host, SPß presumably follows a two-step strategy, adsorbing primarily to teichoic acid and secondarily to a yet unknown factor. Once in the host, SPß-related phages pass through complex lysis-lysogeny decisions and either enter a lytic cycle or integrate as a dormant prophage. As prophages, SPß-related phages integrate at the host chromosome's replication terminus, and frequently into the spsM or kamA gene. As a prophage, it imparts additional properties to its host via phage-encoded proteins. The most notable of these functional proteins is sublancin 168, which is used as a molecular weapon by the host and ensures prophage maintenance. In this review, we summarise the existing knowledge about the biology of the phage regarding its life cycle and discuss its potential as a research object.

Aminoglycosides Antagonize Bacteriophage Proliferation, Attenuating Phage Suppression of Bacterial Growth, Biofilm Formation, and Antibiotic Resistance.

The common cooccurrence of antibiotics and phages in both natural and engineered environments underscores the need to understand their interactions and implications for bacterial control and antibiotic resistance propagation. Here, aminoglycoside antibiotics that inhibit protein synthesis (e.g., kanamycin and neomycin) impeded the replication of coliphage T3 and Bacillus phage BSP, reducing their infection efficiency and mitigating their hindrance of bacterial growth, biofilm formation, and tolerance to antibiotics. For example, treatment with phage T3 reduced subsequent biofilm formation by Escherichia coli liquid cultures to 53% ± 5% of that of the no-phage control, but a smaller reduction of biofilm formation (89% ± 10%) was observed for combined exposure to phage T3 and kanamycin. Despite sharing a similar mode of action with aminoglycosides (i.e., inhibiting protein synthesis) and antagonizing phage replication, albeit to a lesser degree, tetracyclines did not inhibit bacterial control by phages. Phage T3 combined with tetracycline showed higher suppression of biofilm formation than when combined with aminoglycosides (25% ± 6% of the no-phage control). The addition of phage T3 to E. coli suspensions with tetracycline also suppressed the development of tolerance to tetracycline. However, this suppression of antibiotic tolerance development disappeared when tetracycline was replaced with 3 mg/liter kanamycin, corroborating the greater antagonism with aminoglycosides. Overall, this study highlights this overlooked antagonistic effect on phage proliferation, which may attenuate phage suppression of bacterial growth, biofilm formation, antibiotic tolerance, and maintenance of antibiotic resistance genes. IMPORTANCE The coexistence of residual antibiotics and phages is common in many environments, which underscores the need to understand their interactive effects on bacteria and the implications for antibiotic resistance propagation. Here, aminoglycosides acting as bacterial protein synthesis inhibitors impeded the replication of various phages. This alleviated the suppressive effects of phages against bacterial growth and biofilm formation and diminished bacterial fitness costs that suppress the emergence of tolerance to antibiotics. We show that changes in bacteria caused by environmentally relevant concentrations of sublethal antibiotics can affect phage-host dynamics that are commonly overlooked in vitro but can result in unexpected environmental consequences.

A novel deep-sea bacteriophage possesses features of Wbeta-like viruses and prophages.

As the most abundant biological entities, viruses are major players in marine ecosystems. However, our knowledge about virus-host interactions and viral ecology in the deep sea remains very limited. In this study, a novel bacteriophage (designated as phage BVE2) infecting Bacillus cereus group bacteria, was isolated from deep-sea sediments. Phage BVE2 caused host lysis within 1.5 h after infection. However, the presence of two integrase-encoding genes in the BVE2 genome suggested that BVE2 may also follow a temperate strategy. The genome of phage BVE2 is approximately 20 kb in length and is predicted to encode 28 proteins. Genomic and phylogenetic analysis suggested that BVE2 is a highly mosaic phage that has inherited genetic features from Wbeta-like viruses, B. cereus prophages, and its host, suggesting that frequent horizontal gene transfer events occurred during its evolution. This study will help to reveal the evolutionary history of Wbeta-like viruses and improve our understanding of viral diversity and virus-host interactions in the deep sea.

Widespread Utilization of Peptide Communication in Phages Infecting Soil and Pathogenic Bacteria.

