Isolation and Characterization of vB_ArS-ArV2 – First Arthrobacter sp. Infecting Bacteriophage with Completely Sequenced Genome

This is the first report on a complete genome sequence and biological characterization of the phage that infects Arthrobacter. A novel virus vB_ArS-ArV2 (ArV2) was isolated from soil using Arthrobacter sp. 68b strain for phage propagation. Based on transmission electron microscopy, ArV2 belongs to the family Siphoviridae and has an isometric head (∼63 nm in diameter) with a non-contractile flexible tail (∼194×10 nm) and six short tail fibers. ArV2 possesses a linear, double-stranded DNA genome (37,372 bp) with a G+C content of 62.73%. The genome contains 68 ORFs yet encodes no tRNA genes. A total of 28 ArV2 ORFs have no known functions and lack any reliable database matches. Proteomic analysis led to the experimental identification of 14 virion proteins, including 9 that were predicted by bioinformatics approaches. Comparative phylogenetic analysis, based on the amino acid sequence alignment of conserved proteins, set ArV2 apart from other siphoviruses. The data presented here will help to advance our understanding of Arthrobacter phage population and will extend our knowledge about the interaction between this particular host and its phages.     

一株类志贺邻单胞菌噬菌体生物学特性及全基因组分析

[目的]多重耐药菌株的出现给食品安全带来严重威胁.噬菌体是不同于抗生素的一类重要杀菌因子,对其生物学特性及基因组的研究和分析可为噬菌体的抗菌应用提供依据.[方法]对噬菌体phiP4-7的生物学特性、基因组学、分类学进行研究.[结果]经透射电子显微镜观察,确定phiP4-7头部直径为(50.59±1.68) nm,尾部长...     

Characterization and Genome Sequencing of Phage Abp1, a New phiKMV-Like Virus Infecting Multidrug-Resistant Acinetobacter baumannii

While screening for alternative antibiotics against multidrug-resistant Acinetobacter baumannii, we isolated a virulent A. baumannii bacteriophage Abp1. Transmission electron microscopy revealed that the phage had an icosahedral head with a short tail and should be classified as a member of the Podoviridae family. SDS-PAGE showed that Abp1 contained at least one major and nine minor proteins. In a single-step growth test, we demonstrated that Abp1 had a latent period of 10 min and a burst size of 350. Abp1 also had a relatively narrow host range. The entire genome was sequenced, and the final assembly yielded a 42,185 bp, linear, double-stranded DNA molecule with a G+C content of 39.15 % and containing 54 putative genes. Among these genes, 26 were functionally known, leaving 28 unknown putative genes. Abp1 is a new member of the phiKMV-like virus subgroup of the T7 group; its genome sequence is very similar to that of the A. baumannii phage phiAB1.     

一株产L-天冬氨酸酶大肠埃希氏菌的噬菌体分离和生物学特性

【目的】从大肠埃希氏菌CICC 11021S发酵液中分离一株噬菌体,对其生物学特性进行研究。【方法】采用双层平板法分离噬菌体CICC 80003;利用透射电镜观察噬菌体形态;提取噬菌体基因组,核酸内切酶处理并进行凝胶电泳;分析噬菌体最佳感染复数、一步生长曲线、p H和温度稳定性、宿主谱。考察CICC 80003对CICC 11021S生长和L-天冬氨酸酶活力的影响。【结果】CICC 80003噬菌斑圆形透明,有明显晕环;头部规则,直径约50-60 nm,尾部长约120-130 nm;基因组能被核酸内切酶Bam H I和Mlu I切开;最佳感染复数0.1,潜伏期5 min,裂解期25 min,平均裂解量约86个;最适p H值8.0;90°C温育15 min,噬菌体全部失活;能裂解大肠埃希氏菌和沙门氏菌的部分菌株。发生噬菌体污染时,CICC 11021S无法正常生长,基本检测不到L-天冬氨酸酶活力。【结论】CICC 80003属于长尾噬菌体科ds DNA噬菌体,液体环境中能够彻底裂解大肠埃希氏菌CICC 11021S。     

