Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes

PubMed Central

Schmeisser, Falko; Weir, Jerry P

2007-01-01

Background Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC) technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2) BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome. Results Here we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction. Conclusion Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors. PMID:17501993

Rifampin-stimulated uv resistance of phage lambda on Escherichia coli K12

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bronner, C.E.; Fluke, D.J.; Pollard, E.C.

1983-01-01

The plaque survival of uv-irradiated phage lambda on excision-proficient E.coli strain AB1157 is greater if the host cells are exposed to rifampin for 10 minutes prior to infection. This repair is accompanied by little or no clear-plaque mutagenesis. Host cells uv-irradiated and incubated for 30 minutes in growth medium prior to treatment with rifampin show some Weigle-reactivation in addition to the repair stimulated by rifampin. Some clear-plaque Weigle-mutagenesis is also observed in the presence of rifampin: however, the amount is less than that seen in the absence of rifampin treatment. In contrast, the uv sensitivity of lambda on strain AB1886,more » an excision-repair deficient mutant, is unchanged by pre-treating the cells with rifampin, and no Weigle-reactivation is observed. These results suggest that repair of lambda on unirradiated cells in the presence of rifampin is an excision-dependent, error-free phenomenon. Since initiation of replication of lambda DNA requires RNA polymerase, and since rifampin blocks transcription by that polymerase, the effect of rifampin on the survival of lambda may be to delay phage replication, thereby allowing more time for excision repair to operate. 6 figures.« less

Bacteriophage T5 DNA ejection under pressure.

PubMed

Leforestier, A; Brasilès, S; de Frutos, M; Raspaud, E; Letellier, L; Tavares, P; Livolant, F

2008-12-19

The transfer of the bacteriophage genome from the capsid into the host cell is a key step of the infectious process. In bacteriophage T5, DNA ejection can be triggered in vitro by simple binding of the phage to its purified Escherichia coli receptor FhuA. Using electrophoresis and cryo-electron microscopy, we measure the extent of DNA ejection as a function of the external osmotic pressure. In the high pressure range (7-16 atm), the amount of DNA ejected decreases with increasing pressure, as theoretically predicted and observed for lambda and SPP1 bacteriophages. In the low and moderate pressure range (2-7 atm), T5 exhibits an unexpected behavior. Instead of a unique ejected length, multiple populations coexist. Some phages eject their complete genome, whereas others stop at some nonrandom states that do not depend on the applied pressure. We show that contrarily to what is observed for the phages SPP1 and lambda, T5 ejection cannot be explained as resulting from a simple pressure equilibrium between the inside and outside of the capsid. Kinetics parameters and/or structural characteristics of the ejection machinery could play a determinant role in T5 DNA ejection.

Illegitimate recombination mediated by calf thymus DNA topoisomerase II in vitro.

PubMed Central

Bae, Y S; Kawasaki, I; Ikeda, H; Liu, L F

1988-01-01

We have found that purified calf thymus DNA topoisomerase II mediates recombination between two phage lambda DNA molecules in an in vitro system. The enzyme mainly produced a linear monomer recombinant DNA that can be packaged in vitro. Novobiocin and anti-calf thymus DNA topoisomerase II antibody inhibit this ATP-dependent recombination. The recombinant molecules contain duplications or deletions, and most crossovers take place between nonhomologous sequences of lambda DNA, as judged by the sequences of recombination junctions. Therefore, the recombination mediated by the calf thymus DNA topoisomerase II is an illegitimate recombination that is similar to recombination mediated by Escherichia coli DNA gyrase or phage T4 DNA topoisomerase. The subunit exchange model, which has been suggested for the DNA gyrase-mediated recombination, is now generalized as follows: DNA topoisomerase II molecules bind to DNAs, associate with each other, and lead to the exchange of DNA strands through the exchange of topoisomerase II subunits. Illegitimate recombination might be carried out by a general mechanism in organisms ranging from prokaryotes to higher eukaryotes. Images PMID:2832845

Absorption Kinetics of Phage Lambda on Its Host Under Shear Flow

NASA Astrophysics Data System (ADS)

Yip, C. W.; Wu, X. L.

2000-03-01

Classical blender experiment by Hershey and Chase played a seminal role in illustrating the infectious process of bacteriophage to its host, and showed unequivocally that DNA is responsible for the transmission of heredity. Subsequent works by others have established that interaction between phage particles and bacterial cells is a diffusion-limited process in that, statistically speaking, each collision results in an irreversible infection. However, such a result is hard to reconcile with the fact that the infection appears to be independent of the density of phage receptors on the bacterial cell membrane. Thus, quantitative experiments showing how a phage finds its receptor and how long does it take would be valuable to this paradoxical view. Simple calculations based on Brownian motion of the phage particles show that the interaction time between the receptor and the phage is given by tau=b^2/(5D), where b is the length of the phage and D is its diffusion coefficient. Using a shear flow apparatus we study absorption kinetics of lambda phage on E. Coli (strain YMEL) under different flow conditions, and the results are compared with a simple diffusion model taking into account the hydrodynamic convection and the interaction time tau.

