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Cylindrospermopsis raciborskii is a central bloom-forming cyanobacteria. However, despite its ecological significance, little is known of its interactions with the phages that infect it. Currently, only a single sequenced genome of a Cylindrospermopsis-infecting phage is publicly available. Here we describe the isolation and characterization of Cr-LKS3, a second phage infecting Cylindrospermopsis. Cr-LKS3 is a siphovirus with a higher genome similarity to prophages within heterotrophic bacteria genomes than to any other cyanophage/cyano-prophage, suggesting that it represents a novel cyanophage group. The function, order and orientation of the 72 genes in the Cr-LKS3 genome are highly similar to those of Escherichia virus Lambda (hereafter Lambda), despite the very low sequence similarity between these phages, showing high evolutionary convergence despite the substantial difference in host characteristics. Similarly to Lambda, the genome of Cr-LKS3 contains various genes that are known to be central to lysogeny, suggesting it can enter a lysogenic cycle. Cr-LKS3 has a unique ability to infect a host with a dramatically different GC content, without carrying any tRNA genes to compensate for this difference. This ability, together with its potential lysogenic lifestyle shed light on the complex interactions between C. raciborskii and its phages.

In this study, we have focused on the lytic phage proteins encoded by the Stenotrophomonas phages. A total of 60 lytic proteins were identified to be encoded by 19 different phages. Those proteins were characterized under eight classes: amidases, muramidases, pectate lyase, peptidases, holins and spanins. The phages encoding these proteins come under the family of Ackermannviridae, Autographiviridae, Myoviridae, Podoviridae and Siphoviridae. All the phages encoding those proteins were found to infect Stenotrophomonas maltophilia. Among the phages, about 50% were found to undergo a lytic lifecycle. The isolated proteins were clustered according to the similarity in the amino acid sequence. These clusters were used to make their phylogenetic tree. The co-occurrence of the amidase, pectate lyase and lipase genes in the phage genome was found using a correlation analysis.

Viruses can play critical roles in symbioses by initiating horizontal gene transfer, affecting host phenotypes, or expanding their host's ecological niche. However, knowledge of viral diversity and distribution in symbiotic organisms remains elusive. Here we use deep-sequenced metagenomic DNA (PacBio Sequel II; two individuals), paired with a population genomics approach (Pool-seq; 11 populations, 550 individuals) to understand viral distributions in the lichen Umbilicaria phaea. We assess (i) viral diversity in lichen thalli, (ii) putative viral hosts (fungi, algae, bacteria) and (iii) viral distributions along two replicated elevation gradients. We identified five novel viruses, showing 28%-40% amino acid identity to known viruses. They tentatively belong to the families Caulimoviridae, Myoviridae, Podoviridae and Siphoviridae. Our analysis suggests that the Caulimovirus is associated with green algal photobionts (Trebouxia) of the lichen, and the remaining viruses with bacterial hosts. We did not detect viral sequences in the mycobiont. Caulimovirus abundance decreased with increasing elevation, a pattern reflected by a specific algal lineage hosting this virus. Bacteriophages showed population-specific patterns. Our work provides the first comprehensive insights into viruses associated with a lichen holobiont and suggests an interplay of viral hosts and environment in structuring viral distributions.

This report presents the characterization of the first virulent phages infecting Brevibacterium aurantiacum, a bacterial species used during the manufacture of surface-ripened cheeses. These phages were also responsible for flavour and colour defects in surface-ripened cheeses. Sixteen phages (out of 62 isolates) were selected for genome sequencing and comparative analyses. These cos-type phages with a long non-contractile tail currently belong to the Siphoviridae family (Caudovirales order). Their genome sizes vary from 35,637 to 36,825 bp and, similar to their host, have a high GC content (~61%). Genes encoding for an immunity repressor, an excisionase and a truncated integrase were found, suggesting that these virulent phages may be derived from a prophage. Their genomic organization is highly conserved, with most of the diversity coming from the presence of long (198 bp) DNA tandem repeats (TRs) within an open reading frame coding for a protein of unknown function. We categorized these phages into seven genomic groups according to their number of TR, which ranged from two to eight. Moreover, we showed that TRs are widespread in phage genomes, found in more than 85% of the genomes available in public databases.

Viruses can affect microbial dynamics, metabolism and biogeochemical cycles in aquatic ecosystems. However, viral diversity and functions in agricultural soils are poorly known, especially in the rhizosphere. We used virome analysis of eight rhizosphere and bulk soils to study viral diversity and potential biogeochemical impacts in an agro-ecosystem. The order Caudovirales was the predominant viral type in agricultural soils, with Siphoviridae being the most abundant family. Phylogenetic analysis of the terminase large subunit of Caudovirales identified high viral diversity and three novel groups. Viral community composition differed significantly between bulk and rhizosphere soils. Soil pH was the main environmental driver of the viral community structure. Remarkably, abundant auxiliary carbohydrate-active enzyme (CAZyme) genes were detected in viromes, including glycoside hydrolases, carbohydrate esterases and carbohydrate-binding modules. These results demonstrate that virus-encoded putative auxiliary metabolic genes or metabolic genes that may change bacterial metabolism and indirectly contribute to biogeochemical cycling, especially carbon cycling, in agricultural soil.

