Bacteriophage ecology in Escherichia coli and Pseudomonas aeruginosa mixed-biofilm communities

Phage therapy is being reexamined as a strategy for bacterial control in medical and other environments. As microorganisms often live in mixed populations, we examined the effect of Escherichia coli bacteriophage λW60 and Pseudomonas aeruginosa bacteriophage PB-1 infection on the viability of monoculture and mixed-species biofilm and planktonic cultures. In mixed-species biofilm communities, E. coli and P. aeruginosa maintained stable cell populations in the presence of one or both phages. In contrast, E. coli planktonic populations were severely depleted in coculture in the presence of λW60. Both E. coli and P. aeruginosa developed phage resistance in planktonic culture; however, reduced resistance was observed in biofilm communities. Increased phage titers and reduced resistance in biofilms suggest that phage can replicate on susceptible cells in biofilms. Infectious phage could be released from mixed-culture biofilms upon treatment with Tween 20 but not upon treatment with chloroform. Tween 20 and chloroform treatments had no effect on phage associated with planktonic cells, suggesting that planktonic phage were not cell or matrix associated. Transmission electron microscopy showed bacteriophage particles to be enmeshed in the extracellular polymeric substance component of biofilms and that this substance could be removed by Tween 20 treatment. Overall, this study demonstrates how mixed-culture biofilms can maintain a reservoir of viable phage and bacterial populations in the environment.     

The phage tail tape measure protein,an inner membrane protein and a periplasmic chaperone play connected roles in the genome injection process of E. coli phage HK97

Phages play critical roles in the spread of virulence factors and control of bacterial populations through their predation of bacteria. An essential step in the phage lifecycle is genome entry, where the infecting phage must productively interact with the components of the bacterial cell envelope in order to transmit its genome out of the viral particle and into the host cell cytoplasm. In this study, we characterize this process for the Escherichia coli phage HK97. We have discovered that HK97 genome injection requires the activities of the inner membrane glucose transporter protein, PtsG, and the periplasmic chaperone, FkpA. The requirements for PtsG and FkpA are determined by the sequence of the phage tape measure protein (TMP). We also identify a region of the TMP that mediates inhibition of phage genome injection by the HK97 superinfection exclusion protein, gp15. This region of the TMP also determines the PtsG requirement, and we show that gp15‐mediated inhibition requires PtsG. Based on these data, we present a model for the in vivo genome injection process of phage HK97 and postulate a mechanism by which the inhibitory action of gp15 is reliant upon PtsG.     

Effect of the polymerase activity of DNA polymerase I on the development of moderate bacteriophages Mu, lambda red- and lambda red-gam-. II. The effect of the polymerase activity of DNA polymerase I on the development of bacteriophage Mu

The paper reports on the influence of polymerizing activity of DNA-polymerase I on different developmental stages of temperate bacteriophage Mu in Escherichia coli K-12 cells. This activity is shown to be necessary for optimization of phage Mu primary integration into cell chromosomes. The relative frequency of Mu integration into bacterial chromosomes is 5-6 times lower in polA cells than in isogenic polA+ control strains, the phage yield from cells being delayed during the phage infectious development, but not in the course of induction from the prophage state. Data have been obtained that show the process of phage Mu DNA integration into the plasmid pRP1 .2 and the process of Mu transposition from the cell chromosome into the plasmid to be independent of the polymerizing activity of DNA-polymerase I.     

Microtiter plate assays for the measurement of phage adsorption and infection in Lactococcus and Enterococcus

Three easy and rapid microtiter plate assays for determining phage sensitivity of lactococci and enterococci have been developed. In the microlysis assay, the degree of sensitivity was measured on the basis of the ability of the bacterial cells to grow in the presence of various concentrations of phage and to effect a color change of an acid-base indicator as a result of acid production. Two assays that specifically measure phage adsorption to bacterial cells have been developed on the basis of the enzyme-linked immunosorbent assay (ELISA) technique. In the direct phage adsorption ELISA, adsorption of phage particles to cells immobilized onto microtiter plate wells was measured using specific anti-phage antibody. In the competitive phage adsorption ELISA, phage adsorption was assayed by allowing phage to compete with specific antibody binding to the bacterial cell surface. All three assays were quantifiable photometrically.     

