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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.  Life in Science: Abraham Eisenstark 
Bacteriophage  2014;4:e29009.
doi:10.4161/bact.29009
PMCID: PMC4116385  PMID: 25101215
bacteriophage; mentors; bacteria; phage display; history
3.  Inactivation of Streptomyces phage ɸC31 by 405 nm light 
Bacteriophage  2014;4:e32129.
Exposure to narrowband violet-blue light around 405 nm wavelength can induce lethal oxidative damage to bacteria and fungi, however effects on viruses are unknown. As photosensitive porphyrin molecules are involved in the microbicidal inactivation mechanism, and since porphyrins are absent in viruses, then any damaging effects of 405 nm light on viruses might appear unlikely. This study used the bacteriophage ɸC31, as a surrogate for non-enveloped double-stranded DNA viruses, to establish whether 405 nm light can induce virucidal effects. Exposure of ɸC31 suspended in minimal media, nutrient-rich media, and porphyrin solution, demonstrated differing sensitivity of the phage. Significant reductions in phage titer occurred when exposed in nutrient-rich media, with ~3-, 5- and 7-log10 reductions achieved after exposure to doses of 0.3, 0.5 and 1.4 kJ/cm2, respectively. When suspended in minimal media a 0.3-log10 reduction (P = 0.012) occurred after exposure to 306 J/cm2: much lower than the 2.7- and > 2.5-log10 reductions achieved with the same dose in nutrient-rich, and porphyrin-supplemented media, suggesting inactivation is accelerated by the photo-activation of light-sensitive components in the media. This study provides the first evidence of the interaction of narrowband 405 nm light with viruses, and demonstrates that viral susceptibility to 405 nm light can be significantly enhanced by involvement of exogenous photosensitive components. The reduced susceptibility of viruses in minimal media, compared with that of other microorganisms, provides further evidence that the antimicrobial action of 405 nm light is predominantly due to the photo-excitation of endogenous photosensitive molecules such as porphyrins within susceptible microorganisms.
doi:10.4161/bact.32129
PMCID: PMC4116386  PMID: 25101216
405 nm light; bacteriophage; inactivation; photosensitizers; virus; ɸC31
4.  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
5.  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
6.  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
7.  Morphotypes of virus-like particles in two hydrothermal vent fields on the East Scotia Ridge, Antarctica 
Bacteriophage  2014;4:e28732.
Viruses from extreme environments are still largely unexplored and may harbor unseen genetic potential. Here, we present a first glance at the morphological diversity of virus like particles (VLPs) from an environment that is extreme in more than one respect: two recently discovered hydrothermal vent fields on the East Scotia Ridge in the Southern Ocean near Antarctica. They are the southernmost hydrothermal sites found to date and have been shown to present a new biogeographic province, containing several new macrofaunal species and associated microbial organisms. Transmission electron microscopy revealed a range of tailed and untailed VLPs of various morphologies as well as an unusual long rod-shaped VLP with three long filaments. Based on its distant similarity with several known archaeal viruses, we hypothesize that this presents a new viral morphology that most likely infects an archaeon. Notably absent in the samples we analyzed were lemon- or spindle-shaped VLPs that have previously been described in other hydrothermal vent settings.
doi:10.4161/bact.28732
PMCID: PMC4124060  PMID: 25105058
hydrothermal vent; deep sea bacteriophage; East Scotia Ridge; Antarctica; transmission electron microscopy; phage morphology; archaeal phage
8.  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
9.  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
10.  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
11.  Tradeoffs in bacteriophage life histories 
Bacteriophage  2014;4:e28365.
Viruses are the most abundant biological entities on the planet, yet most classical principles of evolutionary biology and ecology were not developed with viruses in mind. Here, the concept of biological tradeoffs, a fundamental tenet of life history theory, is examined in the context of bacteriophage biology. Specifically, several important parameters of phage life histories—replication, persistence, host range, and adsorption—are evaluated for tradeoffs. Available data indicate that replication rate is strongly negatively correlated with both persistence and host range, suggesting that the well-documented tradeoff in macroorganisms between offspring production and offspring quality also applies to phages. The biological tradeoffs that appear to characterize viruses’ life histories have potential importance for viral evolution, ecology, and pathogenesis.
doi:10.4161/bact.28365
PMCID: PMC3942329  PMID: 24616839
adsorption; bacteriophages; biological tradeoffs; ECOR; host range; life history theory; microbial ecology; replication rate; viral evolution
12.  Molecular architecture of tailed double-stranded DNA phages 
Bacteriophage  2014;4:e28281.
The tailed double-stranded DNA bacteriophages, or Caudovirales, constitute ~96% of all the known phages. Although these phages come in a great variety of sizes and morphology, their virions are mainly constructed of similar molecular building blocks via similar assembly pathways. Here we review the structure of tailed double-stranded DNA bacteriophages at a molecular level, emphasizing the structural similarity and common evolutionary origin of proteins that constitute these virions.
doi:10.4161/bact.28281
PMCID: PMC3940491  PMID: 24616838
bacteriophage structure; Caudovirales; tailed double-stranded DNA bacteriophages; virus structure; virus capsids; phage tails; phage proteins
13.  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
14.  Application of bacteriophages for detection of foodborne pathogens 
Bacteriophage  2014;4:e28137.