Chicken breast meat is considered as the main source of Salmonella infection in humans. The aim of this study was to isolate lytic bacteriophages specific for Salmonella enterica serovars Enteritidis and examine their efficacy in a cocktail for the biocontrol of Salmonella spp. in raw chicken breast meat. Four lytic phages belonging to the Myoviridae and Siphoviridae families were isolated from a river proximate to a duck farm. They exhibited broad lytic activities against 11 strains of S. Enteritidis, 11 strains of S. Typhimurium, and one each of S. Paratyphi A, S. San Diego, and S. Typhi. The phages were determined to be stable, exhibited similar degrees of resistance to heat and pH, and had latent periods ranging from 5 to 30 min. In addition, the phage particles were 100% adsorbed within 18 to 40 min. Viable cell counts of bacteria were significantly reduced in raw chicken breast samples (P < 0.05) when treated with a cocktail of all four bacteriophages at 4 °C for 7 days (multiplicities of infection were from 104 to 106 ). These results indicate the potential efficacy of the bacteriophage cocktail as a biological agent against S. Enteritidis in raw chicken breast meat. PRACTICAL APPLICATION: Our findings demonstrate that the phages could be effective in reducing the viability of Salmonella spp. bacteria in chicken breast meat. Therefore, the phage cocktail is a potential bactericidal agent for the biocontrol of Salmonella spp. in raw chicken breast meat and could be used use in various poultry industries in the future.

The discovery of a Salmonella-targeting phage from the waterways of the United Kingdom provided an opportunity to address the mechanism by which Chi-like bacteriophage (phage) engages with bacterial flagellae. The long tail fibre seen on Chi-like phages has been proposed to assist the phage particle in docking to a host cell flagellum, but the identity of the protein that generates this fibre was unknown. We present the results from genome sequencing of this phage, YSD1, confirming its close relationship to the original Chi phage and suggesting candidate proteins to form the tail structure. Immunogold labelling in electron micrographs revealed that YSD1_22 forms the main shaft of the tail tube, while YSD1_25 forms the distal part contributing to the tail spike complex. The long curling tail fibre is formed by the protein YSD1_29, and treatment of phage with the antibodies that bind YSD1_29 inhibits phage infection of Salmonella. The host range for YSD1 across Salmonella serovars is broad, but not comprehensive, being limited by antigenic features of the flagellin subunits that make up the Salmonella flagellum, with which YSD1_29 engages to initiate infection.

Aging is a critical factor affecting physical health and disease in mammals. Emerging evidence indicates that aging may affect the gut bacteriome in cynomolgus macaques, but little is known about whether or how the gut virome changes with age. Here, we compared the DNA gut viral composition of 16 female cynomolgus monkeys (Macaca fascicularis) at three life stages (young, adult, and old) using the shotgun metagenome sequencing method. We found that the DNA gut virome from these monkeys differed substantially among the three groups. The gut viruses were dominated by bacteriophages, the most abundant of which was the Caudovirales order (i.e., Siphoviridae, Myoviridae, and Podoviridae families). Additionally, the co‐occurrence analysis revealed that the age‐related bacteriophages were correlated in an extensive and complex manner with the main intestinal bacteria (i.e., Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria phyla). Furthermore, the age‐related DNA gut viral functions were enriched for genetic information processing, nucleotide, and folate metabolism. Our gut virome analysis provides new insight into how aging influences the gut virome of non‐human primates., We characterized changes of the DNA gut virome associated with age in female crab‐eating macaques. The structural and functional profiles of the gut DNA virome differed significantly among the three age groups. The gut virome was dominated by dsDNA phages, especially the Caudovirales order, which showed strong correlations with the highly prevalent gut bacteria. Our findings suggest that modulating the intestinal virome may be beneficial for the treatment of aging.

The aim of this study was to identify and characterize the SE-P3, P16, P37, and P47 phages infecting Salmonella Enteritidis. Transmission electron microscopy analysis showed that the SE phages belonged to the Myoviridae or Siphoviridae family and had plaque sizes between 0.622 ± 0.027 and 1.630 ± 0.036 mm in diameter. sefC, pefA, spvC, sopE, and gipA virulent gene regions were absent in their genome and their calculated genome sizes were between 35.9 and 37.8 kbp. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the protein profiles of each phage were different. The SE phages had a short latent period (10-20 min), large burst size (76-356 PFU/cell), and a short burst time (25-35 min). The multiplicity of infection values and mutant frequency of the phages were 0.01-0.0001 and 10-7 , respectively. They were very infective against their host bacteria when applied at 20°C, 30°C, or 37°C and adsorbed to their host cells by 96.20-97.65% in the first 5 min of incubation, and also Ca2+ ions did not have a significant effect on their adsorption. The SE phages were resistant to wide pH ranges and high temperatures. These results indicate that the SE phages are good candidates as therapeutic and biocontrol agents against foodborne pathogenic S. Enteritidis.

The present study aims to integrate the benefits of plaque assay using a novel phage mix with phylogenetic and molecular analysis for detecting Pseudomonas aeruginosa in water. Three phages were isolated and the transmission electron microscope related their morphological resemblance to those of Siphoviridae and Podoviridae families, while molecular analysis showed different cp‐gene sizes. The Phage mix was highly specific (86.0%), and data misleading didn't exceed 14.0% compared to membrane filter assay (39.2%). Time elapsed for test completion required 24 h. Identified P. aeruginosa were verified using 16S‐rDNA. Nucleotide sequence data for both phages and bacteria were submitted to the NCBI GenBank database, USA and gained their accession numbers. Concluding remarks highlight the potential of plaque assay as specific, simple and rapid method. The study recommended future efforts to isolate and characterize new phages for detecting other bacterial pathogens of public health concern to control water pollution and maintain adequate hygiene.