Bacteriophage lysins as effective antibacterials

Lysins are highly evolved enzymes produced by bacteriophage (phage for short) to digest the bacterial cell wall for phage progeny release. In Gram-positive bacteria, small quantities of purified recombinant lysin added externally results in immediate lysis causing log-fold death of the target bacterium. Lysins have been used successfully in a variety of animal models to control pathogenic antibiotic resistant bacteria found on mucosal surfaces and infected tissues. The advantages over antibiotics are their specificity for the pathogen without disturbing the normal flora, the low chance of bacterial resistance to lysins, and their ability to kill colonizing pathogens on mucosal surfaces, a capacity previously unavailable. Thus, lysins may be a much needed anti-infective in an age of mounting antibiotic resistance.     

Investigation of bacteriophage MS2 viral dynamics using model discrimination analysis and the implications for phage therapy

Lytic phages infect their bacterial hosts, use the host machinery to replicate, and finally lyse and kill their hosts, releasing progeny phages. Various mathematical models have been developed that describe these phage-host viral dynamics. The aim of this study was to determine which of these models best describes the viral dynamics of lytic RNA phage MS2 and its host Escherichia coli C-3000. Experimental data consisted of uninfected and infected bacterial cell densities, free phage density, and substrate concentration. Parameters of various models were either determined directly through other experimental techniques or estimated using regression analysis of the experimental data. The models were evaluated using a Bayesian-based model discrimination technique. Through model discrimination it was shown that phage-resistant cells inhibited the growth of phage population. It was also shown that the uninfected bacterial population was a quasispecies consisting of phage-sensitive and phage-resistant bacterial cells. When there was a phage attack the phage-sensitive cells died out and the phage-resistant cells were selected for and became the dominant strain of the bacterial population.     

The production of bacteriophage μ2

In three series of experiments, 3-l., 20-l., and 150-l. bacterial cultures were grown in stirred, deep culture vessels to average bacterial cell densities of 71 × 10⁸, 63 × 10⁸, and 43 × 10⁸ viable organisms per milliliter, respectively, and then infected with phage. The average yield of progeny phage in each case was ca. 3000 mpfu (minimum plaque-forming units) per cell. Thus, the average mass of phage obtained in the 3-l. experiments was not less than 124 mg./l., calculated from the plaque counts, assuming a particle size of 3.6 × 10⁶ Daltons for the μ2 phage. This is about twentyfold higher than is obtainable by conventional methods in aerated, shaken culture flasks. The actual phage yields are probably much higher than the minimum values calculated from plaque counts. For example, in the case of one of our culture lysates which was purified at King's College, the efficiency of plating was shown to be only 19%. The carbon dioxide evolution rate of cultures was measured and used as a guide to the time at which phage should be added. In this way, greater control of cultural conditions was obtained than is possible in shaken flasks. For the best yield of phage per milliliter of culture, the optimum time for phage infection was such that bacterial lysis just prevented the carbon dioxide evolution rate from reaching its potential maximum. The major factor influencing the phage yield per milliliter of culture was the aeration capacity of the culture vessel used. All had maximum aeration capacities much higher than those obtainable in shaken culture flasks. Cultures grown and infected in 3-l. Vessel operated under conditions of low aeration gave poor yields of phage. The reason for this are discussed.     

青枯菌特异性噬菌体的研究进展与应用

烟草等茄科植物青枯病的防治是一个世界性难题,传统的化学防治、合理轮作、抗病品种等措施无法有效控制该病的发生。噬菌体用于细菌性病害的防治已有很长历史,近年来利用噬菌体防治青枯菌引发的青枯病方面的研究越来越受重视。我们简要综述了青枯菌噬菌体的研究进展,并对青枯菌噬菌体生物防治的应用前景进行了展望。     

