The entry mechanism of membrane-containing phage Bam35 infecting Bacillus thuringiensis

The temperate double-stranded DNA bacteriophage Bam35 infects gram-positive Bacillus thuringiensis cells. Bam35 has an icosahedral protein coat surrounding the viral membrane that encloses the linear 15-kbp DNA genome. The protein coat of Bam35 uses the same assembly principle as that of PRD1, a lytic bacteriophage infecting gram-negative hosts. In this study, we dissected the process of Bam35 entry into discrete steps: receptor binding, peptidoglycan penetration, and interaction with the plasma membrane (PM). Bam35 very rapidly adsorbs to the cell surface, and N-acetyl-muramic acid is essential for Bam35 binding. Zymogram analysis demonstrated that peptidoglycan-hydrolyzing activity is associated with the Bam35 virion. We showed that the penetration of Bam35 through the PM is a divalent-cation-dependent process, whereas adsorption and peptidoglycan digestion are not.     

The Holin protein of bacteriophage PRD1 forms a pore for small-molecule and endolysin translocation

PRD1 is a bacteriophage with an icosahedral outer protein layer surrounding the viral membrane, which encloses the linear double-stranded DNA genome. PRD1 infects gram-negative cells harboring a conjugative IncP plasmid. Here we studied the lytic functions of PRD1. Using infected cells and plasmid-borne lysis genes, we demonstrated that a two-component lysis system (holin-endolysin) operates to release progeny phage particles from the host cell. Monitoring of ion fluxes and the ATP content of the infected cells allowed us to build a model of the sequence of lysis-related physiological changes. A decrease in the intracellular level of ATP is the earliest indicator of cell lysis, followed by the leakage of K+ from the cytosol approximately 20 min prior to the decrease in culture turbidity. However, the K+ efflux does not immediately lead to the depolarization of the cytoplasmic membrane or leakage of the intracellular ATP. These effects are observed only approximately 5 to 10 min prior to cell lysis. Similar results were obtained using cells expressing the holin and endolysin genes from plasmids.     

A snapshot of viral evolution from genome analysis of the tectiviridae family

The origin, evolution and relationships of viruses are all fascinating topics. Current thinking in these areas is strongly influenced by the tailed double-stranded (ds) DNA bacteriophages. These viruses have mosaic genomes produced by genetic exchange and so new natural isolates are quite dissimilar to each other, and to laboratory strains. Consequently, they are not amenable to study by current tools for phylogenetic analysis. Less attention has been paid to the Tectiviridae family, which embraces icosahedral dsDNA bacterial viruses with an internal lipid membrane. It includes viruses, such as PRD1, that infect Gram-negative bacteria, as well as viruses like Bam35 with Gram-positive hosts. Although PRD1 and Bam35 have closely related virion morphology and genome organization, they have no detectable sequence similarity. There is strong evidence that the Bam35 coat protein has the "double-barrel trimer" arrangement of PRD1 that was first observed in adenovirus and is predicted to occur in other viruses with large facets. It is very likely that a single ancestral virus gave rise to this very large group of viruses. The unprecedented degree of conservation recently observed for two Bam35-like tectiviruses made it important to investigate those infecting Gram-negative bacteria. The DNA sequences for six PRD1-like isolates (PRD1, PR3, PR4, PR5, L17, PR772) have now been determined. Remarkably, these bacteriophages, isolated at distinctly different dates and global locations, have almost identical genomes. The discovery of almost invariant genomes for the two main Tectiviridae groups contrasts sharply with the situation in the tailed dsDNA bacteriophages. Notably, it permits a sequence analysis of the isolates revealing that the tectiviral proteins can be dissected into a slowly evolving group descended from the ancestor, the viral self, and a more rapidly changing group reflecting interactions with the host.     

Seasonal variations of phage life strategies and bacterial physiological states in three northern temperate lakes

The current consensus concerning the prevalence of lytic and lysogenic phage life cycles in aquatic systems is that the host physiological state may influence viral strategies, lysogeny being favoured when hosts have reduced metabolic rates. We explored this hypothesis, by following phage cycle dynamics, host physiological state and metabolic activity over an annual cycle in three lakes subjected to strong seasonal fluctuations, including 4–5 months of ice cover. We observed marked seasonal dynamics of viral and bacterial communities, with low bulk and cell‐specific bacterial metabolism in winter, and a dramatic increase in injured bacteria under the ice cover in all lakes. This period was accompanied by contrasting patterns in the proportion of lysogenic cells. In the eutrophic lake, times of low bacterial metabolic rates and high proportion of damaged cells corresponded to highest levels of lysogeny, supporting the notion that hosts are a ‘refuge’ for viruses. In the two unproductive lakes, peaks of injured cells corresponded to a minimum of lysogeny, suggesting an ‘abandon the sinking ship’ response, where the prophage replicates before the loss of genome. We suggest that these diverging responses to the host physiological state are not contradictory, but rather that there may be thresholds of cell stress and metabolic activity leading to one or the other response.     