Exiguobacterium is a versatile genus with potential in industry and agriculture. No bacteriophage that infects Exiguobacterium has been reported, despite its potential impacts on the utilization of Exiguobacterium. E. indicum EI9 was isolated from Dianchi Lake, a plateau eutrophic lake in China, which can significantly inhibit the growth of Microcystis aeruginosa. To isolate and characterize Exiguobacterium-infecting bacteriophage, a virulent bacteriophage, DCEIV-9 that specifically infects E. indicum EI9 was isolated from Dianchi lake water sample. DCEIV-9 produced tiny, round, and clear plaques with 0.5-1 mm in diameter. Electron microscopy showed that DCEIV-9 is a typical representative of the Siphoviridae, with an icosahedral head (56 nm in diameter) and a non-contractile tail (163 nm in length). Based on a one-step growth curve, latent period of 20 min and burst size of 51 PFU/infected cell were determined. DCEIV-9 was sensitive to temperature over 50 °C and prefers acid environment. DCEIV-9 was extremely sensitive to proteinase K, chloroform, ethanol, Triton X-100 but not sensitive to SDS. Restriction endonucleases analysis indicated that DCEIV-9 is a dsDNA virus. DCEIV-9 can only infect E. indicum, indicates that it has a narrow host range. DCEIV-9 is a potential new species.

SummaryBacteriophages infectingEscherichia colihave been used as a proxy for faecal matter and water quality from a variety of environments. However, the diversity of coliphages that are present in seawater remains largely unknown, with previous studies largely focusing on morphological diversity. Here, we isolated and characterised coliphages from three coastal locations in the UK and Poland. This revealed a surprising genetic diversity, with comparative genomics and phylogenetic analysis of phage isolates facilitating the identification of putative new species within the generaRB69virusandT5virusand a putative new genus within the subfamilyTunavirinae. Furthermore, by combining this genomic data with proteomic and host range analyses a number of phage structural proteins were identified, one of which is likely to be responsible for the observed differences in host range.

Bacteria of the genus Campylobacter are the most common pathogens causing zoonotic diseases in humans. Therefore, the aim of the study was to isolate Campylobacter bacteria from broiler chickens and evaluate their susceptibility to selected antibiotics by determining minimum inhibitory concentrations (MIC), followed by isolation and characterization of bacteriophages specific for Campylobacter spp. The material for the study consisted of field isolates of Campylobacter spp. obtained from the gut (cecum) of broiler chickens directly after slaughter in slaughterhouses, and bacteriophages specific for these strains. We isolated 48 strains from poultry (140 broiler chickens): 31 strains of Campylobacter jejuni and 17 of Campylobacter coli. Identification of the strains was confirmed by multiplex PCR and MALDI‐TOF mass spectrometry. Over 83% of Campylobacter strains were resistant to ciprofloxacin, and over half the isolates were resistant to erythromycin, gentamicin, and tetracycline. Resistance to three or more antibiotics was observed in 91.6% of all strains. Four bacteriophages were obtained, and on the basis of their morphological structure, they were assigned to two families of the order Caudovirales: Myoviridae and Siphoviridae. A high percentage of the Campylobacter strains were resistant to at least three of the antibiotic groups tested. All of the phages exhibited lytic activity against the Campylobacter spp. isolates, but the antibacterial effect of the phages was not observed for all strains.

The resistance to antibiotics in clinically important bacteria is one of the major global health concerns. Phage therapy could be one reliable alternative therapeutic strategy to combat these superbugs. In this study, we assessed host range of a novel bacteriophage, JHP, and characterized for its potential use in phage therapy. The bacteriophage demonstrated infectivity over a broad range of genera including multidrug resistant clinical isolates of Pseudomonas aeruginosa, members of family Enterobacteracae, and other important human pathogens. The antibacterial activity was highest at pH 7, and at temperature of 37 °C. The phage lytic activity gradually decreased till 60 °C and showed no activity when temperature was further raised. The bacteriophage could safely be stored at 4 °C or -20 °C. The latent period of the bacteriophage was 25 min and showed a burst size of 433 virions per cell. The size of JHP genome was approximately 30 kb. Family, Siphoviridae was assigned to JHP based on its icosahedral head with non-contractile tail. The diameter of JHP head and tail length was found 115 and 152 nm, respectively. To sum up, the broad spectrum Siphoviridae phage JHP is an ingenious candidate for phage therapy.

This study investigated the presence of prophages in Lactococcus garvieae isolated from several marine fish species in Japan. Representative strains of 16 bacterial genotypes (S1-S16) selected from more than 400 L. garvieae isolates were used to induce lysogenic bacteriophages. These strains were treated with 500 ng mL(-1) freshly prepared mitomycin C. A cross-spotting assay was performed to validate the lysogenic and indicator strains. The lysogenic strains were selected for isolation and concentration of the phages. Phage DNA was digested with EcoRI for biased sinusoidal field gel electrophoresis analysis. Polymerase chain reaction (PCR) was used to detect integrated prophage DNA. Of the 16 representative bacterial genotypes, 12 strains integrated prophages as indicated by the PCR assay, and 10 phages were detected and isolated using two indicator bacterial strains. Analysis of genomic DNA showed that these phages were homologous and named as PLgT-1. Transmission electron microscopy revealed that the morphology of PLgT-1 was consistent with the virus family Siphoviridae. PCR analysis of the prophage DNA revealed that all of the S1 genotype strains were lysogenic (30/30), but none of the S16 genotype strains were lysogenic (0/30). This is the first study to investigate lysogenic bacteriophages from L. garvieae.

Hypolithic microbial communities are specialized desert communities inhabiting the underside of translucent rocks. Here, we present the first study of the viral fraction of these communities isolated from the hyperarid Namib Desert. The taxonomic composition of the hypolithic viral communities was investigated and a functional assessment of the sequences determined. Phylotypic analysis showed that bacteriophages belonging to the order Caudovirales, in particular the family Siphoviridae, were most prevalent. Functional analysis and comparison with other metaviromes revealed a relatively high frequency of cell wall-degrading enzymes, ribonucleotide reductases (RNRs) and phage-associated genes. Phylogenetic analyses of terL and phoH marker genes indicated that many of the sequences were novel and distinct from known isolates, and the class distribution of the RNRs suggests that this is a novel environment. The composition of the viral hypolith fraction containing many Bacillus-infecting phages was not completely consistent with Namib hypolith phylotypic surveys of the bacterial hosts, in which the cyanobacterial genus Chroococcidiopsis was found to be dominant. This could be attributed to the lack of sequence information about hypolith viruses/bacteria in public databases or the possibility that hypolithic communities incorporate viruses from the surrounding soil.

