On-line monitoring of changes in host cell physiology during the one-step growth cycle of Bacillus phage Bam35.

In this study an on-line electrochemical method was developed to examine the one-step growth cycle (OSGC) of the bacteriophage Bam35. The on-line conditions for monitoring the OSGC and the effect of aeration on the duration of the OSGC were defined. The data indicate that binding of phenyldicarbaundecaborane anions to Bacillus thuringiensis cells infected with Bam35 can be used as a sensitive indicator of cell lysis.

Bacteria, phages and septicemia.

The use of phages is an attractive option to battle antibiotic resistant bacteria in certain bacterial infections, but the role of phage ecology in bacterial infections is obscure. Here we surveyed the phage ecology in septicemia, the most severe type of bacterial infection. We observed that the majority of the bacterial isolates from septicemia patients spontaneously secreted phages active against other isolates of the same bacterial strain, but not to the strain causing the disease. Such phages were also detected in the initial blood cultures, indicating that phages are circulating in the blood at the onset of sepsis. The fact that most of the septicemic bacterial isolates carry functional prophages suggests an active role of phages in bacterial infections. Apparently, prophages present in sepsis-causing bacterial clones play a role in clonal selection during bacterial invasion.

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 linear double-stranded DNA of phage Bam35 enters lysogenic host cells, but the late phage functions are suppressed.

Bam35, a temperate double-stranded DNA bacteriophage with a 15-kb linear genome, infects gram-positive Bacillus thuringiensis cells. Bam35 morphology and genome organization resemble those of PRD1, a lytic phage infecting gram-negative bacteria. Bam35 and PRD1 have an outer protein coat surrounding a membrane that encloses the viral DNA. We used electrochemical methods to investigate physiological changes of the lysogenic and nonlysogenic hosts during Bam35 DNA entry and host cell lysis. During viral DNA entry, there was an early temporal decrease of membrane voltage associated with K+ efflux that took place when either lysogenic or nonlysogenic hosts were infected. Approximately 40 min postinfection, a second strong K+ efflux was registered that was proposed to be associated with the insertion of holin molecules into the plasma membrane. This phenomenon occurred only when nonlysogenic cells were infected. Lysogenic hosts rarely were observed entering the lytic cycle as demonstrated by thin-section electron microscopy.

Penetration of enveloped double-stranded RNA bacteriophages phi13 and phi6 into Pseudomonas syringae cells.

Bacteriophages phi6 and phi13 are related enveloped double-stranded RNA viruses that infect gram-negative Pseudomonas syringae cells. phi6 uses a pilus as a receptor, and phi13 attaches to the host lipopolysaccharide. We compared the entry-related events of these two viruses, including receptor binding, envelope fusion, peptidoglycan penetration, and passage through the plasma membrane. The infection-related events are dependent on the multiplicity of infection in the case of phi13 but not with phi6. A temporal increase of host outer membrane permeability to lipophilic ions was observed from 1.5 to 4 min postinfection in both virus infections. This enhanced permeability period coincided with the fast dilution of octadecyl rhodamine B-labeled virus-associated lipid molecules. This result is in agreement with membrane fusion, and the presence of temporal virus-derived membrane patches on the outer membrane. Similar to phi6, phi13 contains a thermosensitive lytic enzyme involved in peptidoglycan penetration. The phage entry also caused a limited depolarization of the plasma membrane. Inhibition of host respiration considerably decreased the efficiency of irreversible virus binding and membrane fusion. An active role of cell energy metabolism in restoring the infection-induced defects in the cell envelope was also observed.

Comparative analysis of bacterial viruses Bam35, infecting a gram-positive host, and PRD1, infecting gram-negative hosts, demonstrates a viral lineage.

Extra- and intracellular viruses in the biosphere outnumber their cellular hosts by at least one order of magnitude. How is this enormous domain of viruses organized? Sampling of the virosphere has been scarce and focused on viruses infecting humans, cultivated plants, and animals as well as those infecting well-studied bacteria. It has been relatively easy to cluster closely related viruses based on their genome sequences. However, it has been impossible to establish long-range evolutionary relationships as sequence homology diminishes. Recent advances in the evaluation of virus architecture by high-resolution structural analysis and elucidation of viral functions have allowed new opportunities for establishment of possible long-range phylogenic relationships-virus lineages. Here, we use a genomic approach to investigate a proposed virus lineage formed by bacteriophage PRD1, infecting gram-negative bacteria, and human adenovirus. The new member of this proposed lineage, bacteriophage Bam35, is morphologically indistinguishable from PRD1. It infects gram-positive hosts that evolutionarily separated from gram-negative bacteria more than one billion years ago. For example, it can be inferred from structural analysis of the coat protein sequence that the fold is very similar to that of PRD1. This and other observations made here support the idea that a common early ancestor for Bam35, PRD1, and adenoviruses existed.

Two distinct mechanisms ensure transcriptional polarity in double-stranded RNA bacteriophages.

In most double-stranded RNA (dsRNA) viruses, RNA transcription occurs inside a polymerase (Pol) complex particle, which contains an RNA-dependent RNA Pol subunit as a minor component. Only plus- but not minus-sense copies of genomic segments are produced during this reaction. In the case of phi6, a dsRNA bacteriophage from the Cystoviridae family, isolated Pol synthesizes predominantly plus strands using virus-specific dsRNAs in vitro, thus suggesting that Pol template preferences determine the transcriptional polarity. Here, we dissect transcription reactions catalyzed by Pol complexes and Pol subunits of two other cystoviruses, phi8 and phi13. While both Pol complexes synthesize exclusively plus strands over a wide range of conditions, isolated Pol subunits can be stimulated by Mn(2+) to produce minus-sense copies on phi13 dsRNA templates. Importantly, all three Pol subunits become more prone to the native-like plus-strand synthesis when the dsRNA templates (including phi13 dsRNA) are activated by denaturation before the reaction. Based on these and earlier observations, we propose a model of transcriptional polarity in Cystoviridae controlled on two independent levels: Pol affinity to plus-strand initiation sites and accessibility of these sites to the Pol in a single-stranded form.