Mechanisms of coexistence of a bacteria and a bacteriophage in a spatially homogeneous environment

When bacteriophage are added to laboratory bacteria populations, bacteria mutants that are resistant to the phage quickly dominate the population. The phage will only persist in the long‐term if there are sufficient bacteria in the population that show susceptibility to the phage. We investigated the mechanisms allowing for coexistence by adding the virulent bacteriophage φ6 to cultures of the bacterium Pseudomonas syringae pv. phaseolicola in a spatially homogeneous environment. We saw large differences between replicate cultures, in particular when one or both of the species persisted. These differences can be explained by variation in the timing of the appearance of various resistant phenotypes in the bacteria populations before the phage were added, which determines their relative frequencies within the populations. Although these resistant phenotypes have similar fitnesses in the presence and in the absence of the phage, they have a profound effect on the persistence of the phage. Our results give a clearer understanding of the ecological mechanisms that lead to the coexistence of bacteria and virulent phage in environments where there are no spatial refuges available to the bacteria population.     

The tripartite associations between bacteriophage, Wolbachia, and arthropods

By manipulating arthropod reproduction worldwide, the heritable endosymbiont Wolbachia has spread to pandemic levels. Little is known about the microbial basis of cytoplasmic incompatibility (CI) except that bacterial densities and percentages of infected sperm cysts associate with incompatibility strength. The recent discovery of a temperate bacteriophage (WO-B) of Wolbachia containing ankyrin-encoding genes and virulence factors has led to intensifying debate that bacteriophage WO-B induces CI. However, current hypotheses have not considered the separate roles that lytic and lysogenic phage might have on bacterial fitness and phenotype. Here we describe a set of quantitative approaches to characterize phage densities and its associations with bacterial densities and CI. We enumerated genome copy number of phage WO-B and Wolbachia and CI penetrance in supergroup A- and B-infected males of the parasitoid wasp Nasonia vitripennis. We report several findings: (1) variability in CI strength for A-infected males is positively associated with bacterial densities, as expected under the bacterial density model of CI, (2) phage and bacterial densities have a significant inverse association, as expected for an active lytic infection, and (3) CI strength and phage densities are inversely related in A-infected males; similarly, males expressing incomplete CI have significantly higher phage densities than males expressing complete CI. Ultrastructural analyses indicate that approximately 12% of the A Wolbachia have phage particles, and aggregations of these particles can putatively occur outside the Wolbachia cell. Physical interactions were observed between approximately 16% of the Wolbachia cells and spermatid tails. The results support a low to moderate frequency of lytic development in Wolbachia and an overall negative density relationship between bacteriophage and Wolbachia. The findings motivate a novel phage density model of CI in which lytic phage repress Wolbachia densities and therefore reproductive parasitism. We conclude that phage, Wolbachia, and arthropods form a tripartite symbiotic association in which all three are integral to understanding the biology of this widespread endosymbiosis. Clarifying the roles of lytic and lysogenic phage development in Wolbachia biology will effectively structure inquiries into this research topic.     

Dynamics of the interaction between Pseudomonas aeruginosa bacteriophage phimF81 and host bacteria]

The population interactions of Pseudomonas aeruginosa virulent bacteriophage phi mF81 with host bacterial cells were studied in dynamics under the conditions of continuous cultivation in the chemostat regime with glucose limitation. It was detected that a maintenance of the bacterium and its specific bacteriophage in the population was realized due to the successive appearance of bacterial mutants resistant to the phage and of phage mutants overcoming this resistance.     

Phage-plasmid interactions in bacteria]

A review is devoted to consideration of the basic types of interaction of moderate and virulent bacteriophages and extrachromosomal elements in bacterial cells. Various experimental data confirm diversity of such interactions as well as resistance of bacteria to productive phage infection. Role of plasmids in such processes as abortion infection, lysogenic state, reparation of phage DNA as well as the effect of plasmids on the basic stages of phage development in the cells are analyzed. Possible mechanisms for limiting development of phages as a result of plasmids' presence in bacterial cells are considered.     