Temperate phages can adopt either a lytic or lysogenic lifestyle within their host bacteria. It was recently shown that Bacillus-subtilis-infecting phages of the SPbeta group utilize a peptide-based communication system called arbitrium to coordinate the lysogeny decision. The occurrence of peptide-based communication systems among phages more broadly remains to be explored. Here, we uncover a wide array of peptide-based communication systems utilized by phages for lysogeny decisions. These arbitrium-like systems show diverse peptide codes and can be detected in numerous genetically distant phage types and conjugative elements. The pathogens Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are commonly infected by arbitrium-carrying mobile elements, which often carry toxins essential for pathogenicity. Experiments with phages containing these arbitrium-like systems demonstrate their involvement in lysogeny decisions. Finally, our results suggest that the peptide-based decision is executed by an antisense RNA that controls the regulator of the lysogenic state.

Bacterial Endospores as Phage Genome Carriers and Protective Shells.

Bacterial endospores can serve as phage genome protection shells against various environmental stresses to enhance microbial control applications. The genomes of polyvalent lytic Bacillus phages PBSC1 and PBSC2, which infect both B. subtilis subsp. subtilis and B. cereus NRS 248, were incorporated into B. subtilis endospores (without integration into the host chromosome). When PBSC1 and PBSC2 were released from germinating endospores, they significantly inhibited the growth of the targeted opportunistic pathogen B. cereus Optimal endospore entrapment was achieved when phages were introduced to the fast-sporulating prespores at a multiplicity of infection of 1. Longer endospore maturation (48 h versus 24 h) increased both spore yield and efficiency of entrapment. Compared with free phages, spore-protected phage genomes showed significantly higher resistance toward high temperatures (60 to 80°C), extreme pH (pH 2 or pH 12), and copper ions (0.1 to 10 mg/liter). Endospore germination is inducible by low concentrations of l-alanine or by a germinant mixture (l-asparagine, d-glucose, d-fructose, and K+) to trigger the expression, assembly, and consequent release of phage particles within 60 to 90 min. Overall, the superior resiliency of polyvalent phages protected by endospores might enable nonrefrigerated phage storage and enhance phage applications after exposure to adverse environmental conditions.IMPORTANCE Bacteriophages are being considered for the control of multidrug-resistant and other problematic bacteria in environmental systems. However, the efficacy of phage-based microbial control is limited by infectivity loss during phage delivery and/or storage. Here, we exploit the pseudolysogenic state of phages, which involves incorporation of their genome into bacterial endospores (without integration into the host chromosome), to enhance survival in unfavorable environments. We isolated polyvalent (broad-host-range) phages that efficiently infect both benign and opportunistically pathogenic Bacillus strains and encapsulated the phage genomes in B. subtilis endospores to significantly improve resistance to various environmental stressors. Encapsulation by spores also significantly enhanced phage genome viability during storage. We also show that endospore germination can be induced on demand with nutrient germinants that trigger the release of active phages. Overall, we demonstrate that encapsulation of polyvalent phage genomes into benign endospores holds great promise for broadening the scope and efficacy of phage biocontrol.

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.

Rapid Detection of Viable Bacillus anthracis Spores in Environmental Samples by Using Engineered Reporter Phages.

Bacillus anthracis, the causative agent of anthrax, was utilized as a bioterrorism agent in 2001 when spores were distributed via the U.S. postal system. In responding to this event, the Federal Bureau of Investigation used traditional bacterial culture viability assays to ascertain the extent of contamination of the postal facilities within 24 to 48 h of environmental sample acquisition. Here, we describe a low-complexity, second-generation reporter phage assay for the rapid detection of viableB. anthracis spores in environmental samples. The assay uses an engineered B. anthracis reporter phage (Wß::luxAB-2) which transduces bioluminescence to infected cells. To facilitate low-level environmental detection and maximize the signal response, expression of luxABin an earlier version of the reporter phage (Wß::luxAB-1) was optimized. These alterations prolonged signal kinetics, increased light output, and improved assay sensitivity. Using Wß::luxAB-2, detection of B. anthracis spores was 1 CFU in 8 h from pure cultures and as low as 10 CFU/g in sterile soil but increased to 10(5)CFU/g in unprocessed soil due to an unstable signal and the presence of competing bacteria. Inclusion of semiselective medium, mediated by a phage-expressed antibiotic resistance gene, maintained signal stability and enabled the detection of 10(4)CFU/g in 6 h. The assay does not require spore extraction and relies on the phage infecting germinating cells directly in the soil sample. This reporter phage displays promise for the rapid detection of low levels of spores on clean surfaces and also in grossly contaminated environmental samples from complex matrices such as soils.

