Phage Reproduction Research Articles

Characterization and genomic analysis of Stutzerimonas stutzeri phage vB_PstS_ZQG1, representing a novel viral genus

Stutzerimonas stutzeri is an opportunistic pathogenic bacterium belonging to the Gammaproteobacteria, exhibiting wide distribution in the environment and playing significant ecological roles such as nitrogen fixation or pollutant degradation. Despite its ecological importance, only two S. stutzeri phages have been isolated to date. Here, a novel S. stutzeri phage, vB_PstS_ZQG1, was isolated from the surface seawater of Qingdao, China. Transmission electron microscopy analysis indicates that vB_PstS_ZQG1 has a morphology characterized by a long non-contractile tail. The genomic sequence of vB_PstS_ZQG1 contains a linear, double-strand 61,790-bp with the G+C content of 53.24% and encodes 90 putative open reading frames. Two auxiliary metabolic genes encoding TolA protein and nucleotide pyrophosphohydrolase were identified, which are likely involved in host adaptation and phage reproduction. Phylogenetic and comparative genomic analyses demonstrated that vB_PstS_ZQG1 exhibits low similarity with previously isolated phages or uncultured viruses (average nucleotide identity values range from 21.7 to 29.4), suggesting that it represents a novel viral genus by itself, here named as Fuevirus. Biogeographic analysis showed that vB_PstS_ZQG1 was only detected in epipelagic and mesopelagic zone with low abundance. In summary, our findings of the phage vB_PstS_ZQG1 will provide helpful insights for further research on the interactions between S. stutzeri phages and their hosts, and contribute to discovering unknown viral sequences in the metagenomic database.

Graphene oxide affects bacteriophage infection of bacteria by promoting the formation of biofilms

Graphene oxide (GO) is increasingly used in a range of fields, such as electronics, biosensors, drug delivery, and water treatment, and the likelihood of its release into the environment is increasing correspondingly. GO is involved in the formation of biofilms and leads bacteria to over proliferate, but the effects of GO on bacteriophage infection remain unexplored. We noted bacterial overgrowth in experiments when GO was used to treat the bacterial culture medium, leading us to question whether bacterial proliferation caused by GO affects phage infection of target bacteria. Treating Pseudomonas aeruginosa with GO at a low dosage (0.02 mg/mL) led to biofilm expansion in LB medium. Biofilm formation in the presence of GO affected the ability of bacteriophages to kill bacteria and reproduce. Similarly, the presence of GO deposits increased the ratio of bacteria to phage, providing a favorable environment for bacterial growth. Additionally, increasing the positive electrical charge in the culture environment inhibited the rejection of bacteriophages by negatively charged GO, improving phage reproduction. Finally, adding GO to sewage in imitation field experiments significantly increased the bacterial diversity and richness in the sewage, stimulating a significant increase in the variety and number of bacteria. Collectively, these results indicate that GO hinders phage infection by providing a bacterial refuge. The results of this study provide valuable insights into how GO interacts with bacteriophages to explore the effects on bacterial growth.

Эндолизины – перспективный метод борьбы с антибиотико-резистентными микроорганизмами

The article reveals one of the promising methods of combating antimicrobial resistance in agriculture, medicine, food industry and other fields – endolysins (enzymes of bacteriophages). The history of the origin, discovery, and production of endolysins is displayed. The mechanism of action of endolysins is shown – destruction of the peptidoglycan layer at the end of the lytic cycle of phage reproduction. The results of endolysin studies in recent years have been searched using the Internet resources Scopus, Web of science, Elibrary, Pub Med and others. Analysis of the literature showed that endolysins are divided into 5 groups depending on the cleavable installation in the peptidoglycan – acetylmuramidase, transglycosidase, glucosaminidase, amidase, endopeptidase. The introduction of endolysins is important - intravenous, intraperitoneal, transnasal, transnasal, vaginal. To date, endolysins effective against such pathogens as Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterococcus faecalis, Streptococcus suis, Streptococcus equi, Clostridium perfringens, Bacillus anthracis and a number of other pathogens have been studied. Thus, the analysis shows that endolysins can be effective tools in the fight against infectious diseases of animals and safe to use.

Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors

Bacteria encode sophisticated anti-phage systems that are diverse and versatile and display high genetic mobility. How this variability and mobility occurs remains largely unknown. Here, we demonstrate that a widespread family of pathogenicity islands, the phage-inducible chromosomal islands (PICIs), carry an impressive arsenal of defense mechanisms, which can be disseminated intra- and inter-generically by helper phages. These defense systems provide broad immunity, blocking not only phage reproduction, but also plasmid and non-cognate PICI transfer. Our results demonstrate that phages can mobilize PICI-encoded immunity systems to use them against other mobile genetic elements, which compete with the phages for the same bacterial hosts. Therefore, despite the cost, mobilization of PICIs may be beneficial for phages, PICIs, and bacteria in nature. Our results suggest that PICIs are important players controlling horizontal gene transfer and that PICIs and phages establish mutualistic interactions that drive bacterial ecology and evolution.

Timescales modulate optimal lysis-lysogeny decision switches and near-term phage reproduction.

Temperate phage can initiate lysis or lysogeny after infecting a bacterial host. The genetic switch between lysis and lysogeny is mediated by phage regulatory genes as well as host and environmental factors. Recently, a new class of decision switches was identified in phage of the SPbeta group, mediated by the extracellular release of small, phage-encoded peptides termed arbitrium. Arbitrium peptides can be taken up by bacteria prior to infection, modulating the decision switch in the event of a subsequent phage infection. Increasing the concentration of arbitrium increases the chance that a phage infection will lead to lysogeny, rather than lysis. Although prior work has centered on the molecular mechanisms of arbitrium-induced switching, here we focus on how selective pressures impact the benefits of plasticity in switching responses. In this work, we examine the possible advantages of near-term adaptation of communication-based decision switches used by the SPbeta-like group. We combine a nonlinear population model with a control-theoretic approach to evaluate the relationship between a putative phage reaction norm (i.e. the probability of lysogeny as a function of arbitrium) and the extent of phage reproduction at a near-term time horizon. We measure phage reproduction in terms of a cellular-level metric previously shown to enable comparisons of near-term phage fitness across a continuum from lysis to latency. We show the adaptive potential of communication-based lysis–lysogeny responses and find that optimal switching between lysis and lysogeny increases the near-term phage reproduction compared to fixed responses, further supporting both molecular- and model-based analyses of the putative benefits of this class of decision switches. We further find that plastic responses are robust to the inclusion of cellular-level stochasticity, variation in life history traits, and variation in resource availability. These findings provide further support to explore the long-term evolution of plastic decision systems mediated by extracellular decision-signaling molecules and the feedback between phage reaction norms and ecological context.

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.

The arbitrium system controls prophage induction

SummarySome Bacillus-infecting bacteriophages use a peptide-based communication system, termed arbitrium, to coordinate the lysis-lysogeny decision. In this system, the phage produces AimP peptide during the lytic cycle. Once internalized by the host cell, AimP binds to the transcription factor AimR, reducing aimX expression and promoting lysogeny. Although these systems are present in a variety of mobile genetic elements, their role in the phage life cycle has only been characterized in phage phi3T during phage infection. Here, using the B. subtilis SPβ prophage, we show that the arbitrium system is also required for normal prophage induction. Deletion of the aimP gene increased phage reproduction, although the aimR deletion significantly reduced the number of phage particles produced after prophage induction. Moreover, our results indicated that AimR is involved in a complex network of regulation and brought forward two new players in the SPβ lysis-lysogeny decision system, YopN and the phage repressor YopR. Importantly, these proteins are encoded in an operon, the function of which is conserved across all SPβ-like phages encoding the arbitrium system. Finally, we obtained mutant phages in the arbitrium system, which behaved almost identically to the wild-type (WT) phage, indicating that the arbitrium system is not essential in the laboratory but is likely beneficial for phage fitness in nature. In support of this, by possessing a functional arbitrium system, the SPβ phage can optimize production of infective particles while also preserving the number of cells that survive after prophage induction, a strategy that increases phage persistence in nature.

