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1.  Bacteriophage HK022 Nun protein arrests transcription by blocking lateral mobility of RNA polymerase during transcription elongation 
Bacteriophage  2014;4:e32187.
Coliphage HK022 excludes phage λ by subverting the λ antitermination system and arresting transcription on the λ chromosome. The 12 kDa HK022 Nun protein binds to λ nascent transcript through its N-terminal Arginine Rich Motif (ARM), blocking access by λ N and arresting transcription via a C-terminal interaction with RNA polymerase. In a purified in vitro system, we recently demonstrated that Nun arrests transcription by restricting lateral movement of transcription elongation complex (TEC) along the DNA register, thereby freezing the translocation state. We will discuss some of the key experiments that led to this conclusion, as well as present additional results that further support it.
doi:10.4161/bact.32187
PMCID: PMC4124055  PMID: 25105061
Nun protein; bacteriophage HK022; bacteriophage λ; phage exclusion; transcription arrest
2.  Bacteriophage behavioral ecology 
Bacteriophage  2014;4:e29866.
Bacteriophages have an essential gene kit that enables their invasion, replication, and production. In addition to this “core” genome, they can carry “accessory” genes that dramatically impact bacterial biology, and presumably boost their own success. The content of phage genomes continue to surprise us by revealing new ways that viruses impact bacterial biology. The genome of a Clostridium difficile myovirus, phiCDHM1, contains homologs of three bacterial accessory gene regulator (agr) genes. The agr system is a type of quorum sensing (QS), via which the phage may modify C. difficile interactions with its environment. Although their mechanism of action is unknown, mutants in bacterial versions of these genes impact sporulation and virulence. To explore how phage QS genes may influence C. difficile biology, we examine the main categories of bacterial behavior that phages have been shown to influence and discuss how interactions via QS could influence behavior at a wider level.
doi:10.4161/bact.29866
PMCID: PMC4124054  PMID: 25105060
bacteriophage; bacterial physiology; sporulation; Clostridium difficile; accessory gene regulator; microbial ecology; microbial interactions; quorum sensing; autoinducing peptide; accessory gene
3.  Molecular basis of RNA polymerase promoter specificity switch revealed through studies of Thermus bacteriophage transcription regulator 
Bacteriophage  2014;4:e29399.
Transcription initiation is the central point of gene expression regulation. Understanding of molecular mechanism of transcription regulation requires, ultimately, the structural understanding of consequences of transcription factors binding to DNA-dependent RNA polymerase (RNAP), the enzyme of transcription. We recently determined a structure of a complex between transcription factor gp39 encoded by a Thermus bacteriophage and Thermus RNAP holoenzyme. In this addendum to the original publication, we highlight structural insights that explain the ability of gp39 to act as an RNAP specificity switch which inhibits transcription initiation from a major class of bacterial promoters, while allowing transcription from a minor promoter class to continue.
doi:10.4161/bact.29399
PMCID: PMC4124052  PMID: 25105059
bacterial RNA polymerase; bacteriophage; inhibitor; sigma factor; transcription regulation
4.  Rapid Burkholderia pseudomallei identification and antibiotic resistance determination by bacteriophage amplification and MALDI-TOF MS 
Bacteriophage  2014;4:e29011.
Phage amplification detected by MALDI-TOF MS was investigated for rapid and simultaneous Burkholderia pseudomallei identification and ceftazidime resistance determination. B. pseudomallei ceftazidime susceptible and resistant ΔpurM mutant strains Bp82 and Bp82.3 were infected with broadly targeting B. pseudomallei phage ϕX216 and production of the m/z 37.6 kDa phage capsid protein observed by MALDI-TOF MS over the course of 3 h infections. This allowed for repoducible phage-based bacterial ID within 2 h of the onset of infection. MALDI-TOF MS-measured time to detection correlated with in silico modeling, which predicted an approximate 2 h detection time. Ceftazidime susceptible strain Bp82, while detectable in the absence of the drug, owing to the reliance of phage amplification on a viable host, was not detectable when 10 μg/mL ceftazidime was added at the onset of infection. In contrast, resistant strain Bp82.3 was detected in the same 2 h timeframe both with and without the addition of ceftazidime.
doi:10.4161/bact.29011
PMCID: PMC4090906  PMID: 25050191
phage amplification; bacterial identification; antibiotic resistance; Burkholderia; MALDI-TOF MS
5.  The sabotage of the bacterial transcription machinery by a small bacteriophage protein 
Bacteriophage  2014;4:e28520.