A Virulent Phage Infecting Lactococcus garvieae,with Homology to Lactococcus lactis Phages

A new virulent phage belonging to the Siphoviridae family and able to infect Lactococcus garvieae strains was isolated from compost soil. Phage GE1 has a prolate capsid (56 by 38 nm) and a long noncontractile tail (123 nm). It had a burst size of 139 and a latent period of 31 min. Its host range was limited to only two L. garvieae strains out of 73 tested. Phage GE1 has a double-stranded DNA genome of 24,847 bp containing 48 predicted open reading frames (ORFs). Putative functions could be assigned to only 14 ORFs, and significant matches in public databases were found for only 17 ORFs, indicating that GE1 is a novel phage and its genome contains several new viral genes and encodes several new viral proteins. Of these 17 ORFs, 16 were homologous to deduced proteins of virulent phages infecting the dairy bacterium Lactococcus lactis, including previously characterized prolate-headed phages. Comparative genome analysis confirmed the relatedness of L. garvieae phage GE1 to L. lactis phages c2 (22,172 bp) and Q54 (26,537 bp), although its genome organization was closer to that of phage c2. Phage GE1 did not infect any of the 58 L. lactis strains tested. This study suggests that phages infecting different lactococcal species may have a common ancestor.     

Genomic analyses of a novel bacteriophage (VB_PmiS-Isfahan) within Siphoviridae family infecting Proteus mirabilis

Proteus mirabilis is one of the most common causes of complicated urinary tract infections (UTI), especially in catheter-associated UTIs. The increased resistance to antibiotics, among P. mirabilis isolates has led us to search for alternative antibacterial agents. In this study, genome of a lytic Proteus phage VB_PmiS-Isfahan, isolated from wastewater, and active against planktonic and biofilms of P. mirabilis, isolated from UTI, was analyzed. Accordingly, the genome was sequenced and its similarity to other phages was assessed by the Mauve and EasyFig softwares. “One Click” was used for phylogenetic tree construction. The complete genome of VB_PmiS-Isfahan was 54,836 bp, dsDNA with a G+C content of 36.09%. Nighty-one open reading frames (ORFs) was deduced, among them, 23 were considered as functional genes, based on the homology to the previously characterized proteins. The BLASTn of VB_PmiS-Isfahan showed low similarity to complete genome of Salmonella phages VB_SenS_Sasha, 9NA, and VB_SenS-Sergei. A comparison of Nucleic acid and amino acid sequence, and phylogenetic analyses indicated that the phage is novel, significantly differs, and is distant from other genera, within Siphoviridae family. No virulence-associated and antibiotic resistance genes were detected. Thus, VB_PmiS-Isfahan phage is suggested as a potential novel candidate for the treatment of diseases, caused by P. mirabilis.     

Isolation,growth and genome of the Rhodothermus RM378 thermophilic bacteriophage

Several bacteriophages that infect different strains of the thermophilic bacterium Rhodothermus marinus were isolated and their infection pattern was studied. One phage, named RM378 was cultivated and characterized. The RM378 genome was also sequenced and analyzed. The phage was grouped as a member of the Myoviridae family with A2 morphology. It had a moderately elongated head, with dimensions of 85 and 95 nm between opposite apices and a 150 nm long tail, attached with a connector to the head. RM378 showed a virulent behavior that followed a lytic cycle of infection. It routinely gave lysates with 10¹¹ pfu/ml, and sometimes reached titers as high as 10¹³ pfu/ml. The titer remained stable up to 65 °C but the phage lost viability when incubated at higher temperatures. Heating for 30 min at 96 °C lowered the titer by 10⁴. The RM378 genome consisted of ds DNA of 129.908 bp with a GC ratio of 42.0 % and contained about 120 ORFs. A few structural proteins, such as the major head protein corresponding to the gp23 in T4, could be identified. Only 29 gene products as probable homologs to other proteins of known function could be predicted, with most showing only low similarity to known proteins in other bacteriophages. These and other studies based on sequence analysis of a large number of phage genomes showed RM378 to be distantly related to all other known T4-like phages.     

