Pseudomonas PB1-Like Phages: Whole Genomes from Metagenomes Offer Insight into an Abundant Group of Bacteriophages.
ABSTRACT: Despite the abundance, ubiquity and impact of environmental viruses, their inherent genomic plasticity and extreme diversity pose significant challenges for the examination of bacteriophages on Earth. Viral metagenomic studies have offered insight into broader aspects of phage ecology and repeatedly uncover genes to which we are currently unable to assign function. A combined effort of phage isolation and metagenomic survey of Chicago’s nearshore waters of Lake Michigan revealed the presence of Pbunaviruses, relatives of the Pseudomonas phage PB1. This prompted our expansive investigation of PB1-like phages. Genomic signatures of PB1-like phages and Pbunaviruses were identified, permitting the unambiguous distinction between the presence/absence of these phages in soils, freshwater and wastewater samples, as well as publicly available viral metagenomic datasets. This bioinformatic analysis led to the de novo assembly of nine novel PB1-like phage genomes from a metagenomic survey of samples collected from Lake Michigan. While this study finds that Pbunaviruses are abundant in various environments of Northern Illinois, genomic variation also exists to a considerable extent within individual communities.
Project description:The study of bacteriophages continues to generate key information about microbial interactions in the environment. Many phenotypic characteristics of bacteriophages cannot be examined by sequencing alone, further highlighting the necessity for isolation and examination of phages from environmental samples. While much of our current knowledge base has been generated by the study of marine phages, freshwater viruses are understudied in comparison. Our group has previously conducted metagenomics-based studies samples collected from Lake Michigan - the data presented in this study relate to four phages that were extracted from the same samples.Four phages were extracted from Lake Michigan on the same bacterial host, exhibiting similar morphological characteristics as shown under transmission electron microscopy. Growth characteristics of the phages were unique to each isolate. Each phage demonstrated a host-range spanning several phyla of bacteria - to date, such a broad host-range is yet to be reported. Genomic data reveals genomes of a similar size, and close similarities between the Lake Michigan phages and the Pseudomonas phage PB1, however, the majority of annotated genes present were ORFans and little insight was offered into mechanisms for host-range.The phages isolated from Lake Michigan are capable of infecting several bacterial phyla, and demonstrate varied phenotypic characteristics despite similarities in host preference, and at the genomic level. We propose that such a broad host-range is likely related to the oligotrophic nature of Lake Michigan, and the competitive benefit that this characteristic may lend to phages in nature.
Project description:Pseudomonas aeruginosa causes serious intractable infections in humans and animals. Bacteriophage (phage) therapy has been applied to treat P. aeruginosa infections, and phages belonging to the PB1-like virus genus in the Myoviridae family have been used as therapeutic phages. To achieve safer and more effective phage therapy, the use of preadapted phages is proposed. To understand in detail such phage preadaptation, the short-term antagonistic evolution of bacteria and phages should be studied. In this study, the short-term antagonistic evolution of bacteria and PB1-like phage was examined by studying phage-resistant clones of P. aeruginosa strain PAO1 and mutant PB1-like phages that had recovered their infectivity. First, phage KPP22 was isolated and characterized; it was classified as belonging to the PB1-like virus genus in the Myoviridae family. Subsequently, three KPP22-resistant PAO1 clones and three KPP22 mutant phages capable of infecting these clones were isolated in three sets of in vitro experiments. It was shown that the bacterial resistance to phage KPP22 was caused by significant decreases in phage adsorption and that the improved infectivity of KPP22 mutant phages was caused by significant increases in phage adsorption. The KPP22-resistant PAO1 clones and the KPP22 mutant phages were then analyzed genetically. All three KPP22-resistant PAO1 clones, which were deficient for the O5 antigen, had a common nonsense mutation in the wzy gene. All the KPP22 mutant phage genomes showed the same four missense mutations in the open reading frames orf060, orf065, and orf086 The information obtained in this study should be useful for further development of safe and efficient phage therapy.Pseudomonas aeruginosa causes serious intractable infections in humans and animals; bacteriophage (phage) therapy has been utilized to treat P. aeruginosa infections, and phages that belong to the PB1-like virus genus in the family Myoviridae have been used as therapeutic phages. The preadapted phage is trained in advance through the antagonistic evolution of bacteria and phage and is proposed to be used to achieve safer and more effective phage therapy. In this study, to understand the phage preadaptation, the in vitro short-term antagonistic evolution was studied using P. aeruginosa strain PAO1 and the newly isolated PB1-like phage KPP22. Phage KPP22 was characterized, and the molecular framework regarding the phage preadaptation of KPP22 was elucidated. The importance of study of antagonistic evolution of bacteria and phage in phage therapy is discussed.