Many bacteriophages produce small proteins that specifically interfere with the bacterial host transcription machinery and thus contribute to the acquisition of the bacterial cell by the bacteriophage. We recently described how a small protein, called P7, produced by the Xp10 bacteriophage inhibits bacterial transcription initiation by causing the dissociation of the promoter specificity sigma factor subunit from the host RNA polymerase holoenzyme. In this addendum to the original publication, we present the highlights of that research.
doi:10.4161/bact.28520
PMCID: PMC3962504  PMID: 24701369
bacterial RNA polymerase; bacteriophage; inhibitor; sigma factor; transcription
6.  Different approaches for using bacteriophages against antibiotic-resistant bacteria 
Bacteriophage  2014;4:e28491.
Bacterial resistance to antibiotics is an emerging threat requiring urgent solutions. Ever since their discovery, lytic bacteriophages have been suggested as therapeutic agents, but their application faces various obstacles: sequestration of the phage by the spleen and liver, antibodies against the phage, narrow host range, poor accessibility to the infected tissue, and bacterial resistance. Variations on bacteriophage use have been suggested, such as temperate phages as gene-delivery vehicles into pathogens. This approach, which is proposed to sensitize pathogens residing on hospital surfaces and medical personnel's skin, and its prospects are described in this addendum. Furthermore, phage-encoded products have been proposed as weapons against antibiotic resistance in bacteria. We describe a new phage protein which was identified during basic research into T7 bacteriophages. This protein may serendipitously prove useful for treating antibiotic-resistant pathogens. We believe that further basic research will lead to novel strategies in the fight against antibiotic-resistant bacteria.
doi:10.4161/bact.28491
PMCID: PMC3956485  PMID: 24653944
temperate bacteriophage; sensitizing gene; lysin; host takeover; bacterial division
7.  Flap endonuclease of bacteriophage T7 
Bacteriophage  2014;4:e28507.
Gene 6 protein of bacteriophage T7 has 5′-3′-exonuclease activity specific for duplex DNA. We have found that gene 6 protein also has flap endonuclease activity. The flap endonuclease activity is considerably weaker than the exonuclease activity. Unlike the human homolog of gene 6 protein, the flap endonuclease activity of gene 6 protein is dependent on the length of the 5′-flap. This dependency of activity on the length of the 5′-flap may result from the structured helical gateway region of gene 6 protein which differs from that of human flap endonuclease 1. The flap endonuclease activity provides a mechanism by which RNA-terminated Okazaki fragments, displaced by the lagging strand DNA polymerase, are processed. 3′-extensions generated during degradation of duplex DNA by the exonuclease activity of gene 6 protein are inhibitory to further degradation of the 5′-terminus by the exonuclease activity of gene 6 protein. The single-stranded DNA binding protein of T7 overcomes this inhibition.
doi:10.4161/bact.28507
PMCID: PMC4124056  PMID: 25105057
flap endonuclease; gene 6 protein; bacteriophage T7; Okazaki fragment; DNA replication
8.  Tales from a thousand and one phages 
Bacteriophage  2014;4:e28265.