Comparative genomic analysis of Lactococcus garvieae phage WP-2, a new member of Picovirinae subfamily of Podoviridae

To date, a few numbers of bacteriophages that infect Lactococcus garvieae have been identified, but their complete genome sequences have not yet been investigated. For the first time, herein, the complete DNA sequence of a new phage of L. garvieae (phage WP-2) is reported and analyzed. The morphological characteristics indicated that the phage had a small isometric head along with a short and non-contractile tail, suggesting that WP-2 belongs to the family Podoviridae. Bioinformatic analysis revealed that phage WP-2 can be classified as a new member of Ahjdlikevirus in the Picovirinae subfamily because it had a small dsDNA of 18,899 bp with 24 open reading frames and a protein-primed DNA polymerase. The phage nucleotide sequence and predicted protein products have been identified to share very limited evidence of homology with complete genome and proteome of other phages. To our knowledge, this is the first Ahjdlikevirus bacteriophage which can infect a member of the Lactococcus genus.     

Physical characterization of bacteriophage MX4, a generalized transducing phage for Myxococcus xanthus.

A generalized transducing bacteriophage of Myxococcus xanthus has been examined. The phage particle consists of an isometric head and a contractile tail. The genome of the phage is a linear DNA molecule of molecular weight 39 ± 2.1 × 10⁶, which contains the normal DNA bases 70% of which are guanosine + cytosine. No overall heterogeneity of base composition is present. The DNA does not carry easily detectable cohesive ends nor is it cyclically permuted. It does contain a large and somewhat variable terminal redundancy. Heating phage particles in the presence of EDTA causes tail sheath contraction and ejection of DNA, some of which remains attached to the tail. Digestion of tail-bound DNA with restriction enzymes shows that the phage tail can be attached to either end of the DNA. Thus the DNA probably contains recognition sites for the packaging of its DNA at both ends. These results suggest possible mechanisms for the genesis of transducing particles by phage MX4.     

Genome Sequence of Temperate Vibrio parahaemolyticus Bacteriophage vB_VpaS_MAR10

Vibrio parahaemolyticus is recognized as one of the main causes of human gastroenteritis associated with seafood. We have fully sequenced the genome of a newly isolated phage, vB_VpaS_MAR10, which lysed 61.9% of the V. parahaemolyticus strains tested. Phage MAR10 is a temperate siphovirus, and its genome consists of double-stranded DNA (dsDNA) with a size of 78,751 bp, a G+C content of 49.70%, and 104 open reading frames. Bioinformatic analysis shows that phage MAR10 is closely related to Vibrio phage SSP002.     

A Novel Vibriophage vB＿VcaS＿HC Containing Lysogeny-Related Gene Has Strong Lytic Ability against Pathogenic Bacteria

To avoid the negative effects of antibiotics, using phage to prevent animal disease becomes a promising method in aquaculture. Here, a lytic phage provisionally named v B＿VcaS＿HC that can infect the pathogen(i.e., Vibrio campbellii 18)of prawn was isolated. The phage has an isometric head and a non-contractile tail. During phage infection, the induced host mortality in 5.5 h reached ca. 96%, with a latent period of 1.5 h and a burst size of 172 PFU/cell. It has an 81,566 bp circular dsDNA genome containing 121 open reading frames(ORFs), and ca. 71% of the ORFs are functionally unknown.Comparative genomic and phylogenetic analysis revealed that it is a novel phage belonging to Delepquintavirus,Siphoviridae, Caudovirales. In the phage genome, besides the ordinary genes related to structure assembly and DNA metabolism, there are 10 auxiliary metabolic genes. For the first time, the pyruvate phosphate dikinase(PPDK) gene was found in phages whose product is a key rate-limiting enzyme involving Embden-Meyerhof-Parnas(EMP) reaction.Interestingly, although the phage has a strong bactericidal activity and contains a potential lysogeny related gene, i.e., the recombinase(RecA) gene, we did not find the phage turned into a lysogenic state. Meanwhile, the phage genome does not contain any bacterial virulence gene or antimicrobial resistance gene. This study represents the first comprehensive characterization of a lytic V. campbellii phage and indicates that it is a promising candidate for the treatment of V.campbellii infections.     