The phytopathogen Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of bacterial canker of kiwifruit. In the last years, it has caused severe economic losses to Actinidia spp. cultivations, mainly in Italy and New Zealand. Conventional strategies adopted did not provide adequate control of infection. Phage therapy may be a realistic and safe answer to the urgent need for novel antibacterial agents aiming to control this bacterial pathogen. In this study, we described the isolation and characterization of two bacteriophages able to specifically infect Psa. φPSA1, a member of the Siphoviridae family, is a temperate phage with a narrow host range, a long latency, and a burst size of 178; φPSA2 is a lytic phage of Podoviridae family with a broader host range, a short latency, a burst size of 92 and a higher bactericidal activity as determined by the TOD value. The genomic sequence of φPSA1 has a length of 51,090 bp and a low sequence homology with the other siphophages, whereas φPSA2 has a length of 40 472 bp with a 98% homology with Pseudomonas putida bacteriophage gh-1. Of the two phages examined, φPSA2 may be considered as a candidate for phage therapy of kiwifruit disease, while φPSA1 seems specific toward the recent outbreak's isolates and could be useful for Psa typing.

We report on a genomic and functional analysis of a novel marine siphovirus, the Vibrio phage SIO-2. This phage is lytic for related Vibrio species of great ecological interest including the broadly antagonistic bacterium Vibrio sp. SWAT3 as well as notable members of the Harveyi clade (V.harveyi ATTC BAA-1116 and V.campbellii ATCC 25920). Vibrio phage SIO-2 has a circularly permuted genome of 80598 bp, which displays unusual features. This genome is larger than that of most known siphoviruses and only 38 of the 116 predicted proteins had homologues in databases. Another divergence is manifest by the origin of core genes, most of which share robust similarities with unrelated viruses and bacteria spanning a wide range of phyla. These core genes are arranged in the same order as in most bacteriophages but they are unusually interspaced at two places with insertions of DNA comprising a high density of uncharacterized genes. The acquisition of these DNA inserts is associated with morphological variation of SIO-2 capsid, which assembles as a large (80 nm) shell with a novel T=12 symmetry. These atypical structural features confer on SIO-2 a remarkable stability to a variety of physical, chemical and environmental factors. Given this high level of functional and genomic novelty, SIO-2 emerges as a model of considerable interest in ecological and evolutionary studies.

Microbial source tracking (MST) methods allow the identification of specific faecal sources. The aim is to detect the sources of faecal pollution in a water body to allow targeted, efficient and cost-effective remediation efforts in the catchment. Bacteriophages infecting selected host strains of Bacteroides species are used as markers to track faecal contaminants in water. By using a suitable Bacteroides host from a given faecal origin, it is possible to specifically detect bacteriophages of this faecal origin. It can thus be used to detect specific phages of Bacteroides for MST. With this objective, we isolated several Bacteroides strains from pig, cow and poultry faeces by applying a previously optimized methodology used to isolate the host strains from humans. The isolated strains belonged to Bacteroides fragilis and Bacteroides thetaiotaomicron. These strains, like most Bacteroides species, detected phages of the Siphoviridae morphology. Using the newly isolated host strains for phage enumeration in a range of samples, we showed that these detect phages in faecal sources that coincide with their own origin (70-100% of the samples), and show no detection or very low percentages of detection of phages from other animal origins (from 0 to 20% of the samples). Only strains isolated from pig wastewater detected phages in 50% of human sewage samples. Nevertheless, those strains detecting phages from faecal origins other than their own detected fewer phages (2-3 log₁₀ pfu·100 ml⁻¹) than the phages detected by the specific strain of the same origin. On the basis of our results, we propose that faecal source tracking with phages infecting specific Bacteroides host strains is a useful method for MST. In addition, the method presented here is feasible in laboratories equipped with only basic microbiological equipment, it is more rapid and cost-effective than other procedures and it does not require highly qualified staff.

Virulent phages of the Siphoviridae family are responsible for milk fermentation failures worldwide. Here, we report the characterization of the product of the early expressed gene orf35 from Lactococcus lactis phage p2 (936 group). ORF35(p2), also named Sak3, is involved in the sensitivity of phage p2 to the antiviral abortive infection mechanism AbiK. The localization of its gene upstream of a gene coding for a single-strand binding protein as well as its membership to a superfamily of single-strand annealing proteins (SSAPs) suggested a possible role in homologous recombination. Electron microscopy showed that purified ORF35(p2) form a hexameric ring-like structure that is often found in proteins with a conserved RecA nucleotide-binding core. Gel shift assays and surface plasmon resonance data demonstrated that ORF35(p2) interacts preferentially with single-stranded DNA with nanomolar affinity. Atomic force microscopy showed also that it preferentially binds to sticky DNA substrates over blunt ends. In addition, in vitro assays demonstrated that ORF35(p2) is able to anneal complementary strands. Sak3 also stimulates Escherichia coli RecA-mediated homologous recombination. Remarkably, Sak3 was shown to possess an ATPase activity that is required for RecA stimulation. Collectively, our results demonstrate that ORF35(p2) is a novel SSAP stimulating homologous recombination.