Genetical changes in rhizobium bacteria and in their bacteriophages during coexistence

Summary Three systems, each consisting of a virulent phage and a susceptible bacterium, were incubated for 24 weeks in liquid culture and the processes of interaction and genetical change in both partners investigated. The initial stage of interaction in all systems was identical; all susceptible bacterial cells were lysed and only cells that originally were resistant or mutated to resistance, remained alive. In two systems the multiplication of resistant cells was not restricted by large concentration of phage; in the third there was no bacterial multiplication. Phage concentration was maintained at a high titre for the duration of the experiment by the presence of the resistant mutant bacteria. Phage resistant mutants were genetically less stable than the parent forms; some also had changed colony morphology and symbiotic properties. Mutations in these different characteristics were independent. In two systems virulent phage mutated into temperate form; hence some of the phage-resistant mutant cells were genetically suitable for establishment of lysogeny.     

Coevolution of bacteria and phage: Are there endless cycles of bacterial defenses and phage counterdefenses?

The assertion that the coevolution of bacteria and bacteriophage leads to an endless arms race between resistant bacterial mutants and corresponding host-range phage mutants is questioned. In general, structural constraints on the highly site-specific phage adsorption process appear more severe than physiological constraints on resource assimilation by bacteria. Several alternative hypotheses are presented that could account for the persistence of phage, despite this fundamental asymmetry in the coevolutionary potential of bacteria and phage.     

Evidence that Bacillus subtilis Bacteriophage SPO2 is Temperate and Heteroimmune to Bacteriophage φ105

Some characteristics of Bacillus subtilis phage SPO2 which show that it is a temperate phage are presented. Wild-type SPO2 forms turbid plaques, similar to those of other temperate phages. SPO2 lysogenic strains which are resistant to SPO2 can be isolated; these strains remain stable lysogens despite the fact that they can no longer adsorb SPO2. SPO2 lysogenic strains can be grown for many generations in SPO2 antiserum and remain lysogenic. Phage SPO2 plates on phi105 lysogens and phage phi105 plates on SPO2 lysogens; this indicates that SPO2 and phi105 are heteroimmune. Phage phi105 plates on an SPO2-resistant strain; this indicates that SPO2 and phi105 adsorb to different receptor sites on the bacterial surface.     

Use of Real-Time Quantitative PCR for the Analysis of φLC3 Prophage Stability in Lactococci

Bacteriophages are a common and constant threat to proper milk fermentation. It has become evident that lysogeny is widespread in lactic acid bacteria, and in this work the temperate lactococcal bacteriophage LC3 was used as a model to study prophage stability in lactococci. The stability was analyzed in six LC3 lysogenic Lactococcus lactis subsp. cremoris host strains when they were growing at 15 and 30°C. In order to perform these analyses, a real-time PCR assay was developed. The stability of the LC3 prophage was found to vary with the growth phase of its host L. lactis IMN-C1814, in which the induction rate increased during the exponential growth phase and reached a maximum level when the strain was entering the stationary phase. The maximum spontaneous induction frequency of the LC3 prophage varied between 0.32 and 9.1% (28-fold) in the six lysogenic strains. No correlation was observed between growth rates of the host cells and the spontaneous prophage induction frequencies. Furthermore, the level of extrachromosomal phage DNA after induction of the prophage varied between the strains (1.9 to 390%), and the estimated burst sizes varied up to eightfold. These results show that the host cells have a significant impact on the lytic and lysogenic life styles of temperate bacteriophages. The present study shows the power of the real-time PCR technique in the analysis of temperate phage biology and will be useful in work to reveal the impact of temperate phages and lysogenic bacteria in various ecological fields.     