Characterization of Two Virulent Phages of Lactobacillus plantarum

We characterized two Lactobacillus plantarum virulent siphophages, ATCC 8014-B1 (B1) and ATCC 8014-B2 (B2), previously isolated from corn silage and anaerobic sewage sludge, respectively. Phage B2 infected two of the eight L. plantarum strains tested, while phage B1 infected three. Phage adsorption was highly variable depending on the strain used. Phage defense systems were found in at least two L. plantarum strains, LMG9211 and WCSF1. The linear double-stranded DNA genome of the pac-type phage B1 had 38,002 bp, a G+C content of 47.6%, and 60 open reading frames (ORFs). Surprisingly, the phage B1 genome has 97% identity with that of Pediococcus damnosus phage clP1 and 77% identity with that of L. plantarum phage JL-1; these phages were isolated from sewage and cucumber fermentation, respectively. The double-stranded DNA (dsDNA) genome of the cos-type phage B2 had 80,618 bp, a G+C content of 36.9%, and 127 ORFs with similarities to those of Bacillus and Lactobacillus strains as well as phages. Some phage B2 genes were similar to ORFs from L. plantarum phage LP65 of the Myoviridae family. Additionally, 6 tRNAs were found in the phage B2 genome. Protein analysis revealed 13 (phage B1) and 9 (phage B2) structural proteins. To our knowledge, this is the first report describing such high identity between phage genomes infecting different genera of lactic acid bacteria.     

Purified Membrane-Containing Procapsids of Bacteriophage PRD1 Package the Viral Genome

Icosahedral-tailed double-stranded DNA (dsDNA) bacteriophages and herpesviruses translocate viral DNA into a preformed procapsid in an ATP-driven reaction by a packaging complex that operates at a portal vertex. A similar packaging system operates in the tailless dsDNA phage PRD1 (Tectiviridae family), except that there is an internal membrane vesicle in the procapsid. The unit-length linear dsDNA genome with covalently linked 5′-terminal proteins enters the procapsid through a unique vertex. Two small integral membrane proteins, P20 and P22, provide a conduit for DNA translocation. The packaging machinery also contains the packaging ATPase P9 and the packaging efficiency factor P6. Here we describe a method used to obtain purified packaging-competent PRD1 procapsids. The optimized in vitro packaging system allowed efficient packaging of defined DNA substrates. We determined that the genome terminal protein P8 is necessary for packaging and provided an estimation of the packaging rate.     

Heterocyst division in two blue-green algae

Bacteriophage 16-6-12 of Rhizobium lupini has a long, non-contractile tail and a head which is hexagonal in outline. The tail is 140 nm in length, 11 nm in diameter, and carries a short terminal fiber. Analysis of the tail structure by optical diffraction indicates that it is of the helical “stacked disc” type. After phenol-extraction from purified particles, the DNA of phage 16-6-12 can circularize in vitro. No significant difference in contour length was observed between the linear (14.34±0.28 μm) and circular (14.44±0.24 μm) forms of molecules. After partial denaturation with alkali an AT-GC-map was constructed, which shows an asymmetric distribution of AT- and GC-rich regions. It is concluded that this phage DNA can circularize due to the presence of cohesive ends and that it is not circularly permuted.     

Complete genome sequence of the giant Pseudomonas phage Lu11

The complete genome sequence of the giant Pseudomonas phage Lu11 was determined, comparing 454 and Sanger sequencing. The double-stranded DNA (dsDNA) genome is 280,538 bp long and encodes 391 open reading frames (ORFs) and no tRNAs. The closest relative is Ralstonia phage φRSL1, encoding 40 similar proteins. As such, Lu11 can be considered phylogenetically unique within the Myoviridae and indicates the diversity of the giant phages within this family.     