The sequencing of marine metagenomic fosmids led to the discovery of several new complete phage genomes. Among the 21 major sequence groups, 10 totally novel groups of marine phages could be identified. Some of these represent the first phages infecting large marine prokaryotic phyla, such as the Verrucomicrobia and the recently described Ca. Actinomarinales. Coming from a single deep photic zone sample the diversity of phages found is astonishing, and the comparison with a metavirome from the same location indicates that only 2% of the real diversity was recovered. In addition to this large macro-diversity, rich micro-diversity was also found, affecting host-recognition modules, mirroring the variation of cell surface components in their host marine microbes.
doi:10.4161/bact.28265
PMCID: PMC3945994  PMID: 24616837
metagenomics; metavirome; marine phages; deep chlorophyll maximum; constant-diversity; red-queen; phage evolution; pan-selectome; pan-genome
9.  Applying the ResFinder and VirulenceFinder web-services for easy identification of acquired antibiotic resistance and E. coli virulence genes in bacteriophage and prophage nucleotide sequences 
Bacteriophage  2014;4:e27943.
Extensive research is currently being conducted on the use of bacteriophages for applications in human medicine, agriculture and food manufacturing. However, phages are important vehicles of horisontal gene transfer and play a significant role in bacterial evolution. As a result, concern has been raised that this increased use and dissemination of phages could result in spread of deleterious genes, e.g., antibiotic resistance and virulence genes.
Meanwhile, in the wake of the genomic era, several tools have been developed for characterization of bacterial genomes. Here we describe how two of these tools, ResFinder and VirulenceFinder, can be used to identify acquired antibiotic resistance and virulence genes in phage genomes of interest. The general applicability of the tools is demonstrated on data sets of 1,642 phage genomes and 1,442 predicted prophages.
doi:10.4161/bact.27943
PMCID: PMC3926868  PMID: 24575358
antibiotic resistance genes; virulence genes; lysogenic conversion; horizontal gene transfer; web-services; prediction; genomics
10.  Effect of supercoiling on the λ switch 
Bacteriophage  2014;4:e27517.
The lysogenic state of the λ switch is exceptionally stable, still, it is capable of responding to DNA-damage and rapidly enter the lytic state. We invented an assay where PNA mediated tethering of a plasmid allowed for single molecule investigations of the effect of supercoiling on the efficiency of the epigenetic λ switch. Compared with non-supercoiled DNA, the presence of supercoils enhances the CI-mediated DNA looping probability and renders the transition between the looped and unlooped states steeper, thus increasing the Hill coefficient. Interestingly, the transition occurs exactly at the CI concentration corresponding to the minimum number of CI molecules capable of maintaining the pRM-repressed state. Based on these results we propose that supercoiling maintains the pRM-repressible state as CI concentration decline during induction and thus prevent autoregulation of cI from interfering with induction.
doi:10.4161/bact.27517
PMCID: PMC3875608  PMID: 24386605
CI protein; PNA; cooperativity; epigenetics; supercoiling; tethered particle motion; λ switch
11.  Identification of the ssDNA-binding protein of bacteriophage T5 
Bacteriophage  2013;3(4):e27304.
In a recent study, we identified and characterized the long-elusive replicative single-stranded DNA-binding protein of bacteriophage T5, which we showed is related to the eukaryotic transcription coactivator PC4. Here, we provide an extended discussion of these data, report several additional observations and consider implications for the recombination-dependent replication mechanism of the T5 genus, which is still poorly understood.
doi:10.4161/bact.27304
PMCID: PMC3897522  PMID: 24482743
T5; SSB; single-stranded DNA; PC4; replication; recombination; repair; 3R; RDR
12.  Properties and mutation studies of a bacteriophage-derived chimeric recombinant staphylolytic protein P128 
Bacteriophage  2013;3(3):e26564.
P128 is a chimeric anti-staphylococcal protein having a catalytic domain from a Staphylococcus bacteriophage K tail associated structural protein and a cell wall targeting domain from the Staphylococcus bacteriocin-lysostaphin. In this study, we disclose additional properties of P128 and compared the same with lysostaphin. While lysostaphin was found to get inactivated by heat and was inactive on its parent strain S. simulans biovar staphylolyticus, P128 was thermostable and was lytic towards S. simulans biovar staphylolyticus demonstrating a difference in their mechanism of action. Selected mutation studies of the catalytic domain of P128 showed that arginine and cysteine, at 40th and 76th positions respectively, are critical for the staphylolytic activity of P128, although these amino acids are not conserved residues. In comparison to native P128, only the R40S mutant (P301) was catalytically active on zymogram gel and had a similar secondary structure, as assessed by circular dichroism analysis and in silico modeling with similar cell binding properties. Mutation of the arginine residue at 40th position of the P128 molecule caused dramatic reduction in the Vmax (∆OD600 [mg/min]) value (nearly 270 fold) and the recombinant lysostaphin also showed lesser Vmax value (nearly 1.5 fold) in comparison to the unmodified P128 protein. The kinetic parameters such as apparent Km (Km APP) and apparent Kcat (KcatAPP) of the native P128 protein also showed significant differences in comparison to the values observed for P301 and lysostaphin.