The Phage Nucleus and PhuZ Spindle: Defining Features of the Subcellular Organization and Speciation of Nucleus-Forming Jumbo Phages.

Bacteriophages and their bacterial hosts are ancient organisms that have been co-evolving for billions of years. Some jumbo phages, those with a genome size larger than 200 kilobases, have recently been discovered to establish complex subcellular organization during replication. Here, we review our current understanding of jumbo phages that form a nucleus-like structure, or “Phage Nucleus,” during replication. The phage nucleus is made of a proteinaceous shell that surrounds replicating phage DNA and imparts a unique subcellular organization that is temporally and spatially controlled within bacterial host cells by a phage-encoded tubulin (PhuZ)-based spindle. This subcellular architecture serves as a replication factory for jumbo Pseudomonas phages and provides a selective advantage when these replicate in some host strains. Throughout the lytic cycle, the phage nucleus compartmentalizes proteins according to function and protects the phage genome from host defense mechanisms. Early during infection, the PhuZ spindle positions the newly formed phage nucleus at midcell and, later in the infection cycle, the spindle rotates the nucleus while delivering capsids and distributing them uniformly on the nuclear surface, where they dock for DNA packaging. During the co-infection of two different nucleus-forming jumbo phages in a bacterial cell, the phage nucleus establishes Subcellular Genetic Isolation that limits the potential for viral genetic exchange by physically separating co-infection genomes, and the PhuZ spindle causes Virogenesis Incompatibility, whereby interacting components from two diverging phages negatively affect phage reproduction. Thus, the phage nucleus and PhuZ spindle are defining cell biological structures that serve roles in both the life cycle of nucleus-forming jumbo phages and phage speciation.

Characterization of a novel roseophage and the morphological and transcriptional response of the sponge symbiont Ruegeria AU67 to infection.

Sponges have recently been recognized to contain complex communities of bacteriophages; however, little is known about how they interact with their bacterial hosts. Here, we isolated a novel phage, called Ruegeria phage Tedan, and characterized its impact on the bacterial sponge symbiont Ruegeria AU67 on a morphological and molecular level. Phage Tedan was structurally, genomically and phylogenetically characterized to be affiliated with the genus Xiamenvirus of the family Siphoviridae. Through microscopic observations and transcriptomic analysis, we show that phage Tedan upon infection induces a process leading to metabolic and morphological changes in its host. These changes would render Ruegeria AU67 better adapted to inhabit the sponge holobiont due to an improved utilization of ecologically relevant energy and carbon sources as well as a potential impediment of phagocytosis by the sponge through cellular enlargement. An increased survival or better growth of the bacterium in the sponge environment will likely benefit the phage reproduction. Our results point towards the possibility that phages from host-associated environments require, and have thus evolved, different strategies to interact with their host when compared to those phages from free-living or planktonic environments.