Potential Significance of Lysogeny to Bacteriophage Production and Bacterial Mortality in Coastal Waters of the Gulf of Mexico

The potential effect that induction of lysogenic bacteria has on bacteriophage production and bacterial mortality in coastal waters was investigated, and we present estimates for the percentage of lysogenic cells in a natural aquatic bacterial community. Various concentrations of mitomycin C and exposure times to UV C radiation (UV-C) (wavelength of 254 nm) were used to induce the lytic cycle in lysogenic cells of natural communities of marine bacteria. UV-C treatment occasionally resulted in phage production, but phage production induced by UV-C was always less than that caused by the addition of mitomycin C. There was no evidence that high growth rates of bacteria resulted in lysogenic phage production. The burst size of cells induced by mitomycin C was determined by transmission electron microscopy and ranged from 11 to 45. Dividing the induced phage production by the burst size provided an estimate of the number of lysogenic bacterial cells, which ranged from 0.07 to 4.4% (average, 1.5%) of the total bacterial population. The percentages of lysogenic bacteria that were induced by mitomycin C were similar for samples collected nearshore from the pier of the Marine Science Institute (chlorophyll a, 1.6 to 2.9 (mu)g liter(sup-1)) and in relatively oligotrophic water (chlorophyll a, 0.2 to 0.9 (mu)g liter(sup-1)) collected 25 to 100 km offshore. By using a steady-state model, if all lysogenic bacteria were induced simultaneously, 0.14 to 8.8% (average, 3.0%) of the total bacterial mortality would result from induction of lysogenic cells. If mitomycin C induces all or the majority of lysogenized cells, our results imply that lysogenic phage production is generally not an important source of phage production or bacterial mortality in the coastal waters of the western Gulf of Mexico.     

Use of real-time quantitative PCR for the analysis of phiLC3 prophage stability in lactococci

Bacteriophages are a common and constant threat to proper milk fermentation. It has become evident that lysogeny is widespread in lactic acid bacteria, and in this work the temperate lactococcal bacteriophage phi LC3 was used as a model to study prophage stability in lactococci. The stability was analyzed in six phi LC3 lysogenic Lactococcus lactis subsp. cremoris host strains when they were growing at 15 and 30 degrees C. In order to perform these analyses, a real-time PCR assay was developed. The stability of the phi LC3 prophage was found to vary with the growth phase of its host L. lactis IMN-C1814, in which the induction rate increased during the exponential growth phase and reached a maximum level when the strain was entering the stationary phase. The maximum spontaneous induction frequency of the phi LC3 prophage varied between 0.32 and 9.1% (28-fold) in the six lysogenic strains. No correlation was observed between growth rates of the host cells and the spontaneous prophage induction frequencies. Furthermore, the level of extrachromosomal phage DNA after induction of the prophage varied between the strains (1.9 to 390%), and the estimated burst sizes varied up to eightfold. These results show that the host cells have a significant impact on the lytic and lysogenic life styles of temperate bacteriophages. The present study shows the power of the real-time PCR technique in the analysis of temperate phage biology and will be useful in work to reveal the impact of temperate phages and lysogenic bacteria in various ecological fields.     

Isolation, characterization and mapping of the N15 phasmid insertion mutations]

Prophage of N15 temperate bacteriophage is stably maintained in Escherichia coli lysogens as a 46.33 kb linear plasmid. Using different transposons we obtained 18 insertion mutants of the N15 plasmid prophage. They were analysed for plaque formation ability, stability of the plasmid state and lysogenic conversion. Restriction mapping of the insertions allowed us to localize on the map the regions necessary for lytic growth and to map the lysogenic conversion gene. A recombinant phage encoding two antibiotic resistance genes was obtained. The phage contains an additional 4.77 kb DNA fragment (over 10% of the N15 genome).     