Rapid detection of Bacillus anthracis by γ phage amplification and lateral flow immunochromatography.

New, rapid point-of-need diagnostic methods for Bacillus anthracis detection can enhance civil and military responses to accidental or deliberate dispersal of anthrax as a biological weapon. Current laboratory-based methods for clinical identification of B. anthracis require 12 to 120h, and are confirmed by plaque assay using the well-characterized γ typing phage, which requires an additional minimum of 24h for bacterial culture. To reduce testing time, the natural specificity of γ phage amplification was investigated in combination with lateral flow immunochromatography (LFI) for rapid, point-of-need B. anthracis detection. Phage-based LFI detection of B. anthracis Sterne was validated over a range of bacterial and phage concentrations with optimal detection achieved in as little as 2h from the onset of amplification with a threshold sensitivity of 2.5×10(4)cfu/mL. The novel use of γ phage amplification detected with a simple, inexpensive LFI assay provides a rapid, sensitive, highly accurate, and field-deployable method for diagnostic ID of B. anthracis in a fraction of the time required by conventional techniques, and without the need for extensive laboratory culture.

Bacteriophage PBC1 and its endolysin as an antimicrobial agent against Bacillus cereus.

Bacillus cereus is an opportunistic human pathogen responsible for food poisoning and other, nongastrointestinal infections. Due to the emergence of multidrug-resistant B. cereus strains, the demand for alternative therapeutic options is increasing. To address these problems, we isolated and characterized a Siphoviridae virulent phage, PBC1, and its lytic enzymes. PBC1 showed a very narrow host range, infecting only 1 of 22 B. cereus strains. Phylogenetic analysis based on the major capsid protein revealed that PBC1 is more closely related to the Bacillus clarkii phage BCJA1c and phages of lactic acid bacteria than to the phages infecting B. cereus. Whole-genome comparison showed that the late-gene region, including the terminase gene, structural genes, and holin gene of PBC1, is similar to that from B. cereus temperate phage 250, whereas their endolysins are different. Compared to the extreme host specificity of PBC1, its endolysin, LysPBC1, showed a much broader lytic spectrum, albeit limited to the genus Bacillus. The catalytic domain of LysPBC1 when expressed alone also showed Bacillus-specific lytic activity, which was lower against the B. cereus group but higher against the Bacillus subtilis group than the full-length protein. Taken together, these results suggest that the virulent phage PBC1 is a useful component of a phage cocktail to control B. cereus, even with its exceptionally narrow host range, as it can kill a strain of B. cereus that is not killed by other phages, and that LysPBC1 is an alternative biocontrol agent against B. cereus.

An improved method for rapid generation and screening of Bacillus thuringiensis phage-resistant mutants.

A simple method to isolate, screen and select phage-resistant mutants of Bacillus thuringiensis was developed. The traditional double-layer agar method was improved by a combination of the spotting assay using a lytic phage, to generate the bacterial-resistant mutants, with an inverted spotting assay (ISA), to rapidly screen the candidate-resistant mutants.

The product of SPO1 gene 56 inhibits host cell division during infection of Bacillus subtilis by bacteriophage SPO1.

Although cells of Bacillus subtilis continue to grow after being infected by bacteriophage SPO1, they do not undergo cell division. The product of SPO1 gene 56 is necessary and sufficient for this inhibition of cell division. GP56 inhibits cell division when expressed in uninfected B. subtilis, without preventing cell growth, DNA synthesis or chromosome segregation, ultimately causing filamentation and loss of viability. During infection, a gene 56 mutation prevents the inhibition of cell division that occurs in wild-type infection. Under the laboratory conditions used, the gene 56 mutation did not affect burst size, latent period, or other components of the host-takeover process.

Poly-γ-(D)-glutamic acid capsule interferes with lytic infection of Bacillus anthracis by B. anthracis-specific bacteriophages.