doi:10.4161/bact.26564
PMCID: PMC3827070  PMID: 24251076
Staphylococcus aureus; methicillin resistance; arginine mutation; disulfide bonds; in silico modeling; western blot
13.  Identification and characterization of ϕH111-1 
Bacteriophage  2013;3(4):e26649.
Characterization of prophages in sequenced bacterial genomes is important for virulence assessment, evolutionary analysis, and phage application development. The objective of this study was to identify complete, inducible prophages in the cystic fibrosis (CF) clinical isolate Burkholderia cenocepacia H111. Using the prophage-finding program PHAge Search Tool (PHAST), we identified three putative intact prophages in the H111 sequence. Virions were readily isolated from H111 culture supernatants following extended incubation. Using shotgun cloning and sequencing, one of these virions (designated ϕH111-1 [vB_BceM_ϕH111-1]) was identified as the infective particle of a PHAST-detected intact prophage. ϕH111-1 has an extremely broad host range with respect to B. cenocepacia strains and is predicted to use lipopolysaccharide (LPS) as a receptor. Bioinformatics analysis indicates that the prophage is 42,972 base pairs in length, encodes 54 proteins, and shows relatedness to the virion morphogenesis modules of AcaML1 and “Vhmllikevirus” myoviruses. As ϕH111-1 is active against a broad panel of clinical strains and encodes no putative virulence factors, it may be therapeutically effective for Burkholderia infections.
doi:10.4161/bact.26649
PMCID: PMC3829948  PMID: 24265978
prophage identification; PHAST; bioinformatics; phage therapy; Burkholderia cepacia complex
14.  Innate and acquired bacteriophage-mediated immunity 
Bacteriophage  2013;3(3):e25857.
We recently described a novel, non-host-derived, phage-mediated immunity active at mucosal surfaces, the main site of pathogen entry in metazoans. In that work, we showed that phage T4 adheres to mucus glycoproteins via immunoglobulin-like domains displayed on its capsid. This adherence positions the phage in mucus surfaces where they are more likely to encounter and kill bacteria, thereby benefiting both the phage and its metazoan host. We presented this phage-metazoan symbiosis based on an exclusively lytic model of phage infection. Here we extend our bacteriophage adherence to mucus (BAM) model to consider the undoubtedly more complex dynamics in vivo. We hypothesize how mucus-adherent phages, both lytic and temperate, might impact the commensal microbiota as well as protect the metazoan epithelium from bacterial invasion. We suggest that BAM may provide both an innate and an acquired antimicrobial immunity.
doi:10.4161/bact.25857
PMCID: PMC3821666  PMID: 24228227
phage; bacteriophage; immune system; mucus; lysogen; lytic
15.  Phage–host interactions during pseudolysogeny 
Bacteriophage  2013;3(1):e25029.
Although the study of phage infection has a long history and catalyzed much of our current understanding in bacterial genetics, molecular biology, evolution and ecology, it seems that microbiologists have only just begun to explore the intricacy of phage–host interactions. In a recent manuscript by Cenens et al. we found molecular and genetic support for pseudolysogenic development in the Salmonella Typhimurium–phage P22 model system. More specifically, we observed the existence of phage carrier cells harboring an episomal P22 element that segregated asymmetrically upon subsequent divisions. Moreover, a newly discovered P22 ORFan protein (Pid) able to derepress a metabolic operon of the host (dgo) proved to be specifically expressed in these phage carrier cells. In this addendum we expand on our view regarding pseudolysogeny and its effects on bacterial and phage biology.
doi:10.4161/bact.25029
PMCID: PMC3694060  PMID: 23819109
Salmonella Typhimurium; phage P22; phage carrier state; phage–host interactions; pseudolysogeny
16.  Novel group of podovirus infecting the marine bacterium Alteromonas macleodii 
Bacteriophage  2013;3(2):e24766.