Thermus strains are regarded as models to investigate the mechanism of thermostability of thermophiles, and phages from Thermus are particularly interesting because of their way to regulate gene expression. In this research, a Thermus bacteriophage named TSP4 (Thermus Siphoviridae phage) was isolated from Tengchong hot springs in China, and characteristics of morphology, temperature for phage production, pH and organic solvent sensitivity, DNA restriction endonuclease digestion and protein composition of TSP4 were further studied. TSP4 belonged to the Siphoviridae family and had a hexagonal head of 73 nm in diameter, an extremely long and flexible tail of 785 nm in length and 10 nm in width. TSP4 was very stable at 65 °C and pH 7.6. The capsid was apparently devoid of lipid. By SDS-PAGE, six protein bands were found in purified virions. Despite their exceptional habitats separated by thousands of kilometers, the characteristics of this thermophilic phage showed high similarity to Thermus siphoviruses P23-45 and P74-26 isolated from Kamchatka peninsula hot springs in the Far East, Russia. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

SPP1 is a siphophage infecting the gram-positive bacterium Bacillus subtilis. The SPP1 tail electron microscopy (EM) reconstruction revealed that it is mainly constituted by conserved structural proteins such as the major tail proteins (gp17.1), the tape measure protein (gp18), the Distal tail protein (Dit, gp19.1), and the Tail associated lysin (gp21). A group of five small genes (22–24.1) follows in the genome but it remains to be elucidated whether their protein products belong or not to the tail. Noteworthy, an unassigned EM density accounting for ∼245 kDa is present at the distal end of the SPP1 tail-tip. We report here the gp23.1 crystal structure at 1.6 A resolution, a protein that lacks sequence identity to any known protein. We found that gp23.1 forms a hexamer both in the crystal lattice and in solution as revealed by light scattering measurements. The gp23.1 hexamer does not fit well in the unassigned SPP1 tail-tip EM density and we hypothesize that this protein might act as a chaperone.

Given the great biological importance and high diversity of temperate Staphylococcus aureus bacteriophages, a method is needed for the description of their genomic structure. Here we have updated a multiplex PCR strategy for the complex characterization of S. aureus phages of the family Siphoviridae. Based on the comparative genomic analysis of the available phage sequences, a multilocus PCR strategy for typing the major modules of the phage genome was designed. The genomic modules were classified on the basis of the genes for integrase (10 types), anti-repressor (five types), replication proteins polA, dnaC and dnaD (four types), dUTPase (four types), portal protein (eight types), tail appendices (four types) and endolysin (four types) corresponding to the integrase locus, lysogeny control region, and modules for DNA replication, transcription regulation, packaging, tail appendices and lysis respectively. The nine PCR assays designed for the above sequences were shown to be capable to identify the bacteriophage gene pool present both in the phage and bacterial genomes and their extensive mosaic structure. The established multiplex PCR-based multilocus diagnostic scheme is convenient for rapid and reliable phage and prophage classification and for the study of bacteriophage evolution.

We characterize CdSe/ZnS quantum dot (QD) binding to genetically modified bacteriophage as a model for bacterial detection. Interactions among QDs, lambda (lambda) phage, and Escherichia coli are examined by several cross-validated methods. Flow and image-based cytometry clarify fluorescent labeling of bacteria, with image-based cytometry additionally reporting the number of decorated phage bound to cells. Transmission electron microscopy, image-based cytometry, and electrospray differential mobility analysis allow quantization of QDs attached to each phage (4-17 QDs) and show that lambda phage used in this study exhibits enhanced QD binding to the capsid by nearly a factor of four compared to bacteriophage T7. Additionally, the characterization methodology presented can be applied to the quantitative characterization of other fluorescent nanocrystal-biological conjugates.

Introduction: Bacterial viruses play crucial roles in the pathogenesis of many systemic diseases. They are known to inhabit the oral cavity, both as free virions and as prophages in lysogenic bacterial strains; however, there has been no report of bacteriophages in endodontic infections. In this study, we sought to detect, isolate, and describe temperate bacteriophages harbored by Enterococcus faecalis strains isolated from endodontic infections. Methods: Ten E. faecalis strains were isolated from root canals of teeth undergoing retreatment following unsuccessful endodontic therapy. Mitomycin C was used to induce any prophages present in the bacterial isolates. The induced phages were purified and examined using electron microscopy. The DNA extracted from one of the phage isolates was subjected to restriction endonuclease digestion and agarose electrophoresis analysis. Results: Lysogeny was demonstrated in 4 of the 10 E. faecalis strains. Three of the lysogenic strains yielded phages exhibiting a Siphoviridae morphology, with long, non-contractile tails 130 nm in length, and spherical/icosahedral heads 41 nm in diameter. The virus induced from the fourth lysogenic E. faecalis strain had a contractile tail characteristic of Myoviridae. Restriction endonuclease analysis of NsiI and NdeI DNA fragments from one of the Siphoviridae phage isolates (phage φEf11) indicated a genome size of approximately 41 kbp. Conclusion: This is the first report of lysogenic bacteria and their inducible viruses in infected root canals.

The tail apparatus of the bacteriophage SPP1 is an extraordinary approximately 1600-A-long molecular machine. The tail mediates attachment of the virus to the host surface receptor, as well-as ejection of the viral genome into the host. The distal tip of the tail binds the extracellular ectodomain of the Bacillus subtilis receptor YueB, while the tail tube provides a conduit to funnel the viral genome into the host. This process, which culminates with the ejection of the approximately 44 kb of viral DNA across the thick, cell envelope of the Gram-positive bacterial cell, takes place in a time scale of seconds to minutes and represents a remarkable example of biotransformation. In this issue of Molecular Microbiology, Auzat et al. provide compelling evidence that the two major structural proteins of the SPP1 tail, gp17.1 (approximately 19.1 kDa) and gp17.1* (approximately 28 kDa), share a common N-terminal sequence, and that gp17.1* is generated by a translational frameshift in the gene 17.1. The extra domain fused to gp17.1* is synthesized by a +1 programmed translational frameshift at the end of gene 17.1, which leads to the synthesis of approximately one gp17.1* for every three equivalents of gp17.1. This finding extends our current knowledge of translational frameshifts and provides a framework to understand how Siphoviridae phages like SPP1 have developed long-tail machines using only two major structural proteins.

