Application of filamentous phages in environment: A tectonic shift in the science and practice of ecorestoration

Theories in soil biology, such as plant–microbe interactions and microbial cooperation and antagonism, have guided the practice of ecological restoration (ecorestoration). Below‐ground biodiversity (bacteria, fungi, invertebrates, etc.) influences the development of above‐ground biodiversity (vegetation structure). The role of rhizosphere bacteria in plant growth has been largely investigated but the role of phages (bacterial viruses) has received a little attention. Below the ground, phages govern the ecology and evolution of microbial communities by affecting genetic diversity, host fitness, population dynamics, community composition, and nutrient cycling. However, few restoration efforts take into account the interactions between bacteria and phages. Unlike other phages, filamentous phages are highly specific, nonlethal, and influence host fitness in several ways, which make them useful as target bacterial inocula. Also, the ease with which filamentous phages can be genetically manipulated to express a desired peptide to track and control pathogens and contaminants makes them useful in biosensing. Based on ecology and biology of filamentous phages, we developed a hypothesis on the application of phages in environment to derive benefits at different levels of biological organization ranging from individual bacteria to ecosystem for ecorestoration. We examined the potential applications of filamentous phages in improving bacterial inocula to restore vegetation and to monitor changes in habitat during ecorestoration and, based on our results, recommend a reorientation of the existing framework of using microbial inocula for such restoration and monitoring. Because bacterial inocula and biomonitoring tools based on filamentous phages are likely to prove useful in developing cost‐effective methods of restoring vegetation, we propose that filamentous phages be incorporated into nature‐based restoration efforts and that the tripartite relationship between phages, bacteria, and plants be explored further. Possible impacts of filamentous phages on native microflora are discussed and future areas of research are suggested to preclude any potential risks associated with such an approach.     

Three-Membered Parasitic System: a Bacteriophage, Bdellovibrio bacteriovorus, and Escherichia coli

Two bacteriophages for Bdellovibrio bacteriovorus were isolated. One of the phages (VL-1) was isolated on a host-independent Bdellovibrio strain, and the other (VL-2) was isolated on a host-dependent strain. Both phages grew on host-dependent as well as on host-independent Bdellovibrio strains. The development of the phages in host-dependent bdellovibrios occurred only when the phage-infected bdellovibrios parasitized cells of other bacteria. In the absence of other bacteria, the phages adsorbed to the bdellovibrios and killed them and in the process lost their own plaque-forming ability.     

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of phages infecting Arthrobacter hosts has been limited, perhaps due to the low success rate of many previous isolation techniques, resulting in an underrepresented group of Arthrobacter phages available for study. The enrichment technique described here, unlike many others, uses a filtered extract free of contaminating bacteria as the base for indicator bacteria growth, Arthrobactersp. KY3901, specifically. By first removing soil bacteria the target phages are not hindered by competition with native soil bacteria present in initial soil samples. This enrichment method has resulted in dozens of unique phages from several different soil types and even produced different types of phages from the same enriched soil sample isolate. The use of this procedure can be expanded to most nutrient rich aerobic media for the isolation of phages in a vast diversity of interesting host bacteria.     

Regulatory light-chains and scallop myosin. Full dissociation, reversibility and co-operative effects

Lambda duplication phages grown for several rounds on Escherichia coli strains containing arl mutations were recombined at elevated frequencies (3 to 6-fold higher) in subsequent test infections. Enhanced recombination of Arl^? phages (grown on arl bacteria) was demonstrable by assays for altered genetic linkages as well as by the standard assay, which measures the conversion of duplication phages (EDTA-sensitive) to single-copy phages (EDTA-resistant). The accumulated potential for enhanced recombination was lost during subsequent growth of the phages on arl⁺ bacteria. Arl^? phages had the same mutation frequencies, at a variety of loci, as control phages; arl bacteria themselves exhibited normal mutation rates. Arl^? phages had normal plating efficiencies and buoyant densities. DNA extracted from Arl^? phages exhibited the same frequency of strand interruption, the same superhelical density (when circularized in vivo), and the same thermal denaturation profile as DNA from phages grown on arl⁺ bacteria. Recombination of Arl^? phages in the presence of λ repressor was very low, as is the case for normal phages. The recombination frequency of ultraviolet light irradiated (80 J/m²) Arl^? phages was more than twice the sum of the frequencies for unirradiated Arl^? phages and irradiated control phages. Substantially increased recombination of Arl^? phages was observed when either the E. coli RecBC, or RecE (but not RecF) pathway was active.     