Four novel, closely related podoviruses, which displayed lytic activity against the gamma-proteobacterium Alteromonas macleodii, have been isolated and sequenced. Alterophages AltAD45-P1 to P4 were obtained from water recovered near a fish farm in the Mediterranean Sea. Their morphology indicates that they belong to the Podoviridae. Their linear and dsDNA genomes are 100–104 kb in size, remarkably larger than any other described podovirus. The four AltAD45-phages share 99% nucleotide sequence identity over 97% of their ORFs, although an insertion was found in AltAD45-P1 and P2 and some regions were slightly more divergent. Despite the high overall sequence similarity among these four phages, the group with the insertion and the group without it, have different host ranges against the A. macleodii strains tested. The AltAD45-P1 to P4 phages have genes for DNA replication and transcription as well as structural genes, which are similar to the N4-like Podoviridae genus that is widespread in proteobacteria. However, in terms of their genomic structure, AltAD45-P1 to P4 differ from that of the N4-like phages. Some distinguishing features include the lack of a large virion encapsidated RNA polymerase gene, very well conserved among all the previously described N4-like phages, a single-stranded DNA binding protein and different tail protein genes. We conclude that the AltAD45 phages characterized in this study constitute a new genus within the Podoviridae.
doi:10.4161/bact.24766
PMCID: PMC3821669  PMID: 24228219
Alteromonas macleodii; Podoviridae; N4-like virus; lytic phage; marine phages
17.  The moonlighting function of bacteriophage P4 capsid protein, Psu, as a transcription antiterminator 
Bacteriophage  2013;3(2):e25657.
Psu, a 20-kD bacteriophage P4 capsid decorating protein moonlights as a transcription antiterminator of the Rho-dependent termination. Psu forms specific complex with E.coli Rho protein, and affects the latter's ATP-dependent translocase activity along the nascent RNA. It forms a unique knotted dimer to take a V-shaped structure. The C-terminal helix of Psu makes specific contacts with a disordered region of Rho, encompassing the residues 139–153. An energy minimized structural model of the Rho–Psu complex reveals that the V-shaped Psu dimer forms a lid over the central channel of the Rho hexamer. This configuration of Psu causes a mechanical impediment to the translocase activity of Rho. The knowledge of structural and mechanistic basis of inhibition of Rho action by Psu may help to design peptide inhibitors for the conserved Rho-dependent transcription termination process of bacteria.
doi:10.4161/bact.25657
PMCID: PMC3821671  PMID: 24228224
transcription termination; Psu; Rho; bacteriophage
18.  Evolution of genetic switch complexity 
Bacteriophage  2013;3(1):e24186.
The circuitry of the phage λ genetic switch determining the outcome of lytic or lysogenic growth is well-integrated and complex, raising the question as to how it evolved. It is plausible that it arose from a simpler ancestral switch with fewer components that underwent various additions and refinements, as it adapted to vast numbers of different hosts and conditions. We have recently identified a new class of genetic switches found in mycobacteriophages and other prophages, in which immunity is dependent on integration. These switches contain only three genes (integrase, repressor and cro) and represent a major departure from the λ-like circuitry, lacking many features such as xis, cII and cIII. These small self-contained switches represent an unrealized, elegant circuitry for controlling infection outcome. In this addendum, we propose a model of possible events in the evolution of a complex λ-like switch from a simpler integration-dependent switch.
doi:10.4161/bact.24186
PMCID: PMC3694055  PMID: 23819104
genetic switch; genetic circuits; bistable; integration-dependent immunity; lytic and lysogenic growth
19.  Lytic bacteriophages reduce Escherichia coli O157 
Bacteriophage  2013;3(1):e24323.