Characteristics of bacteriophages of the Staphylococcus aureus variant bovis

Bacteriophages may be an alternative method of treatment for antibiotic-resistant bacteria, including mastitis in cows. Our study describes the initial isolation and bacteriological activity of bacteriophages, circulating on dairy farms, the against S. aureus var. bovis. Samples of cow’s milk secretions with signs of mastitis and sewage water were used as the study material. The isolation and production of pure bacteriophage lines were performed according to the double agar method. The method of studying a single cycle of phage reproduction was used to determine the duration of the latency period. Determination of the spectrum of the lytic activity of bacteriophages against the clinical isolates of the microorganisms was carried out by the drop method. As a result of the research, four phages, specific for S. aureus var. bovis were isolated: Phage SAvB07, Phage SAvB08, Phage SAvB12 and Phage SAvB14. The negative colonies of the isolated phages were 1–2 mm in size, rounded with clear edges, with varying degrees of transparency. The latency period of Phage SAvB14 was 35 min, with the number of active virions increasing by 8 orders. In the study on growth curves of other bacteriophages, taken in the experiment, the latency period was more than 35 min, and their titre increased by only two orders. Phage SAvB07, Phage SAvB08 and Phage SAvB12 were able to lyse the bacterial strains of S. aureus var. bovis in 25–45.6% of the cases (low lytic activity), whereas Phage SAvB14 lysed 94.1% of S. aureus strains were isolated from the cows. Studies have shown that among the bacteriophages we have studied, Phage SAvB14 with a short latency period has the best lytic action on the culture S. aureus var. bovis. The resulting bacteriophage strain can be used to create a bacteriophage-based drug for the treatment of mastitis in cows.

Bacteriophage-Derived Endolysins Applied as Potent Biocontrol Agents to Enhance Food Safety.

Endolysins, bacteriophage-encoded enzymes, have emerged as antibacterial agents that can be actively applied in food processing systems as food preservatives to control pathogens and ultimately enhance food safety. Endolysins break down bacterial peptidoglycan structures at the terminal step of the phage reproduction cycle to enable phage progeny release. In particular, endolysin treatment is a novel strategy for controlling antibiotic-resistant bacteria, which are a severe and increasingly frequent problem in the food industry. In addition, endolysins can eliminate biofilms on the surfaces of utensils. Furthermore, the cell wall-binding domain of endolysins can be used as a tool for rapidly detecting pathogens. Research to extend the use of endolysins toward Gram-negative bacteria is now being extensively conducted. This review summarizes the trends in endolysin research to date and discusses the future applications of these enzymes as novel food preservation tools in the field of food safety.

Cyclic GMP-AMP signalling protects bacteria against viral infection.

The cyclic GMP-AMP synthase (cGAS)-STING pathway is a central component of the cell-autonomous innate immune system in animals1,2. The cGAS protein is a sensor of cytosolic viral DNA and, upon sensing DNA, it produces a cyclic GMP-AMP (cGAMP) signalling molecule that binds to the STING protein and activates the immune response3-5. The production of cGAMP has also been detected in bacteria6, and has been shown, in Vibrio cholerae, to activate a phospholipase that degrades the inner bacterial membrane7. However, the biological role of cGAMP signalling in bacteria remains unknown. Here we show that cGAMP signalling is part of an antiphage defence system that is common in bacteria. This system is composed of a four-gene operon that encodes the bacterial cGAS and the associated phospholipase, as well as two enzymes with the eukaryotic-like domains E1, E2 and JAB. We show that this operon confers resistance against a wide variety of phages. Phage infection triggers the production of cGAMP, which-in turn-activates the phospholipase, leading to a loss of membrane integrity and to cell death before completion of phage reproduction. Diverged versions of this system appear in more than 10% of prokaryotic genomes, and we show that variants with effectors other than phospholipase also protect against phage infection. Our results suggest that the eukaryotic cGAS-STING antiviral pathway has ancient evolutionary roots that stem from microbial defences against phages.

Hijacking the Hijackers: Escherichia coli Pathogenicity Islands Redirect Helper Phage Packaging for Their Own Benefit.

SummaryPhage-inducible chromosomal islands (PICIs) represent a novel and universal class of mobile genetic elements, which have broad impact on bacterial virulence. In spite of their relevance, how the Gram-negative PICIs hijack the phage machinery for their own specific packaging and how they block phage reproduction remains to be determined. Using genetic and structural analyses, we solve the mystery here by showing that the Gram-negative PICIs encode a protein that simultaneously performs these processes. This protein, which we have named Rpp (for redirecting phage packaging), interacts with the phage terminase small subunit, forming a heterocomplex. This complex is unable to recognize the phage DNA, blocking phage packaging, but specifically binds to the PICI genome, promoting PICI packaging. Our studies reveal the mechanism of action that allows PICI dissemination in nature, introducing a new paradigm in the understanding of the biology of pathogenicity islands and therefore of bacterial pathogen evolution.