1. This study addressed the prevalence of lysogeny within 19 strains of freshwater Synechococcus sp. rich in phycocyanin (PC) that were isolated from a temperate lake or obtained, in four cases, from a culture collection. 2. Lysis of 16 Synechococcus strains (84%) was inducible with mitomycin C, but the required concentration (20 lg mL)1) was higher than in other studies. Mitomycin C induction yielded burst sizes of 3.5–23.7 phages per cell. 3. Transmission Electron Microscopy of the resultant temperate phages showed that all belonged to the family Siphoviridae with capsid diameters ranging from 40.3 to 53.5 nm and tail lengths from 129 to 186 nm. None of the cyanophages could be maintained in the lytic cycle using the three non-lysogenic strains, or any of the other 16 lysogenic strains of Synechococcus. 4.These results suggest a potentially high level of lysogeny within PC-rich Synechococcus species in natural freshwaters, but that the presence of these temperate cyanosiphoviruses may go undetected with the lower mitomycin C concentrations commonly used.

Summary Cyanophages that infect coastal and oceanic Synechococcus have been studied extensively. However, no cyanophages infecting estuarine Synechococcus have been reported. In this study, seven cyanophages (three podoviruses, three siphoviruses and one myovirus) isolated from four estuarine Synechococcus strains were characterized in terms of their morphology, host range, growth and genetic features. All the podoviruses and siphoviruses were highly host specific. For the first time, the photosynthesis gene (psbA) was found in two podoviruses infecting estuarine Synechococcus. However, the psbA gene was not detected in the three siphoviruses. The psbA sequences from the two Synechococcus podoviruses clustered with some environmental psbA sequences, forming a unique cluster distantly related to previous known psbA clusters. Our results suggest that the psbA among Synechococcus podoviruses may evolve independently from the psbA of Synechococcus myoviruses. All three estuarine Synechococcus podoviruses contained the DNA polymerase (pol) gene, and clustered with other podoviruses that infect oceanic Synechococcus and Prochlorococcus, suggesting that the DNA pol is conserved among marine picocyanobacterial podoviruses. Prevalence of host-specific cyanophages in the estuary suggests that Synechococcus and their phages in the estuarine ecosystem may develop a host–phage relationship different from what have been found in the open ocean.

International audience; The majority of known bacteriophages have long noncontractile tails (Siphoviridae) that serve as a pipeline for genome delivery into the host cytoplasm. The tail extremity distal from the phage head is an adsorption device that recognises the bacterial receptor at the host cell surface. This interaction generates a signal transmitted to the head that leads to DNA release. We have determined structures of the bacteriophage SPP1 tail before and after DNA ejection. The results reveal extensive structural rearrangements in the internal wall of the tail tube. We propose that the adsorption device-receptor interaction triggers a conformational switch that is propagated as a domino-like cascade along the 1600 angstrom-long helical tail structure to reach the head-to-tail connector. This leads to opening of the connector culminating in DNA exit from the head into the host cell through the tail tube.

Seven bacteriophages specific to Vibrio harveyi, the causative agent of luminous vibriosis in shrimp, were isolated from coastal aquaculture systems like shrimp farms, hatcheries and tidal creeks along the east and west coast of India. All the seven phages were found to have the typical head and tail morphology with double-stranded DNA as genetic material. Morphologically, six phages were grouped under family Siphoviridae and one under Myoviridae. These phages were further characterized with respect to host range, morphology and structural proteins. Genomic fingerprinting was carried out using restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD). Major capsid proteins of all the phages detected by SDS-PAGE were distinct from one another. All the phages were found to be highly lytic for V. harveyi and had different lytic spectrum for the large number of isolates tested. Six of the seven phages isolated had a broad lytic spectrum and could be potential candidates for biocontrol of V. harveyi in aquaculture systems.

The predominant morphotype of mycobacteriophage virions has a DNA-containing capsid attached to a long flexible non-contractile tail, features characteristic of the Siphoviridae. Within these phage genomes the tape measure protein (tmp) gene can be readily identified due to the well-established relationship between the length of the gene and the length of the phage tail – because these phages typically have long tails, the tmp gene is usually the largest gene in the genome. Many of these mycobacteriophage Tmp's contain small motifs with sequence similarity to host proteins. One of these motifs (motif 1) corresponds to the Rpf proteins that have lysozyme activity and function to stimulate growth of dormant bacteria, while the others (motifs 2 and 3) are related to proteins of unknown function, although some of the related proteins of the host are predicted to be involved in cell wall catabolism. We show here that motif 3-containing proteins have peptidoglycan-hydrolysing activity and that while this activity is not required for phage viability, it facilitates efficient infection and DNA injection into stationary phase cells. Tmp's of mycobacteriophages may thus have acquired these motifs in order to avoid a selective disadvantage that results from changes in peptidoglycan in non-growing cells.

Analysis of the DNA region upstream of the BF23 hrs gene revealed a genetic organisation similar to that of closely related phage T5. A gene encoding a lipoprotein (llpBF23) is located directly upstream of the gene encoding the receptor binding protein (hrs ) but is transcribed in opposite direction. The gene is followed by four open reading frames transcribed in the same direction. The llpBF23 gene product does not show similarity to the corresponding T5 LlpT5 protein, however, like LlpT5 does for FhuA it blocks the BtuB receptor for BF23 infection. While no similarity between BF23 and T5 was observed for the DNA region encoding Llp and the receptor binding protein, the flanking regions were highly similar. Based on our results we conclude that a genetic module, the receptor-binding/receptor-blocking module, exists in phages BF23 and T5. Due to exclusion of homologous recombination within this module, it is hereditary only as an intact module: separation of the receptor-binding gene from the receptor-blocking gene, which apparently results in reduced fitness of the phage due to inactivation of progeny phage by active receptor proteins in the outer membranes of lysed cells, is thus effectively prevented. (© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