Project description:Bacteriophages of freshwater environments have not been well studied despite their numerical dominance and ecological importance. Currently, very few phages have been isolated for many abundant freshwater bacterial groups, especially for the family Comamonadaceae that is found ubiquitously in freshwater habitats. In this study, we report two novel phages, P26059A and P26059B, that were isolated from Lake Soyang in South Korea, and lytically infected bacterial strain IMCC26059, a member of the family Comamonadaceae. Morphological observations revealed that phages P26059A and P26059B belonged to the family Siphoviridae and Podoviridae, respectively. Of 12 bacterial strains tested, the two phages infected strain IMCC26059 only, showing a very narrow host range. The genomes of the two phages were different in length and highly distinct from each other with little sequence similarity. A comparison of the phage genome sequences and freshwater viral metagenomes showed that the phage populations represented by P26059A and P26059B exist in the environment with different distribution patterns. Presence of the phages in Lake Soyang and Lake Michigan also indicated a consistent lytic infection of the Comamonadaceae bacterium, which might control the population size of this bacterial group. Taken together, although the two phages shared a host strain, they showed completely distinctive characteristics from each other in morphological, genomic, and ecological analyses. Considering the abundance of the family Comamonadaceae in freshwater habitats and the rarity of phage isolates infecting this family, the two phages and their genomes in this study would be valuable resources for freshwater virus research.
Project description:BACKGROUND: Pseudomonas aeruginosa causes lung infections in patients suffering from the genetic disorder Cystic Fibrosis (CF). Once a chronic lung infection is established, P. aeruginosa cannot be eradicated by antibiotic treatment. Phage therapy is an alternative to treat these chronic P. aeruginosa infections. However, little is known about the factors which influence phage infection of P. aeruginosa under infection conditions and suitable broad host range phages. RESULTS: We isolated and characterized a phage, named JG024, which infects a broad range of clinical and environmental P. aeruginosa strains. Sequencing of the phage genome revealed that the phage JG024 is highly related to the ubiquitous and conserved PB1-like phages. The receptor of phage JG024 was determined as lipopolysaccharide. We used an artificial sputum medium to study phage infection under conditions similar to a chronic lung infection. Alginate production was identified as a factor reducing phage infectivity. CONCLUSIONS: Phage JG024 is a suitable broad host range phage which could be used in phage therapy. Phage infection experiments under simulated chronic lung infection conditions showed that alginate production reduces phage infection efficiency.
Project description:Cyanobacteria are one of the dominant autotrophs in tropical freshwater communities, yet phages infecting them remain poorly characterized. Here we present the characterization of cyanophage S-SRP02, isolated from a tropical freshwater lake in Singapore, which infects <i>Synechococcus</i> sp. Strain SR-C1 isolated from the same lake. S-SRP02 represents a new evolutionary lineage of cyanophage. Out of 47 open reading frames (ORFs), only 20 ORFs share homology with genes encoding proteins of known function. There is lack of auxiliary metabolic genes which was commonly found as core genes in marine cyanopodoviruses. S-SRP02 also harbors unique structural genes highly divergent from other cultured phages. Phylogenetic analysis and viral proteomic tree further demonstrate the divergence of S-SRP02 from other sequenced phage isolates. Nonetheless, S-SRP02 shares synteny with phage genes of uncultured phages obtained from the Mediterranean Sea deep chlorophyll maximum fosmids, indicating the ecological importance of S-SRP02 and its related viruses. This is further supported by metagenomic mapping of environmental viral metagenomic reads onto the S-SRP02 genome.
Project description:In this study, we characterize three phages (SL1 SL2, and SL4), isolated from hospital sewage with lytic activity against clinical isolates of multi-drug resistant Pseudomonas aeruginosa (MDR-PA). The host spectrum ranged from 41% to 54%, with all three phages together covering 79% of all tested clinical isolates. Genome analysis revealed that SL1 (65,849 bp, 91 open reading frames ORFs) belongs to PB1-like viruses, SL2 (279,696 bp, 354 ORFs) to phiKZ-like viruses and SL4 (44,194 bp, 65 ORFs) to LUZ24-like viruses. Planktonic cells of four of five selected MDR-PA strains were suppressed by at least one phage with multiplicities of infection (MOIs) ranging from 1 to 10-6 for 16 h without apparent regrowth of bacterial populations. While SL2 was most potent in suppressing planktonic cultures the strongest anti-biofilm activity was observed with SL4. Phages were able to rescue bacteria-infected wax moth larvae (Galleria melonella) for 24 h, whereby highest survival rates (90%) were observed with SL1. Except for the biofilm experiments, the effect of a cocktail with all three phages was comparable to the action of the best phage alone; hence, there are no synergistic but also no antagonistic effects among phages. The use of a cocktail with these phages is therefore expedient for increasing host range and minimizing the development of phage resistance.