Cloning and study of the pectate lyase gene of Erwinia carotovora

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bukanov, N.O.; Fonshtein, M.Yu.; Evtushenkov, A.N.

1986-04-01

The cloning of the gene of a secretable protein of Erwinia carotovora, pectate lyase, in Escherichia coli was described. Primary cloning was conducted using the phage vector lambda 47.1. In the gene library of E. carotovora obtained, eight phages carrying the gene sought were identified according to the appearance of enzymatic activity of the gene product, pectate lyase, in situ. The BamHI fragment of DNA, common to all these phages, was recloned on the plasmid pUC19. It was shown that the cloned pectate lyase gene is represented on the E. carotovora chromosome in one copy. Methods of production of representativemore » gene libraries on phage vectors from no less than 1 ..mu..g of cloned DNA even for the genomes of eukaryotes have now been developed. Vectors have been created, for example, lambda 47.1, permitting the selection only of hybrid molecules. A number of methods have been developed for the search for a required gene in the library, depending on whether the cloned gene can be expressed or not, and if it can, what properties it will impart to the hybrid clone containing it.« less

General transfer matrix formalism to calculate DNA-protein-drug binding in gene regulation: application to OR operator of phage lambda.

PubMed

Teif, Vladimir B

2007-01-01

The transfer matrix methodology is proposed as a systematic tool for the statistical-mechanical description of DNA-protein-drug binding involved in gene regulation. We show that a genetic system of several cis-regulatory modules is calculable using this method, considering explicitly the site-overlapping, competitive, cooperative binding of regulatory proteins, their multilayer assembly and DNA looping. In the methodological section, the matrix models are solved for the basic types of short- and long-range interactions between DNA-bound proteins, drugs and nucleosomes. We apply the matrix method to gene regulation at the O(R) operator of phage lambda. The transfer matrix formalism allowed the description of the lambda-switch at a single-nucleotide resolution, taking into account the effects of a range of inter-protein distances. Our calculations confirm previously established roles of the contact CI-Cro-RNAP interactions. Concerning long-range interactions, we show that while the DNA loop between the O(R) and O(L) operators is important at the lysogenic CI concentrations, the interference between the adjacent promoters P(R) and P(RM) becomes more important at small CI concentrations. A large change in the expression pattern may arise in this regime due to anticooperative interactions between DNA-bound RNA polymerases. The applicability of the matrix method to more complex systems is discussed.

A Novel Approach to the Elucidation of the Mechanism of Development of Androgen-Independent Growth of Prostate Cancer

DTIC Science & Technology

2001-01-01

with 1.0 with differential hybridization to the two probes. Phage plaques corresponding ml of RIPA buffer with protease inhibitors (PBS, 1% NP40, 0.5...hybridized to radiolabeled probe prepared from plaque-purified phage . at 10,000 X g for 20 min; supernatants were collected and centrifuged again to...Screening. Lambda phage plaques from the primary unamplified Differential expression screening of cDNA libraries constructed CWR22 library were plated

Novel DNA packaging recognition in the unusual bacteriophage N15

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feiss, Michael; Geyer, Henriette, E-mail: henriettegeyer@gmail.com; Division of Viral Infections, Robert Koch Institute, Berlin

Phage lambda's cosB packaging recognition site is tripartite, consisting of 3 TerS binding sites, called R sequences. TerS binding to the critical R3 site positions the TerL endonuclease for nicking cosN to generate cohesive ends. The N15 cos (cos{sup N15}) is closely related to cos{sup λ}, but whereas the cosB{sup N15} subsite has R3, it lacks the R2 and R1 sites and the IHF binding site of cosB{sup λ}. A bioinformatic study of N15-like phages indicates that cosB{sup N15} also has an accessory, remote rR2 site, which is proposed to increase packaging efficiency, like R2 and R1 of lambda. N15more » plus five prophages all have the rR2 sequence, which is located in the TerS-encoding 1 gene, approximately 200 bp distal to R3. An additional set of four highly related prophages, exemplified by Monarch, has R3 sequence, but also has R2 and R1 sequences characteristic of cosB–λ. The DNA binding domain of TerS-N15 is a dimer. - Highlights: • There are two classes of DNA packaging signals in N15-related phages. • Phage N15's TerS binding site: a critical site and a possible remote accessory site. • Viral DNA recognition signals by the λ-like bacteriophages: the odd case of N15.« less

Study of genetic effects of high energy radiations with different ionizing capacities on extracellular phages.