The development of bacteriophage lambda and double-stranded DNA viruses in general involves the convergence of two separate pathways: DNA replication and head assembly. Clearly, packaging will proceed only if an empty capsid shell, the prohead, is present to receive the DNA, but genetic evidence suggests that proheads play another role in the packaging process. For example, lambda phages with an amber mutation in any head gene or in FI, the gene encoding the accessory packaging protein gpFI, are able to produce normal amounts of DNA concatemers but they are not cut, or matured, into unit length chromosomes for packaging. Similar observations have been made for herpes simplex 1 virus. In the case of lambda, a negative model proposes that in the amber phages, unassembled capsid components are inhibitory to maturation, and a positive model suggests that assembled proheads are required for cutting. We tested the negative model by using a deletion mutant devoid of all prohead genes and FI in an in vivo cos cleavage assay; in this deleted phage, the cohesive ends were not cut. When lambda proheads and gpFI were provided in vivo via a second prophage, cutting was restored, and gpFI was required, results that support the positive model. Phage 21 is a sister phage of lambda, and although its capsid proteins share approximately 60% residue identity with lambda's, phage 21 proheads did not restore cutting, even when provided with the accessory protein gpFI. Models for the role of proheads and gpFI in cos cutting are discussed.

Summary An essential component in the assembly of nucleocapsids of tailed bacteriophages and of herpes viruses is the portal protein that is located at the unique vertex of the icosahedral capsid through which DNA movements occur. A library of mutations in the bacteriophage SPP1 portal protein (gp6) was generated by random mutagenesis of gene 6. Screening of the library allowed identification of 67 single amino acid substitutions that impair portal protein function. Most of the mutations cluster within stretches of a few amino acids in the gp6 carboxyl-terminus. The mutations were divided into five classes according to the step of virus assembly that they impair: (1) production of stable gp6; (2) interaction of gp6 with the minor capsid protein gp7; (3) incorporation of gp6 in the procapsid structure; (4) DNA packaging; and (5) sizing of the packaged DNA molecule. Most of the mutations fell in classes 3 and 4. This is the first high-resolution functional map of a portal protein, in which its function at different steps of viral assembly can be directly correlated with specific regions of its sequence. The work provides a framework for the understanding of central processes in the assembly of viruses that use specialized portals to govern entry and exit of DNA from the viral capsid.

Constitutive and thermoinducible expression plasmids based on strong PR,PL promoters from phage lambda were compared for production of TNF-alpha and its analogs under various conditions. Much higher accumulation of TNF was obtained in a constitutive system, so the wider applicability of such systems was studied. In constitutive systems, proteolytically susceptible proteins can be produced easily at low cultivation temperatures and the addition of expensive or toxic chemical inducers is not required. On the other hand, toxic proteins cannot be produced and selection pressure must be strictly maintained to ensure segregational stability of plasmids. Accumulation of TNF-alpha and various analogs at levels up to 25% of total soluble protein was repeatedly achieved, which was 2–3-fold higher than in a thermoinducible system. The stable behavior of the constitutive system in laboratory fermentors was also confirmed. We propose the constitutive system described here as a general model for many currently used expression systems containing strong but not completely repressed promoters. Such systems may be considered as constitutive ones with reduced promoter strengths, but still exhibiting all the intrinsic properties of constitutive expression systems. Although all modern expression systems are inducible, wider use of a constitutive system is evidently possible. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 181–190, 2003.

Summary This is the first study on lactic acid bacteria bacteriophages isolated from dairy plants in Slovenia. Over a period of 2 years, thirteen lactococcal phages were isolated from the whey samples taken in three dairy plants. Phages were characterized by morphological properties, host range, restriction patterns, genome size and similarity of phage protein genes. All phages belonged to the Siphoviridae family. Five phages had prolate heads (52–61 · 40–52 nm) and 85–120 nm long non-contractile tails (morphotype B2). Eight phages had small isometric heads (46–63 nm in diameter) and long non-contractile tails (morphotype B1). The phages with the prolate heads showed a broader host range than the small isometric-headed phages. The genome sizes of prolate phages were estimated between 15.9 and 16.3 kb while the genome sizes of small isometric-headed phages were between 20.0 and 39.9 kb. Based on DNA restriction patterns, genome sizes and multiplex PCR analysis prolate phages were classified into the c2 group and were similar to the phage type c6A while all of the phages with small isometric heads were found to be related to the phage species 936.

Gene transfer between separate lineages of a bacterial pathogen can promote recombinational divergence and the emergence of new pathogenic variants. Temperate bacteriophages, by virtue of their ability to carry foreign DNA, are potential key players in this process. Our previous work has shown that representative strains of Salmonella typhimurium (LT2, ATCC14028 and SL1344) are lysogenic for two temperate bacteriophages: Gifsy-1 and Gifsy-2. Several lines of evidence suggested that both elements carry genes that contribute to Salmonella virulence. One such gene, on the Gifsy-2 prophage, codes for the [Cu, Zn] superoxide dismutase SodCI. Other putative pathogenicity determinants were uncovered more recently. These include genes for known or presumptive type III-translocated proteins and a locus, duplicated on both prophages, showing sequence similarity to a gene involved in Salmonella enteropathogenesis (pipA). In addition to Gifsy-1 and Gifsy-2, each of the above strains was found to harbour a specific set of prophages also carrying putative pathogenicity determinants. A phage released from strain LT2 and identified as phage Fels-1 carries the nanH gene and a novel sodC gene, which was named sodCIII. Strain ATCC14028 releases a lambdoid phage, named Gifsy-3, which contains the phoP/phoQ-activated pagJ gene and the gene for the secreted leucine-rich repeat protein SspH1. Finally, a phage specifically released from strain SL1344 was identified as SopEPhi. Most phage-associated loci transferred efficiently between Salmonella strains of the same or different serovars. Overall, these results suggest that lysogenic conversion is a major mechanism driving the evolution of Salmonella bacteria.