Project description:Bacteriophages have a significant impact on the structure and function of marine microbial communities. Phages of some major bacterial lineages have recently been shown to dominate the marine viral communities. However, phages that infect many important bacterial clades still remained unexplored. Members of the marine OM43 clade are methylotrophs that play important roles in C1 metabolism. OM43 phages (phages that infect the OM43 bacteria) represent an understudied viral group with only one known isolate. In this study, we describe the genomic characterization and biogeography of an OM43 phage that infects the strain HTCC2181, designated MEP301. MEP301 has a genome size of 34,774 bp. We found that MEP301 is genetically distinct from other known phage isolates and only displays significant sequence similarity with some metagenomic viral genomes (MVGs). A total of 12 MEP301-type MVGs were identified from metagenomic datasets. Comparative genomic and phylogenetic analyses revealed that MEP301-type phages can be separated into two subgroups (subgroup I and subgroup II). We also performed a metagenomic recruitment analysis to determine the relative abundance of reads mapped to these MEP301-type phages, which suggested that subgroup I MEP301-type phages are present predominantly in the cold upper waters with lower salinity. Notably, subgroup II phages have an inverse different distribution pattern, implying that they may infect hosts from a distinct OM43 subcluster. Our study has expanded the knowledge about the genomic diversity of marine OM43 phages and identified a new phage group that is widespread in the ocean.
Project description:<i>Pseudomonas</i> phage MD8 is a temperate phage isolated from the freshwater lake Baikal. The organisation of the MD8 genome resembles the genomes of lambdoid bacteriophages. However, MD8 gene and protein sequences have little in common with classified representatives of lambda-like phages. Analysis of phage genomes revealed a group of other <i>Pseudomonas</i> phages related to phage MD8 and the genomic layout of MD8-like phages indicated extensive gene exchange involving even the most conservative proteins and leading to a high degree of genomic mosaicism. Multiple horizontal transfers and mosaicism of the genome of MD8, related phages and other λ-like phages raise questions about the principles of taxonomic classification of the representatives of this voluminous phage group. Comparison and analysis of various bioinformatic approaches applied to λ-like phage genomes demonstrated different efficiency and contradictory results in the estimation of genomic similarity and relatedness. However, we were able to make suggestions for the possible origin of the MD8 genome and the basic principles for the taxonomic classification of lambdoid phages. The group comprising 26 MD8-related phages was proposed to classify as two close genera belonging to a big family of λ-like phages.
Project description:Phages are viruses that infect bacteria. The phages can be classified into two different categories based on their lifestyles: temperate and lytic. Now, the metavirome can generate a large number of fragments from the viral genomic sequences of entire environmental community, which makes it impossible to determine their lifestyles through experiments. Thus, there is a need to development computational methods for annotating phage contigs and making prediction of their lifestyles. Alignment-based methods for classifying phage lifestyle are limited by incomplete assembled genomes and nucleotide databases. Alignment-free methods based on the frequencies of <i>k</i>-mers were widely used for genome and metagenome comparison which did not rely on the completeness of genome or nucleotide databases. To mimic fragmented metagenomic sequences, the temperate and lytic phages genomic sequences were split into non-overlapping fragments with different lengths, then, I comprehensively compared nine alignment-free dissimilarity measures with a wide range of choices of <i>k</i>-mer length and Markov orders for predicting the lifestyles of these phage contigs. The dissimilarity measure, d2S , performed better than other dissimilarity measures for classifying the lifestyles of phages. Thus, I propose that the alignment-free method, d2S , can be used for predicting the lifestyles of phages which derived from the metagenomic data.
Project description:Bacteriophages, as the most abundant biological entities on Earth, place significant predation pressure on their hosts. This pressure plays a critical role in the evolution, diversity, and abundance of bacteria. In addition, phages modulate the genetic diversity of prokaryotic communities through the transfer of auxiliary metabolic genes. Various studies have been conducted in diverse ecosystems to understand phage-host interactions and their effects on prokaryote metabolism and community composition. However, hypersaline environments remain among the least studied ecosystems and the interaction between the phages and prokaryotes in these habitats is poorly understood. This study begins to fill this knowledge gap by analyzing bacteriophage-host interactions in the Great Salt Lake, the largest prehistoric hypersaline lake in the Western Hemisphere. Our metagenomics analyses allowed us to comprehensively identify the bacterial and phage communities with Proteobacteria, Firmicutes, and Bacteroidetes as the most dominant bacterial species and Siphoviridae, Myoviridae, and Podoviridae as the most dominant viral families found in the metagenomic sequences. We also characterized interactions between the phage and prokaryotic communities of Great Salt Lake and determined how these interactions possibly influence the community diversity, structure, and biogeochemical cycles. In addition, presence of prophages and their interaction with the prokaryotic host was studied and showed the possibility of prophage induction and subsequent infection of prokaryotic community present in the Great Salt Lake environment under different environmental stress factors. We found that carbon cycle was the most susceptible nutrient cycling pathways to prophage induction in the presence of environmental stresses. This study gives an enhanced snapshot of phage and prokaryote abundance and diversity as well as their interactions in a hypersaline complex ecosystem, which can pave the way for further research studies.