Bacteriophages applications in agriculture

The bacteriophages life cycle has two stages: a lytic stage where the phages reproduce inside the bacteria and lyse bacteria and a lysogenic stage where the phage is in a stationary stage where do not exist phage reproduction. The understanding of the life cycle of phages is fundamental to understand the advantages of phage offers as biological control applications and how engineered phages work. The bacteriophages are an alternative to fight against the antimicrobial or pesticides because phages offer advantages such as high host specificity, the ability of long term effect, are active against dividing or not dividing bacterial cells, effective elimination of biofilms and are capable vehicles for nucleic acids delivery. Phages have been isolated from water or soil samples in different parts of the world and for specific bacterial pathogens. In the following review, in the main topics in bacteriophages and its applications in agriculture: the bacteriophages life cycle, advantages of phages in biological control applications, the last isolated phages and described for different pathogens and the last advances in phage engineering applications for biological control.

Analysis of Host-Takeover During SPO1 Infection of Bacillus subtilis.

When Bacillus subtilis is infected by bacteriophage SPO1, the phage directs the remodeling of the host cell, converting it into a factory for phage reproduction. Much synthesis of host DNA, RNA, and protein is shut off, and cell division is prevented. Here I describe the protocols by which we have demonstrated those processes, and identified the roles played by specific SPO1 gene products in causing those processes.

Analysis of the CRISPR-Cas system in bacteriophages active on epidemic strains of Vibrio cholerae in Bangladesh

CRISPR-Cas (clustered regularly interspersed short palindromic repeats-CRISPR-associated proteins) are microbial nuclease systems involved in defense against phages. Bacteria also resist phages by hosting phage-inducible chromosomal islands (PICI) which prevent phage reproduction. Vibrio cholerae which causes cholera epidemics, interacts with numerous phages in the environment and in cholera patients. Although CRISPR-Cas systems are usually carried by bacteria and archea, recently V. cholerae specific ICP1 phages were found to host a CRISPR-Cas system that inactivates PICI-like elements (PLE) in V. cholerae. We analyzed a collection of phages and V. cholerae isolated during seasonal cholera epidemics in Bangladesh, to study the distribution, and recent evolution of the phage-encoded CRISPR-Cas system. Five distinct but related phages carrying the CRISPR-Cas system, and possible CRISPR-Cas negative progenitor phages were identified. Furthermore, CRISPR arrays in the phages were found to have evolved by acquisition of new spacers targeting diverse regions of PLEs carried by the V. cholerae strains, enabling the phages to efficiently grow on PLE positive strains. Our results demonstrate a continuing arms-race involving genetic determinants of phage-resistance in V. cholerae, and the phage-encoded CRISPR-Cas system in the co-evolution of V. cholerae and its phages, presumably fostered by their enhanced interactions during seasonal epidemics of cholera.

Sak and Sak4 recombinases are required for bacteriophage replication in Staphylococcus aureus.

DNA-single strand annealing proteins (SSAPs) are recombinases frequently encoded in the genome of many bacteriophages. As SSAPs can promote homologous recombination among DNA substrates with an important degree of divergence, these enzymes are involved both in DNA repair and in the generation of phage mosaicisms. Here, analysing Sak and Sak4 as representatives of two different families of SSAPs present in phages infecting the clinically relevant bacterium Staphylococcus aureus, we demonstrate for the first time that these enzymes are absolutely required for phage reproduction. Deletion of the genes encoding these enzymes significantly reduced phage replication and the generation of infectious particles. Complementation studies revealed that these enzymes are required both in the donor (after prophage induction) and in the recipient strain (for infection). Moreover, our results indicated that to perform their function SSAPs require the activity of their cognate single strand binding (Ssb) proteins. Mutational studies demonstrated that the Ssb proteins are also required for phage replication, both in the donor and recipient strain. In summary, our results expand the functions attributed to the Sak and Sak4 proteins, and demonstrate that both SSAPs and Ssb proteins are essential for the life cycle of temperate staphylococcal phages.