Phage endolysins are adapted to specific hosts and are evolutionarily dynamic

Endolysins are produced by (bacterio)phages to rapidly degrade the bacterial cell wall and release new viral particles. Despite sharing a common function, endolysins present in phages that infect a specific bacterial species can be highly diverse and vary in types, number, and organization of their catalytic and cell wall binding domains. While much is now known about the biochemistry of phage endolysins, far less is known about the implication of their diversity on phage–host adaptation and evolution. Using CRISPR-Cas9 genome editing, we could genetically exchange a subset of different endolysin genes into distinct lactococcal phage genomes. Regardless of the type and biochemical properties of these endolysins, fitness costs associated to their genetic exchange were marginal if both recipient and donor phages were infecting the same bacterial strain, but gradually increased when taking place between phage that infect different strains or bacterial species. From an evolutionary perspective, we observed that endolysins could be naturally exchanged by homologous recombination between phages coinfecting a same bacterial strain. Furthermore, phage endolysins could adapt to their new phage/host environment by acquiring adaptative mutations. These observations highlight the remarkable ability of phage lytic systems to recombine and adapt and, therefore, explain their large diversity and mosaicism. It also indicates that evolution should be considered to act on functional modules rather than on bacteriophages themselves. Furthermore, the extensive degree of evolvability observed for phage endolysins offers new perspectives for their engineering as antimicrobial agents.

Endolysins are produced by bacteriophages to degrade the host cell wall and release new particles, but the implications of their diversity on phage-host adaptation and evolution is unknown. This study uses CRISPR-Cas9 genome editing to reveal novel insights into bacteriophage endolysin diversity and phage-bacteria interactions as well as into endolysin adaptation towards a new bacterial host.     

Ecological and Evolutionary Benefits of Temperate Phage: What Does or Doesn't Kill You Makes You Stronger

Infection by a temperate phage can lead to death of the bacterial cell, but sometimes these phages integrate into the bacterial chromosome, offering the potential for a more long‐lasting relationship to be established. Here we define three major ecological and evolutionary benefits of temperate phage for bacteria: as agents of horizontal gene transfer (HGT), as sources of genetic variation for evolutionary innovation, and as weapons of bacterial competition. We suggest that a coevolutionary perspective is required to understand the roles of temperate phages in bacterial populations.     

鼠疫菌噬菌体效价噬菌斑测定法的建立

目的建立鼠疫菌噬菌体噬菌斑效价测定方法。方法通过分析细菌接种浓度、孵育吸附时间及培养温度等参数,建立鼠疫菌噬菌体效价测定方法,并分析其精密性;建立鼠疫活疫苗鉴别及纯菌检查用噬菌体效价质量标准。结果经优化后确定细菌接种浓度为7×108/mL,不需孵育吸附,培养温度为29℃,所建立的检测方法精密性较好,用于鼠疫活疫苗鉴别及纯菌检查用噬菌体效价质量标准应不低于1×106PFU/mL。结论建立了鼠疫菌噬菌体噬菌斑效价测定方法,为鼠疫菌噬菌体及疫苗质量控制奠定了基础。     

Genetic and physiological control of host cell lysis by bacteriophage lambda.

The timing of host cell lysis at the end of the lytic cycle of phage lambda is under complex control. The lambda S protein stimulates lysis. Another physiological system, the lysis regulator, inhibitis lysis from occurring prematurely. The effects of a series of phage and bacterial mutations on these controls are described. They show that the lambda rex gene plays a role in regulating lysis under suboptimal growth conditions. In certain mutant cells, and especially under anaerobic culture conditions, the rex gene aids in the scheduling of host cell lysis. The data also suggest that the lysis regulator may control the transition of the lambda S protein from an inactive to an active state.     

Biotechnological exploitation of bacteriophage research

The experimentally amenable nature of phage and their use in testing fundamental biological questions have meant that phage research has had a profound effect on modern molecular biology. Phage research has also fuelled multiple biotechnological developments. For example, phage display has recently been harnessed in a multidisciplinary approach for the generation of novel nanotechnologies. In addition, with the emerging threat of antibiotic-resistant bacterial infections, phage have begun to provide technologies to combat these problems. Finally, recent data acquired from genome sequencing and advances in phage biology research have aided the development of phage-derived bacterial detection and treatment strategies in addition to methods to control the detrimental effects of phage in industry. Here, we examine the promising uses of phage in these important areas of biotechnology.     