The poly-γ-d-glutamic acid capsule of Bacillus anthracis is a barrier to infection by B. anthracis-specific bacteriophages. Capsule expression was found to completely inhibit lytic infection by γ phage, an observation supported by the demonstration that this phage does not elaborate a hydrolase that would facilitate penetration through the protective capsule outer layer.

Single-stranded DNA binding protein Gp5 of Bacillus subtilis phage Φ29 is required for viral DNA replication in growth-temperature dependent fashion.

In the absence of viral single-stranded DNA binding protein gp5, Bacillus subtilis phage φ29 failed to grow and to replicate its genome at 45 °C, while it grew and replicated normally at 30 °C and 42 °C. This indicates that gp5 is dispensable for φ29 DNA replication at 42 °C and lower temperatures.

Phage-borne factors and host LexA regulate the lytic switch in phage GIL01.

The Bacillus thuringiensis temperate phage GIL01 does not integrate into the host chromosome but exists stably as an independent linear replicon within the cell. Similar to that of the lambdoid prophages, the lytic cycle of GIL01 is induced as part of the cellular SOS response to DNA damage. However, no CI-like maintenance repressor has been detected in the phage genome, suggesting that GIL01 uses a novel mechanism to maintain lysogeny. To gain insights into the GIL01 regulatory circuit, we isolated and characterized a set of 17 clear plaque (cp) mutants that are unable to lysogenize. Two phage-encoded proteins, gp1 and gp7, are required for stable lysogen formation. Analysis of cp mutants also identified a 14-bp palindromic dinBox1 sequence within the P1-P2 promoter region that resembles the known LexA-binding site of Gram-positive bacteria. Mutations at conserved positions in dinBox1 result in a cp phenotype. Genomic analysis identified a total of three dinBox sites within GIL01 promoter regions. To investigate the possibility that the host LexA regulates GIL01, phage induction was measured in a host carrying a noncleavable lexA (Ind(-)) mutation. GIL01 formed stable lysogens in this host, but lytic growth could not be induced by treatment with mitomycin C. Also, mitomycin C induced ß-galactosidase expression from GIL01-lacZ promoter fusions, and induction was similarly blocked in the lexA (Ind(-)) mutant host. These data support a model in which host LexA binds to dinBox sequences in GIL01, repressing phage gene expression during lysogeny and providing the switch necessary to enter lytic development.

Prevalence of Bacillus anthracis-like organisms and bacteriophages in the intestinal tract of the earthworm Eisenia fetida.

Stable infection of Bacillus anthracis laboratory strains with environmental bacteriophages confers survival phenotypes in soil and earthworm intestinal niches (R. Schuch and V. A. Fischetti, PLoS One 4:e6532, 2009). Here, the natural occurrence of two such B. anthracis-infective bacteriophages, Wip1 and Wip4, was examined in the intestines of Eisenia fetida earthworms as part of a 6-year longitudinal study at a Pennsylvania forest site. The Wip1 tectivirus was initially dominant before being supplanted by the Wip4 siphovirus, which was then dominant for the next 3 years. In a host range analysis of a wide-ranging group of Bacillus species and related organisms, Wip1 and Wip4 were both infective only toward B. anthracis and certain B. cereus strains. The natural host of Wip4 remained constant for 3 years and was a B. cereus strain that expressed a B. anthracis-like surface polysaccharide at septal positions on the cell surface. Next, a novel metagenomic approach was used to determine the extent to which such B. cereus- and B. anthracis-like strains are found in worms from two geographical locations. Three different enrichment strategies were used for metagenomic DNA isolation, based either on the ability of B. cereus sensu lato to form heat-resistant spores, the sensitivity of B. anthracis to the PlyG lysin, or the selective amplification of environmental phages cocultured with B. anthracis. Findings from this work indicate that B. cereus sensu lato and its phages are common inhabitants of earthworm intestines.

A passion for research.

During my postdoctoral training in Severo Ochoa's laboratory, I determined the direction of reading of the genetic message and I discovered two proteins in Escherichia coli involved in the initiation of protein synthesis. After my return to Spain, I have been working with the Bacillus subtilis phage varphi29. We discovered a protein covalently linked to the 5' DNA ends that is the primer for the initiation of varphi29 DNA replication. We also found that the phage-encoded DNA polymerase has unique properties such as processivity and strand displacement activity. These properties, in addition to its high fidelity, have made the varphi29 DNA polymerase the ideal enzyme for DNA amplification, both for rolling circle and whole-genome amplification. I am happy to say that the work carried out in my laboratory has been possible thanks to many brilliant students and collaborators, most of whom have become high quality independent scientists.