PubMed

Bresler, S E; Kalinin, V L; Kopylova, Y U; Krivisky, A S; Rybchin, V N; Shelegedin, V N

1975-07-01

The inactivating and mutagenic action of high-energy radiations with different ionizing capacities (gamma-rays, protons, alpha-particles and accelerated ions of 12C and 20Ne) was studied by using coliphages lambda11 and SD as subjects. In particular the role of irradiation conditions (broth suspension, pure buffer, dry samples) and of the host functions recA, exrA and polA was investigated. The dose-response curve of induced mutagenesis was studied by measuring the yield of vir mutants in lambda11 and plaque mutants in SD. The following results were obtained. (1) The inactivation kinetics of phages under the action of gamma-rays and protons was first order to a survival of 10(-7). Heavy ions also showed exponential inactivation kinetics to a survival of 10(-4). At higher doses of 20Ne ion bombardment some deviation from one-hit kinetics was observed. For dry samples of phages the dimensions of targets for all types of radiation were approximately proportional to the molecular weights of phage DNA's. For densely ionizing radiation (heavy ions) the inactivating action was 3-5 times weaker than for gamma-rays and protons. (2) Mutagenesis was observed for all types of radiation, but heavy ions were 1-5-2 times less efficient than gamma-rays. For both phages studied the dose-response curve of mutagenesis was non-linear. The dependence on the dose was near to parabolic for lambda11. For SD a plateau or maximum of mutagenesis was observed for the relative number of mutants at a survival of about 10(-4). (3) Host-cell functions recA and exrA were practically indifferent for survival of gamma-irradiated phage lambda11, but indispensable for mutagenesis. Mutation recAI3 abolished induced vir mutations totally and exrA- reduced them significantly. The absence of the function polA had a considerable influence on phage survival, but no effect on vir mutation yield (if compared at the same survival level). (4) In conditions of indirect action of gamma-rays no vir mutations were induced. This is regarded as evidence that the single-strand breaks formed under indirect action conditions cannot serve as pre-mutational damage in DNA.

Analysis of Endonuclease R·EcoRI Fragments of DNA from Lambdoid Bacteriophages and Other Viruses by Agarose-Gel Electrophoresis

PubMed Central

Helling, Robert B.; Goodman, Howard M.; Boyer, Herbert W.

1974-01-01

By means of agarose-gel electrophoresis, endonuclease R·EcoRI-generated fragments of DNA from various viruses were separated, their molecular weights were determined, and complete or partial fragment maps for lambda, φ80, and hybrid phages were constructed. Images PMID:4372397

Content and persistence of extracellular DNA in native soils

NASA Astrophysics Data System (ADS)

Blagodatskaya, Evgenia; Blagodatsky, Sergey; Anderson, Traute-Heidi; Kuzyakov, Yakov

2014-05-01

The long-term persistence of soil extracellular DNA is questionable because of high potential activity of nucleases produced by soil microorganisms. By the other hand, the relative persistence of DNA-like biopolymers could be due to their adsorption on clay minerals and humus substances in soil. High-specific and ultra sensitive reagent PicoGreenTM (Molecular Probes) permits the quantitative assessment of microbial dsDNA in diluted soil extracts giving a good tool for tracing the DNA fate in soil. Our goal was to determine intracellular and extracellular DNA content in cambisol (loamy sand) and in chernozem (silty loam) soils and to investigate the possible adsorption and degradation of extracellular DNA in soil. Optimized procedure of mechanical and enzymatic destruction of cell walls was used for direct extraction of microbial DNA with Tris-EDTA buffer (Blagodatskaya et al., 2003). Extracellular dsDNA was determined in distilled water and in Tris-EDTA extracts without enzymatic or mechanical treatments. DNA content was determined after addition of PicoGreen to diluted soil extracts. Degradation of extracellular DNA was traced during 24 h incubation of 2 µg lambda-phage DNA in soil. Possible DNA adsorption to soil matrix was determined by recovery of lambda -phage DNA added to autoclaved soil. Extracellular dsDNA was absent in water extracts of both soils. The content of extracellular dsDNA extracted by Tris-EDTA buffer was 0.46 µg/g in chernozem and 1.59 µg/g in cambisol amounting 0.43 and 2.8% of total dsDNA content in these soils, respectively. 100% and 64.8% of added extracellular lambda -phage dsDNA was found in cambisol and chernozem soils, respectively, in 5 h after application. 39% and 73.5% of added DNA disappeared in cambisol and in chernozem, respectively, during 24 h incubation. Degradation rate of extracellular DNA depended on microbial biomass content, which was 2.5 times higher in chernozem as compared to cambisol. Maximum adsorption of DNA by soils was observed in cambisol and reached 2.7% of added amount. We speculate that probability of gene transfer could be rather high in soils, taking into account possible increase of extracellular DNA content after transient environmental events (i.e. drying - rewetting and freezing - thawing).