Immune electron microscopy in the study of biological matter

A brief review of literature data and our investigations on the antibodies used for specific labeling in electron microscopy is presented. Considered are the problems connected with structure and function of separate components of bacterial viruses revealed by means of specific antibodies. The results of fine differentiation of antigenic components in the case of phages of the colidysentery group allowed to elucidate the functional role of the adsorption apparatus in the course of phage interaction with the bacterial cell. The topology of structural proteins (gene-products 35, 36, 37) of the tail's long strands for phages T4, DDVI+h and DDVIh is determined. Antigenic properties of proteins that are found in the composition of two forms of Bacillus mycoides are demonstrated immune-electronmicroscopically. On the basis of this finding, a conclusion was made that one of these phages acted as precursor, the other--as satellite during the simultaneous development of these phages in the bacterial cell. It was also established that temperate and virulent phages are related antigenically, which proves that lysogenic bacteria can be one of the phage sources on the environmental conditions. Visualization of non-ribosomal genes of procaryots that code for structural proteins of a defective phage proves the efficiency of the immune-electronmicroscopic method for investigating of biological objects.     

Isolation and morphology of temperature Agrobacterium tumefaciens bacteriophage

Zimmerer, Robert P. (The Pennsylvania State University, University Park), Robert H. Hamilton, and Christine Pootjes. Isolation and morphology of temperate Agrobacterium tumefaciens bacteriophage. J. Bacteriol. 92:746-750. 1966.-Lysogeny was detected in 14 strains of Agrobacterium tumefaciens among 130 bacterial strains tested with strain B-6 used as the host. Partial lysis was observed with 13 additional bacterial strains. Morphological studies of five strains showed that the phage had similar features. A typical phage (Lv-1) had a polyhedralshaped head, approximately 71 by 63 mmu, and a tail, approximately 211 mmu by 9.5 mmu. The phage nucleic acid was found to be deoxyribonucleic acid. The bacteriophage have been designated L (for lysogenic) followed by the bacterial strain designation in lower case letters.     

Bacteriophage phi297, a new species of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> temperate phages with a mosaic genome: Potential use in phage therapy

The genome of halo-forming temperate Pseudomonas aeruginosa phage phi297 and lytic activity of its virulent mutant were studied. A mosaic structure was revealed for phi297 genome by its complete sequencing. The phi297 genome was partly homologous to the genomes of phages D3 and F116. High lytic activity was assumed for temperate P. aeruginosa bacteriophage phi297 on the basis of morphological features of negative colonies. Virulent mutant phi297vir, which was capable of lysing the wild-type phage bacteria, was isolated. Lytic activity was compared for phi297 and the phages from commercial mixtures of two manufacturers (facilities of Nizhnii Novgorod and Perm’). Phage phi297 caused lysis of the mutant PAO1 bacteria that were resistant to the phages from commercial preparations, but the lytic activity spectrum of phi297 was narrower that the spectra of the commercial phages. The use of nonreverting virulent mutants of certain temperate bacteriophages was proposed for the treatment of P. aeruginosa infections.     

Bacteriophage phi297--the new species of temperate phages Pseudomonas aeruginosa with a mosaic genome: potential use in phagotherapy

The genome of halo-forming temperate Pseudomonas aeruginosa phage phi297 and lytic activity of its virulent mutant were studied. A mosaic structure was revealed for phi297 genome by its complete sequencing. The phi97 genome was partly homologous to the genomes of phages D3 and F116. High lytic activity was assumed for temperate P. aeruginosa bacteriophage phi297 on the basis of morphological features of negative colonies. Virulent mutant phi297vir, which was capable oflysing bacteria, while the wild-type phage induced lysogeny, was isolated. Lytic activity was compared for phi297 and the phages from commercial mixtures of two manufacturers (facilities of Nizhnii Novgorod and Perm'). Phage phi297 caused lysis of the mutant PAO1 bacteria that were resistant to the phages from commercial preparations, but the lystic activity spectrum of phi297 was narrower that the spectra of the commercial phages. The use of nonreverting virulent mutants of certain temperate bacteriophages was proposed for the treatment of P. aeruginosa infections.     