The role of lytic bacteriophages in preventing cross contamination of produce has not been evaluated. A cocktail of three lytic phages specific for E. coli O157:H7 (EcoShield™) or a control (phosphate buffered saline, PBS) was applied to lettuce by either; (1) immersion of lettuce in 500 ml of EcoShield™ 8.3 log PFU/ml or 9.8 log PFU/ml for up to 2 min before inoculation with E. coli O157:H7; (2) spray-application of EcoShield™ (9.3 log PFU/ml) to lettuce after inoculation with E. coli O157:H7 (4.10 CFU/cm2) following exposure to 50 μg/ml chlorine for 30 sec. After immersion studies, lettuce was spot-inoculated with E. coli O157:H7 (2.38 CFU/cm2). Phage-treated, inoculated lettuce pieces were stored at 4°C for and analyzed for E. coli O157:H7 populations for up to 7 d. Immersion of lettuce in 9.8 log PFU/ml EcoShield™ for 2 min significantly (p < 0.05) reduced E. coli O157:H7 populations after 24 h when stored at 4°C compared with controls. Immersion of lettuce in suspensions containing high concentrations of EcoShield™ (9.8 log PFU/ml) resulted in the deposition of high concentrations (7.8 log log PFU/cm2) of bacteriophages on the surface of fresh cut lettuce, potentially contributing to the efficacy of the lytic phages on lettuce. Spraying phages on to inoculated fresh cut lettuce after being washed in hypochlorite solution was significantly more effective in reducing E. coli O157:H7 populations (2.22 log CFU/cm2) on day 0 compared with control treatments (4.10 log CFU/cm2). Both immersion and spray treatments provided protection from E. coli O157:H7 contamination on lettuce, but spray application of lytic bacteriophages to lettuce was more effective in immediately reducing E. coli O157:H7 populations fresh cut lettuce.
doi:10.4161/bact.24323
PMCID: PMC3694057  PMID: 23819106
bacteriophages; E. coli O157:H7; lettuce; processing
20.  Biocontrol of Escherichia coli O157 
Bacteriophage  2013;3(1):e24620.
The effect of a bacteriophage cocktail (EcoShield™) that is specific against Escherichia coli O157:H7 was evaluated against a nalidixic acid-resistant enterohemorrhagic E. coli O157:H7 RM4407 (EHEC) strain on leafy greens stored under either (1) ambient air or (2) modified atmosphere (MA; 5% O2/35% CO2/60% N2). Pieces (~2 × 2 cm2) of leafy greens (lettuce and spinach) inoculated with 4.5 log CFU/cm2 EHEC were sprayed with EcoShield™ (6.5 log PFU/cm2). Samples were stored at 4 or 10°C for up to 15 d. On spinach, the level of EHEC declined by 2.38 and 2.49 log CFU/cm2 at 4 and 10°C, respectively, 30 min after phage application (p ≤ 0.05). EcoShield™ was also effective in reducing EHEC on the surface of green leaf lettuce stored at 4°C by 2.49 and 3.28 log units in 30 min and 2 h, respectively (p ≤ 0.05).
At 4°C under atmospheric air, the phage cocktail significantly (p ≤ 0.05) lowered the EHEC counts in one day by 1.19, 3.21 and 3.25 log CFU/cm2 on spinach, green leaf and romaine lettuce, respectively compared with control (no bacteriophage) treatments. When stored under MA at 4°C, phages reduced (p ≤ 0.05) EHEC populations by 2.18, 3.50 and 3.13 log CFU/cm2, on spinach, green leaf and romaine lettuce. At 10°C, EHEC reductions under atmospheric air storage were 1.99, 3.90 and 3.99 log CFU/cm2 (p ≤ 0.05), while population reductions under MA were 3.08, 3.89 and 4.34 logs on spinach, green leaf and romaine lettuce, respectively, compared with controls (p ≤ 0.05). The results of this study showed that bacteriophages were effective in reducing the levels of E. coli O157:H7 on fresh leafy produce, and that the reduction was further improved when produce was stored under the MA conditions.
doi:10.4161/bact.24620
PMCID: PMC3694058  PMID: 23819107
E. coli O157:H7; bacteriophage; modified atmosphere packaging; MAP; leafy green
21.  Biochemical insights into the function of phage G1 gp67 in Staphylococcus aureus 
Bacteriophage  2013;3(1):e24767.