IMPACT OF BACTERIAL MUTATION RATE ON COEVOLUTIONARY DYNAMICS BETWEEN BACTERIA AND PHAGES

Mutator bacteria are frequently found in natural populations of bacteria and although coevolution with parasitic viruses (phages) is thought to be one reason for their persistence, it remains unclear how the presence of mutators affects coevolutionary dynamics. We hypothesized that phages must themselves adapt more rapidly or go extinct, in the face of rapidly evolving mutator bacteria. We compared the coevolutionary dynamics of wild‐type Pseudomonas fluorescens SBW25 with a lytic phage to the dynamics of an isogenic mutator of P. fluorescens SBW25 together with the same phage. At the beginning of the experiment both wild‐type bacteria and mutator bacteria coevolved with phages. However, mutators rapidly evolved higher levels of sympatric resistance to phages. The phages were unable to “keep‐up” with the mutator bacteria, and these rates of coevolution declined to less than the rates of coevolution between the phages and wild‐type bacteria. By the end of the experiment, the sympatric resistance of the mutator bacteria was not significantly different to the sympatric resistance of the wild‐type bacteria. This suggests that the importance of mutators in the coevolutionary interactions with a particular phage population is likely to be short‐lived. More generally, the results demonstrate that coevolving enemies may escape from Red‐Queen dynamics.     

Partially deficient methylation of cytosine in DNA at CCATGG sites stimulates genetic recombination of bacteriophage lambda

Lambda bacteriophages grown on arl mutants of Escherichia coli ("Arl-" phages) display intermediate levels of cytosine methylation: less 5-methylcytosine (m5C) than phages grown on wild-type bacteria ("Arl+" phages) but more than phages grown on dcm mutants, and thus lacking the methylated sequences (Cm5CATGG) characteristic of E. coli K-12 bacteria ("Dcm-" phages). "Arl-" phages are one twelfth as resistant to Eco RII restriction (recognition site CCATGG) as "Arl+" phages, but 40-fold more resistant than "Dcm-" phages. Chromatographic analyses show the 5-methylcytosine content of "Arl-" DNA to be one third that of "Arl+" DNA. Altered cytosine methylation frequency correlates with two previously described properties of "Arl-" phages, increased genetic recombination and unusual sensitivity of phage DNA to endonuclease S1, which are absent in phages grown on dcm or dcm arl bacteria. Methylated/unmethylated heteroduplex DNA prepared in vitro (one strand from Eco RII-modified phages/one from "Dcm-" phages) is highly recombinogenic but not S1-sensitive. We hypothesize that hemimethylated CCATGG sites in "Arl-" DNA are necessary and sufficient for enhanced recombination, and necessary but not sufficient for S1 sensitivity.     

CRISPR-Cas系统与细菌和噬菌体的共进化

细菌在适应噬菌体攻击的过程中,进化了多种防御系统,噬菌体在细菌的选择压力下,也在不断进化反防御策略,双方的这种进化关系与发生机制一直尚不完全清楚。近年在细菌和古细菌中发现一种新的免疫防御系统,即CRISPR-Cas(clustered regularly interspaced short palindromic repeats-CRISPR-associated system)系统。在对其功能和作用机制深入研究的同时,也不断地揭示了细菌和噬菌体之间的共进化关系。为此,文章在介绍原核细胞中CRISPR-Cas系统介导的免疫机制基础上,重点综述了CRISPR系统在细菌和噬菌体进化中的作用。     

Bacteriological activity in unfiltered calf sera collected for tissue culture use.