Induction of Prophage SPO2 in Bacillus subtilis: Prophage Excision in the Absence of Bacterial or Bacteriophage DNA Synthesis

Bacillus subtilis lysogenic for SPO2 wild type was induced under conditions preventing synthesis of both bacterial and phage DNA. The infectivity of phage DNA in transfection is strongly decreased under these conditions, whereas the activity of single phage genes as measured by marker rescue with superinfecting phage is unaffected. DNA from induced cells was sedimented in neutral sucrose gradients. After induction, phage DNA was detected at a position in the gradients, which was different from the bulk of the bacterial DNA, corresponding to linear double-stranded DNA of about 25 x 10(6) daltons. Similar results were obtained with bacteria lysogenic for a SPO2 prophage carrying a DNA-negative mutation. No separation of phage and bacterial DNA activity was detected when chloramphenicol was present during the induction period. These experiments show that prophage SPO2 can excise from the bacterial chromosome without previous replication.     

Social evolution in micro-organisms and a Trojan horse approach to medical intervention strategies

Medical science is typically pitted against the evolutionary forces acting upon infective populations of bacteria. As an alternative strategy, we could exploit our growing understanding of population dynamics of social traits in bacteria to help treat bacterial disease. In particular, population dynamics of social traits could be exploited to introduce less virulent strains of bacteria, or medically beneficial alleles into infective populations. We discuss how bacterial strains adopting different social strategies can invade a population of cooperative wild-type, considering public good cheats, cheats carrying medically beneficial alleles (Trojan horses) and cheats carrying allelopathic traits (anti-competitor chemical bacteriocins or temperate bacteriophage viruses). We suggest that exploitation of the ability of cheats to invade cooperative, wild-type populations is a potential new strategy for treating bacterial disease.     

The evolution of bacterial resistance against bacteriophages in the horse chestnut phyllosphere is general across both space and time

Insight to the spatial and temporal scales of coevolution is key to predicting the outcome of host–parasite interactions and spread of disease. For bacteria infecting long-lived hosts, selection to overcome host defences is just one factor shaping the course of evolution; populations will also be competing with other microbial species and will themselves be facing infection by bacteriophage viruses. Here, we examine the temporal and spatial patterns of bacterial adaptation against natural phage populations from within leaves of horse chestnut trees. Using a time-shift experiment with both sympatric and allopatric phages from either contemporary or earlier points in the season, we demonstrate that bacterial resistance is higher against phages from the past, regardless of spatial sympatry or how much earlier in the season phages were collected. Similarly, we show that future bacterial hosts are more resistant to both sympatric and allopatric phages than contemporary bacterial hosts. Together, our results suggest the evolution of relatively general bacterial resistance against phages in nature and are contrasting to previously observed patterns of phage adaptation to bacteria from the same tree hosts over the same time frame, indicating a potential asymmetry in coevolutionary dynamics.     

Phage-host interactions in soil

Abstract Phages are abundant and ubiquitous in nature, and are therefore important components of microbial communities. They can impact on host populations in several ways, including predation and alteration of host phenotype by genetic interactions. The dynamic survival of phage populations in soil requires infective interactions with host populations which must be undergoing growth. Hence survival is limited by the activity of soil bacteria, and phage populations must adopt strategies to overcome periods of inactivity. One of the most effective strategies is the lysogenic cycle of temperate phages. It is argued here that lysogeny in soil has a distinct advantage over virulence for phage and host survival, as opposed to aquatic ecosystems where virulence seems a more successful strategy for phage populations.     

Linked Transformation of Bacterial and Prophage Markers in Bacillus subtilis 168 Lysogenic for Bacteriophage φ105

Level of competence reached by Bacillus subtilis 168 lysogenic for temperate phage phi 105 was reduced compared to that reached by nonlysogenic cells. This effect was probably related to an alteration of the bacterial surface. Deoxyribonucleic acid extracted from phi 105 lysogenic bacteria was used to transform other lysogenic bacteria. About 25% linkage was found between the bacterial phe-1 marker and prophage marker ts N15. The order of a few prophage markers relative to phe-1 was established in three-factor crosses. The usefulness of this system for a study of the linkage between an integrated prophage genome and that of its host was discussed.