GIL16, a new gram-positive tectiviral phage related to the Bacillus thuringiensis GIL01 and the Bacillus cereus pBClin15 elements

One of the most notable characteristics of Tectiviridae resides in their double-layer coats: the double-stranded DNA is located within a flexible lipoprotein vesicle covered by a rigid protein capsid. Despite their apparent rarity, tectiviruses have an extremely wide distribution compared to other phage groups. Members of this family have been found to infect gram-negative (PRD1 and relatives) as well as gram-positive (Bam35, GIL01, AP50, and phiNS11) hosts. Several reports have shown that tectiviruses infecting gram-negative bacteria are closely related, whereas no information is currently available on the genetic relationship among those infecting gram-positive bacteria. The present study reports the sequence of GIL16, a new isolate originating from Bacillus thuringiensis, and a genetic comparison of this isolate with the tectiviral bacteriophages Bam35 and GIL01, which originated from B. thuringiensis serovars Alesti and Israelensis, respectively. In contrast to PRD1 and its relatives, these are temperate bacteriophages existing as autonomous linear prophages within the host cell. Mutations in a particular motif in both the GIL01 and GIL16 phages are also shown to correlate with a switch to the lytic cycle. Interestingly, both bacterial viruses displayed narrow, yet slightly different, host spectrums. We also explore the hypothesis that pBClin15, a linear plasmid hosted by the Bacillus cereus reference strain ATCC 14579, is also a prophage. Sequencing of its inverted repeats at both extremities and a comparison with GIL01 and GIL16 emphasize its relationship to the Tectiviridae.     

Isolation of nonlysogenic bacteria from lambda-lysogenized strains using antibiotic-resistant phage

A simple and efficient method for obtaining nonlysogenic bacteria from lambda-lysogenized strains using phage with an inserted antibiotic-resistant plasmid is described. When the lysogenic culture is infected with antibiotic-resistant phage, single non-lysogenic cells are lysogenized, isolated on selective medium, and cured of phage during incubation at 37 degrees C.     

Isolation of nonlysogenic bacteria from lysogenic ones using a phage carrying resistance to antibiotics

The method for obtaining nonlysogenic bacteria from lysogenic ones by means of a phage carrying resistance to antibiotics is proposed. Solitary nonlysogenic cells in a lysogenic culture are lysogenized after the infection of the culture with a labeled phage and then harvested on a selective medium: under special conditions the phage is eliminated from the cells.     

Transformation and Transfection in Lysogenic Strains of Bacillus subtilis 168

Strains of Bacillus subtilis lysogenic for either temperate bacteriophage phi105 or SPO2 were reduced to less than 1.0% of the level of transformation of the nonlysogenic strains. Strains lysogenic for both phi105 and SPO2 are virtually nontransformable, indicating that the effect of lysogeny is additive. Lysogenic cultures transfected at essentially wild-type levels with deoxyribonucleic acid (DNA) isolated from bacteriophages phi29 and SPO1. The residual transformation and transfection achieved by the lysogenic cultures changed dramatically during growth in SPII medium, whereas nonlysogenic strains remained competent for 5 hr in SPII medium. Despite a marked reduction in transformation, lysogenic cultures initially irreversibly bound as much DNA as nonlysogenic cultures. After 60 min in SPII medium, there was a rapid decrease in the capacity of lysogenic cells to bind DNA irreversibly. These results, as discussed, indicate that the inhibition of transformation is probably due to an alteration of the cell surface or a differential inactivation of bacterial genes after lysogenic conversion.     

The Lytic Enzyme of Bacteriophage PRD1 Is Associated with the Viral Membrane

Bacteriophage PRD1 encodes two proteins (P7 and P15) that are associated with a muralytic activity. Protein P15 is a soluble beta-1,4-N-acetylmuramidase that causes phage-induced host cell lysis. We demonstrate here that P15 is also a structural component of the PRD1 virion and that it is connected to the phage membrane. Small viral membrane proteins P20 and P22 modulate incorporation of P15 into the virion and may connect it to the phage membrane. The principal muralytic protein involved in PRD1 DNA entry seems to be the putative lytic transglycosylase protein P7, as the absence of protein P15 did not delay initiation of phage DNA replication in the virus-host system used. The incorporation of two different lytic enzymes into virions may reflect the broad host range of bacteriophage PRD1.     