The role of interactions between phage and bacterial proteins within the infected cell: a diverse and puzzling interactome

Interactions between bacteriophage proteins and bacterial proteins are important for efficient infection of the host cell. The phage proteins involved in these bacteriophage–host interactions are often produced immediately after infection. A survey of the available set of published bacteriophage–host interactions reveals the targeted host proteins are inhibited, activated or functionally redirected by the phage protein. These interactions protect the bacteriophage from bacterial defence mechanisms or adapt the host-cell metabolism to establish an efficient infection cycle. Regrettably, a large majority of bacteriophage early proteins lack any identified function. Recent research into the antibacterial potential of bacteriophage–host interactions indicates that phage early proteins seem to target a wide variety of processes in the host cell – many of them non-essential. Since a clear understanding of such interactions may become important for regulations involving phage therapy and in biotechnological applications, increased scientific emphasis on the biological elucidation of such proteins is warranted.     

Prophage as a genetic reservoir: Promoting diversity and driving innovation in the host community

Sequencing of bacterial genomes has revealed an abundance of prophage sequences in many bacterial species. Since these sequences are accessible, through recombination, to infecting phages, bacteria carry an arsenal of genetic material that can be used by these viruses. We develop a mathematical model to isolate the effects of this phenomenon on the coevolution of temperate phage and bacteria. The model predicts that prophage sequences may play a key role in maintaining the phage population in situations that would otherwise favor host cell resistance. In addition, prophage recombination facilitates the existence of multiple phage types, thus promoting diverse co‐existence in the phage‐host ecosystem. Finally, because the host carries an archive of previous phage strategies, prophage recombination can drive waves of innovation in the host cell population.     

Phage-inducible chromosomal islands promote genetic variability by blocking phage reproduction and protecting transductants from phage lysis

Phage-inducible chromosomal islands (PICIs) are a widespread family of highly mobile genetic elements that disseminate virulence and toxin genes among bacterial populations. Since their life cycle involves induction by helper phages, they are important players in phage evolution and ecology. PICIs can interfere with the lifecycle of their helper phages at different stages resulting frequently in reduced phage production after infection of a PICI-containing strain. Since phage defense systems have been recently shown to be beneficial for the acquisition of exogenous DNA via horizontal gene transfer, we hypothesized that PICIs could provide a similar benefit to their hosts and tested the impact of PICIs in recipient strains on host cell viability, phage propagation and transfer of genetic material. Here we report an important role for PICIs in bacterial evolution by promoting the survival of phage-mediated transductants of chromosomal or plasmid DNA. The presence of PICIs generates favorable conditions for population diversification and the inheritance of genetic material being transferred, such as antibiotic resistance and virulence genes. Our results show that by interfering with phage reproduction, PICIs can protect the bacterial population from phage attack, increasing the overall survival of the bacterial population as well as the transduced cells. Moreover, our results also demonstrate that PICIs reduce the frequency of lysogenization after temperate phage infection, creating a more genetically diverse bacterial population with increased bet-hedging opportunities to adapt to new niches. In summary, our results identify a new role for the PICIs and highlight them as important drivers of bacterial evolution.     

Isolation and characteristics of mutation pfm affecting the penetration of phage Mu into Escherichia coli K-12 cells

A mutant of Escherichia coli K-12 strain with the destroyed process of establishment of lysogenic state for phage Mu in the course of zygotic induction has been obtained. The mutation revealed, designated pfm (penetration factor for Mu), interferes with adsorption of phage Mu to the surface of E. coli K-12 cells. On the basis of data obtained, there is every reason to believe that the phage Mu DNA connection with the membrane components of the bacterial cell provides optimizing condition for the primary integrative transposition of phage Mu at the stage of Mu DNA introduction into the cell.     

Mini-review: efficacy of lytic bacteriophages on multispecies biofilms

There is potential for phages to prevent and control bacterial biofilms, but few studies have examined the effect of phages on the multispecies biofilms that characterize most bacterial infections. This paper reviews the mechanism of action of phages, the evidence supporting the view that phage therapy will be effective against bacterial targets and the opposite viewpoint, phage application approaches, and the comparative advantage of phage therapy in multispecies biofilms. The few reports measuring the actions of lytic phages against multispecies biofilms are also reviewed. The authors are cautiously optimistic about the application of phages against their targets when in multispecies biofilms because some lysis mechanisms do not require species specificity.     

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.