Bacteriophage (phage) are among the most diverse and abundant life forms on Earth. Studies have recently used phage diversity to identify novel antimicrobial peptides and proteins. We showed that one such phage protein, Staphylococcus aureus (Sau) phage G1 gp67, inhibits cell growth in Sau by an unusual mechanism. Gp67 binds to the host RNA polymerase (RNAP) through an interaction with the promoter specificity σ subunit, but unlike many other σ-binding phage proteins, gp67 does not disrupt transcription at most promoters. Rather, gp67 prevents binding of another RNAP domain, the α-C-terminal domain, to upstream A/T-rich elements required for robust transcription at rRNA promoters. Here, we discuss additional biochemical insights on gp67, how phage promoters escape the inhibitory function of gp67, and methodological advancements that were foundational to our work.
doi:10.4161/bact.24767
PMCID: PMC3694059  PMID: 23819108
Staphylococcus aureus; RNA polymerase; bacteriophage; gp67; transcription
22.  Soil-based systemic delivery and phyllosphere in vivo propagation of bacteriophages 
Bacteriophage  2012;2(4):215-224.
Soil-based root applications and attenuated bacterial strains were evaluated as means to enhance bacteriophage persistence on plants for bacterial disease control. In addition, the systemic nature of phage applied to tomato roots was also evaluated. Several experiments were conducted applying either single phages or phage mixtures specific for Ralstonia solanacearum, Xanthomonas perforans or X. euvesicatoria to soil surrounding tomato plants and measuring the persistence and translocation of the phages over time. In general, all phages persisted in the roots of treated plants and were detected in stems and leaves; although phage level varied and persistence in stems and leaves was at a much lower level compared with persistence in roots. Bacterial wilt control was typically best if the phage or phage mixtures were applied to the soil surrounding tomatoes at the time of inoculation, less effective if applied 3 days before inoculation, and ineffective if applied 3 days after inoculation. The use of an attenuated X. perforans strain was also evaluated to improve the persistence of phage populations on tomato leaf surfaces. In greenhouse and field experiments, foliar applications of an attenuated mutant X. perforans 91-118:∆OPGH strain prior to phage applications significantly improved phage persistence on tomato foliage compared with untreated tomato foliage. Both the soil-based bacteriophage delivery and the use of attenuated bacterial strains improved bacteriophage persistence on respective root and foliar tissues, with evidence of translocation with soil-based bacteriophage applications. Both strategies could lead to improved control of bacterial pathogens on plants.
doi:10.4161/bact.23530
PMCID: PMC3594209  PMID: 23532156
bacteriophage; biocontrol; phage; tomato
23.  Development of a high throughput assay for indirectly measuring phage growth using the OmniLogTM system 
Bacteriophage  2012;2(3):159-167.
The conventional and most accepted method of measuring the lytic activity of a phage against its bacterial host is the plaque assay. This method is laborious, time consuming and expensive, especially in high throughput analyses where multiple phage-bacterial interactions are required to be monitored simultaneously. It can also vary considerably with the experimenter and by the growth and plating conditions. Alternatively, the lytic activity can be measured indirectly by following the decrease in optical density of the bacterial cultures owing to lysis. Here we describe an automated, high throughput, indirect liquid lysis assay to evaluate phage growth using the OmniLogTM system. The OmniLogTM system uses redox chemistry, employing cell respiration as a universal reporter. During active growth of bacteria, cellular respiration reduces a tetrazolium dye and produces a color change that is measured in an automated fashion. On the other hand, successful phage infection and subsequent growth of the phage in its host bacterium results in reduced bacterial growth and respiration and a concomitant reduction in color. Here we show that microtiter plate wells inoculated with Bacillus anthracis and phage show decreased or no growth, compared with the wells containing bacteria only or phage resistant bacteria plus phage. Also, we show differences in the kinetics of bacterial growth and the timing of appearance of phage resistant bacteria in the presence of individual phages or a cocktail of B. anthracis specific phages. The results of these experiments indicate that the OmniLogTM system could be used reliably for indirectly measuring phage growth in high throughput host range and phage and antibiotics combination studies.