In vitro DNA packaging of PRD1: a common mechanism for internal-membrane viruses

PRD1 is the type virus of the Tectiviridae family. Its linear double-stranded DNA genome has covalently attached terminal proteins and is surrounded by a membrane, which is further enclosed within an icosahedral protein capsid. Similar to tailed bacteriophages, PRD1 packages its DNA into a preformed procapsid. The PRD1 putative packaging ATPase P9 is a structural protein located at a unique vertex of the capsid. An in vitro system for packaging DNA into preformed empty procapsids was developed. The system uses cell extracts of overexpressed P9 protein and empty procapsids from a P9-deficient mutant virus infection and PRD1 DNA containing a LacZalpha-insert. The in vitro packaged virions produce distinctly blue plaques when plated on a suitable host. This is the first time that a viral genome is packaged in vitro into a membrane vesicle. Comparison of PRD1 P9 with putative packaging ATPase sequences from bacterial, archaeal and eukaryotic viruses revealed a new packaging ATPase-specific motif. Surprisingly the viruses having this packaging ATPase motif, and thus considered to be related, were the same as those recently grouped together using the coat protein fold and virion architecture. Our finding here strongly supports the idea that all these viruses infecting hosts in all domains of life had a common ancestor.     

Changes in host cell energetics in response to bacteriophage PRD1 DNA entry.

Double-stranded DNA bacteriophage PRD1 infects a variety of gram-negative bacteria harboring an IncP-type conjugative plasmid. The plasmid codes for the DNA transfer phage receptor complex in the cell envelope. Our goal was, by using a collection of mutant phage particles for which the variables are the DNA content and/or the presence of the receptor-binding protein, to obtain information on the energy requirements for DNA entry as well as on alterations in the cellular energetics taking place during the first stages of infection. We studied the fluxes of tetraphenylphosphonium (TPP+), phenyldicarbaundecaborane (PCB-), and K+ ions as well as ATP through the envelope of Salmonella typhimurium cells. The final level of the membrane voltage (delta psi) indicator TPP+ accumulated by the infected cells exceeds the initial level before the infection. Besides the effects on TPP+ accumulation, PRD1 induces the leakage of ATP and K+ from the cytosol. All these events were induced only by DNA-containing infectious particles and were cellular ATP and delta psi dependent. PRD1-caused changes in delta psi and in PCB- binding differ considerably from those observed in other bacteriophage infections studied. These results are in accordance with the presence of a specific channel engaged in phage PRD1 DNA transport.     

Sequential model of phage PRD1 DNA delivery: active involvement of the viral membrane

DNA translocation across the barriers of recipient cells is not well understood. Viral DNA delivery mechanisms offer an opportunity to obtain useful information in systems in which the process can be arrested to a number of stages. PRD1 is an icosahedral double-stranded (ds)DNA bacterial virus with an internal membrane. It is an atypical dsDNA phage, as any of the vertex spikes can be used for receptor recognition. In this report, we dissect the PRD1 DNA entry into a number of steps: (i) outer membrane (OM) penetration; (ii) peptidoglycan digestion; (iii) cytoplasmic membrane (CM) penetration; and (iv) DNA translocation. We present a model for PRD1 DNA entry proposing that the initial stage of entry is powered by the pressure build-up during DNA packaging. The viral protein P11 is shown to function as the first DNA delivery protein needed to penetrate the OM. We also report a DNA translocation machinery composed of at least three viral integral membrane proteins, P14, P18 and P32.     

Temperate Myxococcus xanthus phage Mx8 encodes a DNA adenine methylase, Mox.

Temperate bacteriophage Mx8 of Myxococcus xanthus encapsidates terminally repetitious DNA, packaged as circular permutations of its 49-kbp genome. During both lytic and lysogenic development, Mx8 expresses a nonessential DNA methylase, Mox, which modifies adenine residues in occurrences of XhoI and PstI recognition sites, CTCGAG and CTGCAG, respectively, on both phage DNA and the host chromosome. The mox gene is necessary for methylase activity in vivo, because an amber mutation in the mox gene abolishes activity. The mox gene is the only phage gene required for methylase activity in vivo, because ectopic expression of mox as part of the M. xanthus mglBA operon results in partial methylation of the host chromosome. The predicted amino acid sequence of Mox is related most closely to that of the methylase involved in the cell cycle control of Caulobacter crescentus. We speculate that Mox acts to protect Mx8 phage DNA against restriction upon infection of a subset of natural M. xanthus hosts. One natural isolate of M. xanthus, the lysogenic source of related phage Mx81, produces a restriction endonuclease with the cleavage specificity of endonuclease BstBI.     