Bacteriological tests were made on 24 lots of unfiltered calf serum collected for subsequent use as a component of tissue culture media. The examination included the isolation and identification of bacteria, assay of phages, and demonstration of endotoxin material. Only Gram-positive bacteria were isolated and 96% of the sera were contaminated with bacteria. The prevalent strains of bacteria found were Bacillus species and streptococci and 63% of the sera coagulated Limulus amebocyte lysate. More than 90% of the lots contained phages demonstrable with the C-3000 strain of Escherichia coli. Only one lot of the serum was found to be free from bacteria, phages, and endotoxin by the tests used.     

Using experimental evolution to explore natural patterns between bacterial motility and resistance to bacteriophages

Resistance of bacteria to phages may be gained by alteration of surface proteins to which phages bind, a mechanism that is likely to be costly as these molecules typically have critical functions such as movement or nutrient uptake. To address this potential trade-off, we combine a systematic study of natural bacteria and phage populations with an experimental evolution approach. We compare motility, growth rate and susceptibility to local phages for 80 bacteria isolated from horse chestnut leaves and, contrary to expectation, find no negative association between resistance to phages and bacterial motility or growth rate. However, because correlational patterns (and their absence) are open to numerous interpretations, we test for any causal association between resistance to phages and bacterial motility using experimental evolution of a subset of bacteria in both the presence and absence of naturally associated phages. Again, we find no clear link between the acquisition of resistance and bacterial motility, suggesting that for these natural bacterial populations, phage-mediated selection is unlikely to shape bacterial motility, a key fitness trait for many bacteria in the phyllosphere. The agreement between the observed natural pattern and the experimental evolution results presented here demonstrates the power of this combined approach for testing evolutionary trade-offs.     

Bacteriophages that infect the cellulolytic ruminal bacterium Ruminococcus albus AR67

AIM: To isolate bacterial viruses that infect the ruminal cellulolytic bacterium Ruminococcus albus. METHODS: Four phages infecting R. albus AR67 were isolated under anaerobic conditions using the soft-agar overlay technique. The phages were characterized on morphology, solvent stability, nucleic acid type and digestion characteristics. Two phages, phiRa02 and phiRa04 comprised icosahedral virions with linear double-stranded DNA and appeared to belong to the family Podoviridae [corrected] The other two phages are most likely filamentous phages with circular single-stranded DNA of the family Inoviridae. SIGNIFICANCE OF THE STUDY: Viruses of the family Inoviridae [corrected] have not previously been isolated from rumen bacteria. The phages isolated in this study are the first phages shown to infect the cellulolytic bacteria of the rumen. This suggests that the cellulolytic populations of the rumen are subject to lytic events that may impact on the ability of these bacteria to degrade plant fibre and on the nutrition of the animal.     

Establishment of phage and bacteria in a sterilized compost in a glasshouse

Summary Four strains ofRhizobium trifolii were individually inoculated to pots containing sterilized sand vermiculite mixture, half of which were seeded with red clover and half not. Pots were maintained in an ordinary glasshouse and watered with tap water.Phage was first detected after 4 months, and almost all pots contained one or more phages againstRhizobium trifolii after 9 months. The presence of plants increased the titer of phages in some pots inoculated withR. trifolii, but had no effect on the number of different phages.The pots also contained phages against soil bacteria other than Rhizobium indicating that phages are spread readily and constitute a normal part of the life cycle of soil bacteria.The number of different phages isolated from the pots was affected by the strain of Rhizobium used as inoculum.     

Adsorption of Staphylococcal Bacteriophage by Milk Proteins

Propagation of homologous bacteriophage in a culture of Staphylococcus aureus (1:1 ratio of phage to bacteria) in Trypticase Soy Broth (TSB) and in skim milk indicated more activity of phage in TSB. Early lysis of bacteria in skim milk followed by a pronounced rise in bacterial population suggested that staphylococcal phages were being inactivated by milk. Titration of phages from skim milk, whey, and TSB indicated about 90% adsorption of phages by acid- and heat-precipitable proteins of skim milk, whereas numbers recovered from whey were quite comparable to those recovered from TSB. Reducing the pH from 6.5 to 4.0 increased the percentage of phages recoverable from skim milk from 10 to 56%. Apparently, the changes in electrical charges on the casein micelles at this low pH were responsible for release of many phages from their complex with casein.     