Phage-inducible islands in the Gram-positive cocci.

The SaPIs are a cohesive subfamily of extremely common phage-inducible chromosomal islands (PICIs) that reside quiescently at specific att sites in the staphylococcal chromosome and are induced by helper phages to excise and replicate. They are usually packaged in small capsids composed of phage virion proteins, giving rise to very high transfer frequencies, which they enhance by interfering with helper phage reproduction. As the SaPIs represent a highly successful biological strategy, with many natural Staphylococcus aureus strains containing two or more, we assumed that similar elements would be widespread in the Gram-positive cocci. On the basis of resemblance to the paradigmatic SaPI genome, we have readily identified large cohesive families of similar elements in the lactococci and pneumococci/streptococci plus a few such elements in Enterococcus faecalis. Based on extensive ortholog analyses, we found that the PICI elements in the four different genera all represent distinct but parallel lineages, suggesting that they represent convergent evolution towards a highly successful lifestyle. We have characterized in depth the enterococcal element, EfCIV583, and have shown that it very closely resembles the SaPIs in functionality as well as in genome organization, setting the stage for expansion of the study of elements of this type. In summary, our findings greatly broaden the PICI family to include elements from at least three genera of cocci.

Differential Effects of Physical and Chemical Factors on Infectivity of Three Coliphages used as Water Quality Indicator

The effect of temperature, pH, UV, petroleum ether (60-80), acetone (99 %), ethyl alcohol (95 %) and chloroform (99 %) on the infectivity of local coliphage isolates was studied. Three E. coli phages (ECP) were isolated from Ismailia canal at Mostorod segment. Ability of local E. coli isolates to serve as a host was evaluated after storage at 4-6o C, 25-28o C and 35-38o C for six months. E. coli isolate number 1 of seven tested served as host for phage reproduction and plaque formation (PF). Phage isolates were identified by plaque size and electron microscopy. ECP 1 and ECP 2 are Podoviridae and ECP3 is a Siphoviridae. The infectivity of coliphages decreased with increasing storage periods and incubation temperature. Exposure of somatic coliphages to acidic or alkaline pH, organic solvents or UV radiation (240 nm) or heating (≥ 60o C) diminished infectivity. Ability of E. coli 1 to serve as a host for somatic coliphages inversely related to its age. No detectable effect was found for lower temperature ≤ 5o C on the infectivity of coliphage isolates during storage periods

Structural characterizations of phage antitoxin Dmd and its interactions with bacterial toxin RnlA

Toxin–antitoxin (TA) loci are widespread in bacteria plasmids and chromosomes, and target various cellular functions to regulate cell growth and death. A type II TA system RnlA–RnlB from Escherichia coli is associated with phage-resistance. After the infection of bacteriophage T4 with Dmd defection, RnlA is activated by the disappearance of RnlB, resulting in the rapid degradation of T4 mRNAs. Dmd can bind to RnlA directly and neutralize RnlA toxicity to allow phage reproduction. Dmd represent a heterogenous antitoxin of RnlA replacing antitoxin RnlB. Here, we reported two structures of Dmd from T4 phage and RB69 phage. Both Dmd structures are high similar with a compacted domain composed of a four-stranded anti-parallel β-sheet and an α-helix. Chromatography and SAXS suggest Dmd forms a dimer in solution consistent with that in crystal. Structure-based mutagenesis of Dmd reveals key residues involved in RnlA-binding. Possibility cavities in Dmd used for compounds design were modeled. Our structural study revealed the recognition and inhibition mechanism of RnlA by Dmd and providing a potential laboratory phage prevention target for drug design.