Characterization of ion channels involved in the penetration of phage T4 DNA into Escherichia coli cells

The hypothesis of a channel-mediated transport of phage DNA into Escherichia coli cytoplasmic membrane has been formulated for a long time. In this paper, we present experimental evidence in favor of this proposal. We have analyzed the kinetics of the K+ efflux induced by T4 phage and ghosts (phage depleted of DNA) using a potassium selective electrode. We show that the K+ efflux is not catalyzed by the K+ transport systems. The Km of K+ efflux is the same for phage and ghosts. The rate of K+ efflux is linearly related to the multiplicity of infection. This suggests that phage and ghosts induce the formation of similar channels and that one channel is induced by one virion. The K+ efflux is associated with an influx of H+ and Na+ or Li+ which compete for entry through the channel. These ion fluxes may be responsible for the cell depolarization. The phage-induced channels allow the passage of DNA. They are only transiently opened, and their closing leads to cellular repolarization. The ghost-induced channels remain open. The insertion and conformation of the channels in the membrane depend on the temperature and their confirmation is voltage-dependent. We give an estimate of their size.     

Molecular basis for the exploitation of spore formation as survival mechanism by virulent phage phi29

Phage phi29 is a virulent phage of Bacillus subtilis with no known lysogenic cycle. Indeed, lysis occurs rapidly following infection of vegetative cells. Here, we show that phi29 possesses a powerful strategy that enables it to adapt its infection strategy to the physiological conditions of the infected host to optimize its survival and proliferation. Thus, the lytic cycle is suppressed when the infected cell has initiated the process of sporulation and the infecting phage genome is directed into the highly resistant spore to remain dormant until germination of the spore. We have also identified two host-encoded factors that are key players in this adaptive infection strategy. We present evidence that chromosome segregation protein Spo0J is involved in spore entrapment of the infected phi29 genome. In addition, we demonstrate that Spo0A, the master regulator for initiation of sporulation, suppresses phi29 development by repressing the main early phi29 promoters via different and novel mechanisms and also by preventing activation of the single late phi29 promoter.     

Resources drive trade‐off between viral lifestyles in the plankton: evidence from freshwater microbial microcosms

Viruses have evolved different life strategies for coping with environmental challenges and this is a key explanation for their omnipresence in aquatic systems. However, factors that determine the balance between lytic versus lysogenic decision within natural virioplankton are poorly documented, primarily in freshwaters. This study was designed to investigate the experimental short‐term (24 h incubation) effects of added organic and inorganic nutrients on the two viral lifestyles in nutrient‐depleted freshwater microbial (i.e. < 0.8 μm fraction) microcosms, using mitomycin C as prophage inductor agent. In the absence of mitomycin, viral lytic production increased as a functional response to the strong stimulation of bacterial growth rates (0.7–0.8 day^?1) by the added nutrients, primarily the organic nutrients which appeared scarcer than inorganic nutrients and was related to the sampling period and the geomorphological peculiarities of Lake Pavin. In the presence of mitomycin, temperate phage production (frequency of lysogenically infected bacterial cells, FLC = 17–19% of total cells) significantly exceeded lytic production (frequency of lytically infected bacterial cells, FIC = 9–11%) in natural samples (i.e. without nutrient additions) as a result of enhanced prophage induction, which relatively increased with the decreasing contact probability between viruses and their potential hosts. In contrast, addition of nutrients drastically reduced FLC (< 4%) and increased FIC (> 22%). Both variables were antagonistically correlated as was the correlation between FLC and bacterial growth rates, supporting the idea that lysogeny may represent a maintenance strategy for viruses in harsh nutrient/host conditions which appeared as major instigators of the trade‐off between the two viral lifestyles. Overall, at the community level, we reject the hypothesis that nutrients but mitomicyn C stimulate temperate phage induction, and retained the hypotheses that nutrients rather (i) stimulate lytic viruses via enhanced host growth and (ii) when limiting, promote lysogenic conversion in natural waters.     

Participation of temperate phages LP52 and BL20 in the control of bacitracin synthesis in Bacillus licheniformis

The synthesis of the antibiotic bacitracin in lysogenic and nonlysogenic strains of Bacillus licheniformis 1001 and ATCC10716 has been studied. The antibiotic activity was shown to be about 20% less in lysogens, as compared to nonlysogens. However, the level of bacitracin production was completely restored when temperate bacteriophages BL20 and LP52 were reintroduced into the nonlysogenic strains by virtue of genetic transformation with DNA from lysogenic strains or by transduction with LP52. This may indicate that both phages take part in control of the synthesis of bacitracin. For the time being, the mechanism of regulation is not known. It is likely to be either direct (provided that prophage DNA contains "bacitracin" genes), or indirect.