Bacteriological activity in unfiltered calf sera collected for tissue culture use

Summary Bacteriological tests were made on 24 lots of unfiltered calf serum collected for subsequent use as a component of tissue culture media. The examination included the isolation and identification of bacteria, assay of phages, and demonstration of endotoxin material. Only Gram-positive bacteria were isolated and 96% of the sera were contaminated with bacteria. The prevalent strains of bacteria found wereBacillus species and streptococci and 63% of the sera coagulatedLimulus amebocyte lysate. More than 90% of the lots contained phages demonstrable with the C-3000 strain ofEscherichia coli. Only one lot of the serum was found to be free from bacteria, phages, and endotoxin by the tests used.     

Sustainable Microbiome: a symphony orchestrated by synthetic phages

We are surrounded by microbes, mostly bacteria and their viruses or phages, on the inside and outside of our bodies. These bacteria in constant interactions with phages are   regulating multiple functions critical to our health. Luckily, they are amenable, but we need precise tools for their safe manipulation and improving human health. Here, we argue that recent advances in single-cell technologies, culturomics and synthetic biology offer exciting opportunities to create these tools as well as revealing specific phages–bacteria interactions in the body.     

Comparison of responses by bacteriophages and bacteria to pressures on the base composition of open reading frames

Differences in the base composition of genomes can occur because of GC pressure, purine-loading pressure (AG pressure) and RNY pressure, for which there are possible functional explanations, and because of the more abstract pressures exerted by individual bases. The graphical approach of Muto and Osawa was used to analyse how bacteriophages and bacteria balance potentially conflicting pressures on their genomes. Phages generally respond to AG pressure by increasing A while keeping T constant, and by decreasing C while keeping G constant. In contrast, bacteria generally increase both A and T, the former more so, and decrease both G and C, the latter more so. These differences largely occur at third codon positions, which are more responsive than first and second codon positions to AG pressure and GC pressure. Phages respond to AG pressure more in the third codon position than bacteria, whereas bacteria respond more in the first codon position than phages. Conversely, bacteria respond to GC pressure more in the third codon position than phages, whereas phages respond more in the first codon position than bacteria. As GC pressure increases, A is traded for C and AG pressure decreases; first and second codon positions, having more A than T, are most responsive to this negative effect of increased GC pressure; third positions either do not respond (phages) or respond weakly (bacteria). In a set of 48 phage-host pairs, degrees of purine loading were less correlated between phage and host than were GC percentages. These results suggest that pressures on conventional and genome phenotypes operate differentially in phages and bacteria, generating both general differences in base composition and specific differences characteristic of particular phage-host pairs. The reciprocal relationship between GC pressure and AG pressure implies that effects attributed to GC pressure may actually be due to AG pressure, and vice versa.     

Pre‐adapting parasitic phages to a pathogen leads to increased pathogen clearance and lowered resistance evolution with Pseudomonas aeruginosa cystic fibrosis bacterial isolates

Recent years have seen renewed interest in phage therapy – the use of viruses to specifically kill disease‐causing bacteria – because of the alarming rise in antibiotic resistance. However, a major limitation of phage therapy is the ease at with bacteria can evolve resistance to phages. Here, we determined whether in vitro experimental coevolution can increase the efficiency of phage therapy by limiting the resistance evolution of intermittent and chronic cystic fibrosis Pseudomonas aeruginosa lung isolates to four different phages. We first pre‐adapted all phage strains against all bacterial strains and then compared the efficacy of pre‐adapted and nonadapted phages against ancestral bacterial strains. We found that evolved phages were more efficient in reducing bacterial densities than ancestral phages. This was primarily because only 50% of bacterial strains were able to evolve resistance to evolved phages, whereas all bacteria were able to evolve some level of resistance to ancestral phages. Although the rate of resistance evolution did not differ between intermittent and chronic isolates, it incurred a relatively higher growth cost for chronic isolates when measured in the absence of phages. This is likely to explain why evolved phages were more effective in reducing the densities of chronic isolates. Our data show that pathogen genotypes respond differently to phage pre‐adaptation, and as a result, phage therapies might need to be individually adjusted for different patients.     