Thermal, chemical, and photocatalytic inactivation of Lactobacillus plantarum bacteriophages.

The effect of several biocides, thermal treatments, and photocatalysis on the viability of four Lactobacillus plantarum phages was investigated. Times to achieve 99% inactivation (T99) of phages at 63, 72, and 90 degrees C were evaluated in four suspension media: deMan Rogosa Sharpe broth, reconstituted skim milk, a commercial EM-glucose medium, and Tris magnesium gelatin buffer. The four phages studied were highly resistant to 63 degrees C (T99 > 45 min); however, counts < 10 PFU/ml were achieved by heating at 90 degrees C for 5 min. Higher thermal resistance at 72 degrees C was observed when reconstituted skim milk and EM-glucose medium were assayed. Peracetic acid (0.15%, vol/vol) was an effective biocide for the complete inactivation of all phages studied within 5 min of exposure. Sodium hypochlorite (800 ppm) inactivated the phages completely within 30 min. Ethanol (100%) did not destroy phage particles even after 45 min. Isopropanol did not have any effect on phage viability. Phage counts < 50 PFU/ml were obtained within 180 min of photocatalytic treatment. The results obtained in this work are important for establishing adequate methods for inactivating phages in industrial plants and laboratory environments.

Different responses to Spo0A-mediated suppression of the related Bacillus subtilis phages Nf and phi29.

The phi29 family of phages is divided in three groups. Members of groups 1 and 2 infect the spore-forming bacterium Bacillus subtilis. Previous studies showed that group 1 phage phi29 adapts its infection strategy to the physiological state of the host. Thus, the lytic cycle of phi29 is suppressed when cells are infected during the early stages of sporulation and the infecting genome becomes trapped into the spore. A major element of this adaptive strategy is a very sensitive response to the host-encoded Spo0A protein, the key regulator for sporulation activation, which is directly responsible for suppression of phi29 development. Here we analysed if this adaptation is conserved in phage Nf belonging to group 2. The results obtained show that although Nf also possesses the alternative infection strategy, it is clearly less sensitive to Spo0A-mediated suppression than phi29. Sequence determination of the Nf genome revealed striking differences in the number of Spo0A binding site sequences. The results provide evidence that the life style of two highly related phages is distinctly tuned by differences in binding sites for a host-encoded regulatory protein, being a good example of how viruses have evolved to optimally exploit features of their host.

Phage phi29 protein p56 prevents viral DNA replication impairment caused by uracil excision activity of uracil-DNA glycosylase.

Protein p56 encoded by the Bacillus subtilis phage phi29 inhibits host uracil-DNA glycosylase (UDG) activity. In previous studies, we suggested that this inhibition is likely a defense mechanism developed by phage phi29 to prevent the action of UDG if uracilation occurs in DNA either from deamination of cytosine or the incorporation of dUMP during viral DNA replication. In this work, we analyzed the ability of phi29 DNA polymerase to insert dUMP into DNA. Primer extension analysis showed that viral DNA polymerase incorporates dU opposite dA with a catalytic efficiency only 2-fold lower than that for dT. Using the phi29 DNA amplification system, we found that phi29 DNA polymerase is also able to carry out the extension of the dA:dUMP pair and replicate past uracil. Additionally, UDG and apurinic-apyrimidinic endonuclease treatment of viral DNA isolated from phi29-infected cells revealed that uracil residues arise in phi29 DNA during replication, probably as a result of misincorporation of dUMP by the phi29 DNA polymerase. On the other hand, the action of UDG on uracil-containing phi29 DNA impaired in vitro viral DNA replication, which was prevented by the presence of protein p56. Furthermore, transfection activity of uracil-containing phi29 DNA was significantly higher in cells that constitutively synthesized p56 than in cells lacking this protein. Thus, our data support a model in which protein p56 ensures an efficient viral DNA replication, preventing the deleterious effect caused by UDG when it eliminates uracil residues present in the phi29 genome.