The integrase (Int) proteins encoded by bacteriophages HK022 and lambda catalyse similar site-specific integration and excision reactions between specific DNA regions known as attachment (att) sites. However, the Int proteins of HK022 and lambda are unable to catalyse recombination between non-cognate att sites. The att sites of both phages contain weak binding sites for Int, known as 'core-type' sites. Negatively acting nucleotide determinants associated with specific core sites (lambda B', HK022 B', HK022 C) are responsible for the barrier to non-cognate recombination. In this study, we used challenge phages to demonstrate that the lambda and HK022 Ints cannot bind to core sites containing non-cognate specificity determinants in vivo. We isolated mutants of the HK022 Int, which bind the lambda B' core site. Two mutants, D99N and D99A, have changed a residue in the core-binding (CB) domain, which may be directly contacting the core site DNA. We suggest that binding to the lambda B' site was accomplished by removing the negatively charged aspartate residue, which normally participates in a conflicting interaction with the G4 nucleotide of the lambda B' site. We showed that, although our mutants retain the ability to recombine their cognate att sites, they are unable to recombine lambda att sites.

Two different bacteriophages, FE5-B1 and Z63-B1, active against strains of Lactobacillus fermentum were isolated from a sample of sour dough of a regional wheat bread. They showed different host specificities when tested against 58 strains of obligately heterofermentative lactic acid bacteria, as well as differences in adsorption and one-step growth kinetics. The burst size of FE5-B1 was about 100 pfu cell−1. This phage belonged to the A1 morphotype of Myoviridae family, having an icosahedral head (83 nm diam.) and a sheathed contractile tail (170 nm in length). The phage consisted of five major structural proteins and had a genome of 86 kbp. Z63-B1 showed a burst size of 10 pfu cell−1 and belonged to the B1 morphotype or Siphoviridae family. Z63-B1 had an isometric head (60 nm diam.) and a non-contractile tail (160 nm in length), with eight major different structural proteins and a genome of 32 kbp.

Listeria monocytogenes bacteriophages A118, A500 and A511 are members of three distinct phage groups with characteristic host ranges. Their endolysin (ply) genes were cloned and expressed in Escherichia coli as demonstrated by the conferred lytic phenotype when colonies of recombinant cells were overlaid with a lawn of Listeria cells. The nucleotide sequences of the cloned DNA fragments were determined and the individual enzymes (PLY118, 30.8 kDa; PLY500, 33.4 kDa; PLY511, 36.5 kDa) were shown to have varying degrees of homology within their N-terminal or C-terminal domains. Transcriptional analysis revealed them to be 'late' genes with transcription beginning 15-20 min post-infection. The enzymes were overexpressed and partially purified and their individual specificities examined. When applied exogenously, the lysins induced rapid lysis of Listeria strains from all species but generally did not affect other bacteria. Using hydrolysis of purified listerial cell walls, PLY511 was characterized as an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) and shows homology in its N-terminal domain to other enzymes of this type. In contrast, PLY118 and PLY500 were shown to represent a new class of cell wall lytic enzymes which cleave between the L-alanine and D-glutamate residues of listerial peptidoglycan; these were designated as L-alanoyl-D-glutamate peptidases. These two enzymes share homology in the N-terminal domain which we propose determines hydrolytic specificity. Highly conserved holin (hol) gene sequences are present upstream of ply118 and ply500. They encode proteins of structural similarity to the product of phage lambda gene S, and are predicted to be membrane proteins which form pores to allow access of the lysins to their peptidoglycan substrates. This arrangement of conserved holin genes with downstream lysin genes among the siphoviral lysis cassettes explains why the cytoplasmic endolysins alone are not lethal, since they require a specific transport function across the cell membrane.

Twenty-four bacteriophages of Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris were classified. Two groups of bacteriophages morphologically defined as prolate or isometric types by electron microscopy were examined for their genome sizes, protein patterns and DNA homologies. These criteria showed that prolate phages are quite homogeneous. In contrast, isometric phages exhibit more differences, particularly in particle sizes and protein compositions. Analysis of DNA hybridizations confirmed that prolate phages can be grouped together as can be isometric phages but for one exception, phage I52. These two families were clearly defined. The unique phage which does not fit in either group probably belongs to a third one which is much less represented. No obvious relationships between these criteria and the lytic spectra were detected. Evidence of the presence of cohesive ends in phage genomes is also presented in this study. A more detailed analysis performed on one member of the prolate group revealed 3' protruding ends made up of around 13 nucleotides on complementary single strands.

Sixteen phage suspensions isolated from the sputum of sixteen cystic fibrosis patients and five phages from the present phage typing set were studied by electron microscopy. All sputum samples contained at least one type of bacteriophage (range: 1-4) which could be classified by the morphology and dimensions of the virion. All phages isolated from sputum as well as the four typing phages were tailed phages. The clinical phages belonged either to the Myoviridae, Siphoviridae or Podoviridae family. The four typing phages belonged to the Myoviridae family. The detection of the presence of tailed phages in sputum further supports the prevailing theory that phages play a role in the phenotypical change of Pseudomonas aeruginosa during the chronic lung infection of cystic fibrosis patients, since only tailed phages are known to be temporate and thus mediate transduction and conversion.

Exposure of lambda phage to 658 nm light in the presence of 5,10,15,20-tetrakis-(1-methyl-4-pyridyl)-21H,23H-porphine, tetra-p-tosylate leads to complete (greater than 7 logs) inactivation as measured by the plaque assay. The sensitizer without light and 658 nm photolysis of lambda phage in the absence of sensitizer do not lead to a measurable decrease in viral inactivity. Viral inactivation is not dependent upon the presence of oxygen.