Impact of phages on two-species bacterial communities

A long history of experimental work has shown that addition of bacteriophages to a monoculture of bacteria leads to only a temporary depression of bacterial levels. Resistant bacteria usually become abundant, despite reduced growth rates relative to those of phage-sensitive bacteria. This restoration of high bacterial density occurs even if the phages evolve to overcome bacterial resistance. We believe that the generality of this result may be limited to monocultures, in which the resistant bacteria do not face competition from bacterial species unaffected by the phage. As a simple case, we investigated the impact of phages attacking one species in a two-species culture of bacteria. In the absence of phages, Escherichia coli B and Salmonella enterica serovar Typhimurium were stably maintained during daily serial passage in glucose minimal medium (M9). When either of two E. coli-specific phages (T7 or T5) was added to the mixed culture, E. coli became extinct or was maintained at densities that were orders of magnitude lower than those before phage introduction, even though the E. coli densities with phage reached high levels when Salmonella was absent. In contrast, the addition of a phage that attacked only Salmonella (SP6) led to transient decreases in the bacterial number whether E. coli was absent or present. These results suggest that phages can sometimes, although not always, provide long-term suppression of target bacteria.     

Pseudolysogeny of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> bacteria infected with φKZ-like bacteriophages

In this work, a final piece of evidence proving that bacteria Pseudomonas aeruginosa are capable of transition to the pseudolysogenic state after infection with φKZ-like phages has been produced. It was shown that the decisive factor in this process is multiple infection of bacteria with bacteriophages belonging to this genus. In the course of this work, stable clinical isolates of bacteria liberating novel bacteriophages of this genus (Che2/2 and Che21/5) were detected and attributed to species φKZ and EL, respectively, according to their phenotypic characters and the results of DNA analysis. For three bacteriophages belonging to species EL (EL, RU, and Che21/5), mutants with disorders in the capability for pseudolysogenization were isolated. One of the mutants of phage EL possesses properties of virulent mutants of typical temperate phages (vir mutant). This mutant fails to form pseudolysogens and, moreover, provides the effect of dominance upon coinfection of bacteria with the wild-type phage EL, but however is unable to exhibit this effect upon joint infection of bacteria with wild-type phages of species φKZ and Lin68. It is assumed that the effect of pseudolysogeny may be connected with functioning of φKZ and EL genes that control the products similar to repressors of other phages. Because earlier wild-type φKZ-like phages were shown to be present in commercial phage-therapeutic preparations (which represents certain problems), it is expedient to use virulent mutants of phages belonging to this genus rather than phages of the wild type.     

Coevolutionary dynamics shape the structure of bacteria‐phage infection networks

Coevolution—reciprocal evolutionary change among interacting species driven by natural selection—is thought to be an important force in shaping biodiversity. This ongoing process takes place within tangled networks of species interactions. In microbial communities, evolutionary change between hosts and parasites occurs at the same time scale as ecological change. Yet, we still lack experimental evidence of the role of coevolution in driving changes in the structure of such species interaction networks. Filling this gap is important because network structure influences community persistence through indirect effects. Here, we quantified experimentally to what extent coevolutionary dynamics lead to contrasting patterns in the architecture of bacteria–phage infection networks. Specifically, we look at the tendency of these networks to be organized in a nested pattern by which the more specialist phages tend to infect only a proper subset of those bacteria infected by the most generalist phages. We found that interactions between coevolving bacteria and phages become less nested over time under fluctuating dynamics, and more nested under arms race dynamics. Moreover, when coevolution results in high average infectivity, phages and bacteria differ more from each other over time under arms race dynamics than under fluctuating dynamics. The tradeoff between the fitness benefits of evolving resistance/infectivity traits and the costs of maintaining them might explain these differences in network structure. Our study shows that the interaction pattern between bacteria and phages at the community level depends on the way coevolution unfolds.