Bacterial contamination of food products presents a challenge for the food industry and poses a high risk for the consumer. Despite increasing awareness and improved hygiene measures, foodborne pathogens remain a threat for public health, and novel methods for detection of these organisms are needed. Bacteriophages represent ideal tools for diagnostic assays because of their high target cell specificity, inherent signal-amplifying properties, easy and inexpensive production, and robustness. Every stage of the phage lytic multiplication cycle, from the initial recognition of the host cell to the final lysis event, may be harnessed in several ways for the purpose of bacterial detection. Besides intact phage particles, phage-derived affinity molecules such as cell wall binding domains and receptor binding proteins can serve for this purpose. This review provides an overview of existing phage-based technologies for detection of foodborne pathogens, and highlights the most recent developments in this field, with particular emphasis on phage-based biosensors.
doi:10.4161/bact.28137
PMCID: PMC3919822  PMID: 24533229
foodborne pathogens; bacterial detection; diagnostics; reporter phage; biosensor; phage amplification; cell wall binding domain; receptor binding protein
15.  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
16.  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
17.  Life in Science: William C Summers 
Bacteriophage  2014;4:e26691.
doi:10.4161/bact.26691
PMCID: PMC3895083  PMID: 24478937
18.  Bacteriophage as instructional organisms in introductory biology labs 
Bacteriophage  2014;4:e27336.
Designing lab exercises for introductory biology classes requires balancing the need for students to obtain results with a desire to provide unpredictable outcomes to better approximate actual research. Bacteriophage are particularly well suited for this as many species are well-understood but, with their hosts, represent a relatively complex interacting system. I have designed a seven week series of lab exercises that allow students to select bacteriophage resistant mutant hosts, isolate and sequence the corresponding receptor gene to identify the specific bacterial mutation from a large number of potential mutations. I also examined the possibility of collecting useful mutant strains for other studies. After two semesters, the lab series is working well with over 90% of students successfully isolating mutant bacteria and about half identifying the specific mutation. Here I discuss the advantages of using bacteriophage in an introductory class, the specific labs in this series and future plans.
doi:10.4161/bact.27336
PMCID: PMC3895413  PMID: 24478938
teaching; laboratory teaching; bacteriophage T5; host range; receptor; receptor binding protein
19.  The XXIIIrd Phage/Virus Assembly Meeting 
Bacteriophage  2014;4:e27272.
The XXIIIrd Phage/Virus Assembly (PVA) meeting returned to its birthplace in Lake Arrowhead, CA on September 8–13, 2013 (Fig. 1). The original meeting occurred in 1968, organized by Bob Edgar (Caltech, Pasadena, CA USA), Fred Eiserling (University of California, Los Angeles, Los Angeles, CA USA) and Bill Wood (Caltech, Pasadena, CA USA). The organizers of the 2013 meeting were Bill Gelbart (University of California, Los Angeles, Los Angeles, CA USA) and Jack Johnson (Scripps Research Institute, La Jolla, CA USA). This meeting specializes in an egalitarian format. Students are distinguished from senior faculty primarily by the signs of age. With the exception of historically based introductory talks, all talks were allotted the same time and freedom. This tradition began when the meeting was phage-only and has been continued now that all viruses are included. Many were the animated conversations about basic questions. New and international participants were present, a sign that the field has significant attraction, as it should, based on details below. The meeting was also characterized by a sense of humor and generally good times, a chance to both enjoy the science and forget the funding malaise to which many participants are exposed. I will present some of the meeting content, without attempting to be comprehensive.
doi:10.4161/bact.27272
PMCID: PMC3906323  PMID: 24498537
capsid assembly; viral structure; viral drug delivery vehicle; DNA injection; viral tail assembly; bacteriophage; nucleic acid packaging; viral vaccines; nanoparticle
20.  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
21.  Listeria phages 
Bacteriophage  2013;3(3):e26861.
Listeria is an important foodborne pathogen and the causative agent of Listeriosis, a potentially fatal infection. Several hundred Listeria bacteriophages have been described over the past decades, but only few have actually been characterized in some detail, and genome sequences are available for less than twenty of them. We here present an overview of what is currently known about Listeria phage genomics, their role in host evolution and pathogenicity, and their various applications in biotechnology and diagnostics.
doi:10.4161/bact.26861
PMCID: PMC3827098  PMID: 24251077
CRISPR; Mosaic genomes; biocontrol; comK; endolysin; homologous recombination; pathogen detection; reporter phage
22.  Bacteriophage K for reduction of Staphylococcus aureusbiofilm on central venous catheter material 
Bacteriophage  2013;3(4):e26825.
The purpose of this project was to determine whether bacteriophage can reduce bacterial colonization and biofilm formation on central venous catheter material. Twenty silicone discs were inoculated for 24 h with broth culture of Methicillin sensitive staphylococcus aureus (0.5 McFarland standard). The inoculate was aspirated and discs placed into two equal groups for 24 h: (1) untreated controls; (2) bacteriophage treatment (staphylococcal bacteriophage K, propagated titer > 108). At the completion of the experiment discs were processed for quantitative culture. Statistical testing was performed using the rank sum test. Mean colony forming units (CFU) were significantly decreased in experimental compared with controls (control 6.3 × 105 CFU, experimental 6.7 × 101, P ≤ 0.0001). Application of bacteriophage to biofilm infected central venous catheter material significantly reduced bacterial colonization and biofilm presence. Our data suggests that bacteriophage treatment may be a feasible strategy for addressing central venous catheter staph aureus biofilm infections.
doi:10.4161/bact.26825
PMCID: PMC3829956  PMID: 24265979
central venous catheters; biofilm; phage therapy; staph aureus; bacteriophage k
23.  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
24.  Life in Science: Björn H Lindqvist 
Bacteriophage  2013;3(4):e26673.
doi:10.4161/bact.26673
PMCID: PMC3827068  PMID: 24251078
25.  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

Results 1-25 (105)