Novel Virulent and Broad-Host-Range Erwinia amylovora Bacteriophages Reveal a High Degree of Mosaicism and a Relationship to Enterobacteriaceae Phages

A diverse set of 24 novel phages infecting the fire blight pathogen Erwinia amylovora was isolated from fruit production environments in Switzerland. Based on initial screening, four phages (L1, M7, S6, and Y2) with broad host ranges were selected for detailed characterization and genome sequencing. Phage L1 is a member of the Podoviridae, with a 39.3-kbp genome featuring invariable genome ends with direct terminal repeats. Phage S6, another podovirus, was also found to possess direct terminal repeats but has a larger genome (74.7 kbp), and the virus particle exhibits a complex tail fiber structure. Phages M7 and Y2 both belong to the Myoviridae family and feature long, contractile tails and genomes of 84.7 kbp (M7) and 56.6 kbp (Y2), respectively, with direct terminal repeats. The architecture of all four phage genomes is typical for tailed phages, i.e., organized into function-specific gene clusters. All four phages completely lack genes or functions associated with lysogeny control, which correlates well with their broad host ranges and indicates strictly lytic (virulent) lifestyles without the possibility for host lysogenization. Comparative genomics revealed that M7 is similar to E. amylovora virus ΦEa21-4, whereas L1, S6, and Y2 are unrelated to any other E. amylovora phage. Instead, they feature similarities to enterobacterial viruses T7, N4, and ΦEcoM-GJ1. In a series of laboratory experiments, we provide proof of concept that specific two-phage cocktails offer the potential for biocontrol of the pathogen.     

Characterization of Bacteriophages Cp1 and Cp2, the Strain-Typing Agents for Xanthomonas axonopodis pv. citri

The strains of Xanthomonas axonopodis pv. citri, the causative agent of citrus canker, are historically classified based on bacteriophage (phage) sensitivity. Nearly all X. axonopodis pv. citri strains isolated from different regions in Japan are lysed by either phage Cp1 or Cp2; Cp1-sensitive (Cp1^s) strains have been observed to be resistant to Cp2 (Cp2^r) and vice versa. In this study, genomic and molecular characterization was performed for the typing agents Cp1 and Cp2. Morphologically, Cp1 belongs to the Siphoviridae. Genomic analysis revealed that its genome comprises 43,870-bp double-stranded DNA (dsDNA), with 10-bp 3′-extruding cohesive ends, and contains 48 open reading frames. The genomic organization was similar to that of Xanthomonas phage phiL7, but it lacked a group I intron in the DNA polymerase gene. Cp2 resembles morphologically Escherichia coli T7-like phages of Podoviridae. The 42,963-bp linear dsDNA genome of Cp2 contained terminal repeats. The Cp2 genomic sequence has 40 open reading frames, many of which did not show detectable homologs in the current databases. By proteomic analysis, a gene cluster encoding structural proteins corresponding to the class III module of T7-like phages was identified on the Cp2 genome. Therefore, Cp1 and Cp2 were found to belong to completely different virus groups. In addition, we found that Cp1 and Cp2 use different molecules on the host cell surface as phage receptors and that host selection of X. axonopodis pv. citri strains by Cp1 and Cp2 is not determined at the initial stage by binding to receptors.     