doi:10.4161/bact.21440
PMCID: PMC3530525  PMID: 23275867
Bacillus anthracis; OmniLogTM; bacteriophage; in vitro lytic assay; phage
24.  A Yersinia pestis-specific, lytic phage preparation significantly reduces viable Y. pestis on various hard surfaces experimentally contaminated with the bacterium 
Bacteriophage  2012;2(3):168-177.
Five Y. pestis bacteriophages obtained from various sources were characterized to determine their biological properties, including their taxonomic classification, host range and genomic diversity. Four of the phages (YpP-G, Y, R and YpsP-G) belong to the Podoviridae family, and the fifth phage (YpsP-PST) belongs to the Myoviridae family, of the order Caudovirales comprising of double-stranded DNA phages. The genomes of the four Podoviridae phages were fully sequenced and found to be almost identical to each other and to those of two previously characterized Y. pestis phages Yepe2 and φA1122. However, despite their genomic homogeneity, they varied in their ability to lyse Y. pestis and Y. pseudotuberculosis strains. The five phages were combined to yield a “phage cocktail” (tentatively designated “YPP-100”) capable of lysing the 59 Y. pestis strains in our collection. YPP-100 was examined for its ability to decontaminate three different hard surfaces (glass, gypsum board and stainless steel) experimentally contaminated with a mixture of three genetically diverse Y. pestis strains CO92, KIM and 1670G. Five minutes of exposure to YPP-100 preparations containing phage concentrations of ca. 109, 108 and 107 PFU/mL completely eliminated all viable Y. pestis cells from all three surfaces, but a few viable cells were recovered from the stainless steel coupons treated with YPP-100 diluted to contain ca. 106 PFU/mL. However, even that highly diluted preparation significantly (p = < 0.05) reduced Y. pestis levels by ≥ 99.97%. Our data support the idea that Y. pestis phages may be useful for decontaminating various hard surfaces naturally- or intentionally-contaminated with Y. pestis.
doi:10.4161/bact.22240
PMCID: PMC3530526  PMID: 23275868
bacteriophage; phage; Yersinia pestis; surface decontamination
25.  Bacteriophage cocktail significantly reduces Escherichia coli O157 
Bacteriophage  2012;2(3):178-185.
Foods contaminated with Escherichia coli O157:H7 cause more than 63,000 foodborne illnesses in the United States every year, resulting in a significant economic impact on medical costs and product liabilities. Efforts to reduce contamination with E. coli O157:H7 have largely focused on washing, application of various antibacterial chemicals, and gamma-irradiation, each of which has practical and environmental drawbacks. A relatively recent, environmentally-friendly approach proposed for eliminating or significantly reducing E. coli O157:H7 contamination of foods is the use of lytic bacteriophages as biocontrol agents. We found that EcoShield™, a commercially available preparation composed of three lytic bacteriophages specific for E. coli O157:H7, significantly (p < 0.05) reduced the levels of the bacterium in experimentally contaminated beef by ≥ 94% and in lettuce by 87% after a five minute contact time. The reduced levels of bacteria were maintained for at least one week at refrigerated temperatures. However, the one-time application of EcoShield™ did not protect the foods from recontamination with E. coli O157:H7. Our results demonstrate that EcoShield™ is effective in significantly reducing contamination of beef and lettuce with E. coli O157:H7, but does not protect against potential later contamination due to, for example, unsanitary handling of the foods post processing.
doi:10.4161/bact.22825
PMCID: PMC3530527  PMID: 23275869
EcoShield™; Escherichia coli O157:H7; bacteriophage; beef; food safety; genomics; ground beef; lettuce; phage; phylogeny

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