Comparison of Five Bacteriophages as Models for Viral Aerosol Studies

Bacteriophages are perceived to be good models for the study of airborne viruses because they are safe to use, some of them display structural features similar to those of human and animal viruses, and they are relatively easy to produce in large quantities. Yet, only a few studies have investigated them as models. It has previously been demonstrated that aerosolization, environmental conditions, and sampling conditions affect viral infectivity, but viral infectivity is virus dependent. Thus, several virus models are likely needed to study their general behavior in aerosols. The aim of this study was to compare the effects of aerosolization and sampling on the infectivity of five tail-less bacteriophages and two pathogenic viruses: MS2 (a single-stranded RNA [ssRNA] phage of the Leviviridae family), Φ6 (a segmented double-stranded RNA [dsRNA] phage of the Cystoviridae family), ΦX174 (a single-stranded DNA [ssDNA] phage of the Microviridae family), PM2 (a double-stranded DNA [dsDNA] phage of the Corticoviridae family), PR772 (a dsDNA phage of the Tectiviridae family), human influenza A virus H1N1 (an ssRNA virus of the Orthomyxoviridae family), and the poultry virus Newcastle disease virus (NDV; an ssRNA virus of the Paramyxoviridae family). Three nebulizers and two nebulization salt buffers (with or without organic fluid) were tested, as were two aerosol sampling devices, a liquid cyclone (SKC BioSampler) and a dry cyclone (National Institute for Occupational Safety and Health two-stage cyclone bioaerosol sampler). The presence of viruses in collected air samples was detected by culture and quantitative PCR (qPCR). Our results showed that these selected five phages behave differently when aerosolized and sampled. RNA phage MS2 and ssDNA phage ΦX174 were the most resistant to aerosolization and sampling. The presence of organic fluid in the nebulization buffer protected phages PR772 and Φ6 throughout the aerosolization and sampling with dry cyclones. In this experimental setup, the behavior of the influenza virus resembled that of phages PR772 and Φ6, while the behavior of NDV was closer to that of phages MS2 and ΦX174. These results provide critical information for the selection of appropriate phage models to mimic the behavior of specific human and animal viruses in aerosols.     

Characterization and classification of Bo4 as a cluster G mycobacteriophage that can infect and lyse M. tuberculosis

Mycobacteriophage therapy is a potential alternative treatment for Mycobacterium tuberculosis infection. Here, we further characterized a mycobacteriophage, Bo4, and evaluated its ability to infect and kill M. tuberculosis. We first found that Bo4 can infect M. tuberculosis and Mycobacterium smegmatis. The observed clear plaques created by Bo4 infection indicated that Bo4 might be a lytic phage able to lyse mycobacterial strains, which was confirmed by phage antimicrobial activity. Bo4 formed clear zones in a medium with pH values of 7.4 or 5.0, suggesting the possibility that Bo4 could lyse mycobacteria, such as M. tuberculosis, in blood as well as in lysosomal macrophages. Further investigation into the Bo4 genome revealed that Bo4 had a dsDNA genome. Moreover, Bo4 contained ~39,318 bp comprised of 66.76 % G+C content. Complete genome sequencing showed high nucleotide identity with cluster G mycobacteriophages, thus classifying Bo4 as a member of the cluster G family. Additionally, annotation of the Bo4 genome indicated that it was a lytic bacteriophage and did not contain any harmful genes that increased mycobacterial virulence or decreased human immunity. Overall, the results of investigation indicate that the Bo4 possesses the potential to destroy M. tuberculosis, making it a potentially useful tool for diagnosing and treating tuberculosis.     

Signatures of selection in natural populations adapted to chronic pollution

Background

Ectocarpus siliculosus virus-1 (EsV-1) is a lysogenic dsDNA virus belonging to the super family of nucleocytoplasmic large DNA viruses (NCLDV) that infect Ectocarpus siliculosus, a marine filamentous brown alga. Previous studies indicated that the viral genome is integrated into the host DNA. In order to find the integration sites of the viral genome, a genomic library from EsV-1-infected algae was screened using labelled EsV-1 DNA. Several fragments were isolated and some of them were sequenced and analyzed in detail.

Results

Analysis revealed that the algal genome is split by a copy of viral sequences that have a high identity to EsV-1 DNA sequences. These fragments are interspersed with DNA repeats, pseudogenes and genes coding for products involved in DNA replication, integration and transposition. Some of these gene products are not encoded by EsV-1 but are present in the genome of other members of the NCLDV family. Further analysis suggests that the Ectocarpus algal genome contains traces of the integration of a large dsDNA viral genome; this genome could be the ancestor of the extant NCLDV genomes. Furthermore, several lines of evidence indicate that the EsV-1 genome might have originated in these viral DNA pieces, implying the existence of a complex integration and recombination system. A protein similar to a new class of tyrosine recombinases might be a key enzyme of this system.

Conclusion

Our results support the hypothesis that some dsDNA viruses are monophyletic and evolved principally through genome reduction. Moreover, we hypothesize that phaeoviruses have probably developed an original replication system.