A CI-Independent Form of Replicative Inhibition: Turn Off of Early Replication of Bacteriophage Lambda

PubMed Central

Hayes, Sidney; Horbay, Monique A.; Hayes, Connie

2012-01-01

Several earlier studies have described an unusual exclusion phenotype exhibited by cells with plasmids carrying a portion of the replication region of phage lambda. Cells exhibiting this inhibition phenotype (IP) prevent the plating of homo-immune and hybrid hetero-immune lambdoid phages. We have attempted to define aspects of IP, and show that it is directed to repλ phages. IP was observed in cells with plasmids containing a λ DNA fragment including oop, encoding a short OOP micro RNA, and part of the lambda origin of replication, oriλ, defined by iteron sequences ITN1-4 and an adjacent high AT-rich sequence. Transcription of the intact oop sequence from its promoter, pO is required for IP, as are iterons ITN3–4, but not the high AT-rich portion of oriλ. The results suggest that IP silencing is directed to theta mode replication initiation from an infecting repλ genome, or an induced repλ prophage. Phage mutations suppressing IP, i.e., Sip, map within, or adjacent to cro or in O, or both. Our results for plasmid based IP suggest the hypothesis that there is a natural mechanism for silencing early theta-mode replication initiation, i.e. the buildup of λ genomes with oop + oriλ+ sequence. PMID:22590552

A new and fast method for preparing high quality lambda DNA suitable for sequencing.

PubMed Central

Manfioletti, G; Schneider, C

1988-01-01

A method is described for the rapid purification of high quality lambda DNA. The method can be used from either liquid or plate lysates and on a small scale or a large scale. It relies on the preadsobtion of all polyanions present in the lysate to an "insoluble" anion-exchange matrix (DEAE or TEAE). Phage particles are then disrupted by combined treatment with EDTA/proteinase K and the resulting DNA is precipitated by the addition of the cationic detergent cetyl (or hexadecyl)-trimethyl ammonium bromide-CTAB ("soluble" anion-exchange matrix). The precipitated CTAB-DNA complex is then exchanged to Na-DNA and ethanol precipitated. The resultant purified DNA is suitable for enzymatic reactions and provides a high quality template for dideoxy-sequence analysis. Images PMID:2966928

Use of Lambda Phage DNA as a Hybrid Internal Control in a PCR-Enzyme Immunoassay To Detect Chlamydia pneumoniae

PubMed Central

Pham, Dien G.; Madico, Guillermo E.; Quinn, Thomas C.; Enzler, Mark J.; Smith, Thomas F.; Gaydos, Charlotte A.

1998-01-01

An inherent problem in the diagnostic PCR assay is the presence of ill-defined inhibitors of amplification which may cause false-negative results. Addition of an amplifiable fragment of foreign DNA in the PCR to serve as a hybrid internal control (HIC) would allow for a simple way to identify specimens containing inhibitors. Two oligonucleotide hybrid primers were synthesized to contain nucleic acid sequences of the Chlamydia pneumoniae 16S rRNA primers in a position flanking two primers that target the sequences of a 650-bp lambda phage DNA segment. By using the hybrid primers, hybrid DNA comprising a large sequence of lambda phage DNA flanked by short pieces of chlamydia DNA was subsequently generated by PCR, cloned into a plasmid vector, and purified. Plasmids containing the hybrid DNA were diluted and used as a HIC by adding them to each C. pneumoniae PCR test. Consequently, C. pneumoniae primers were able to amplify both chlamydia DNA and the HIC DNA. The production of a 689-bp HIC DNA band on an acrylamide gel indicated that the specimen contained no inhibitors and that internal conditions were compatible with PCR. Subsequently, a biotinylated RNA probe for the HIC was transcribed from a nested sequence of the HIC and was used for its hybridization. Detection of the HIC DNA-RNA hybrid was achieved by enzyme immunoassay (EIA). This PCR-EIA system with a HIC was initially tested with 12 previously PCR-positive and 14 previously PCR-negative specimens. Of the 12 PCR-positive specimens, 11 were reconfirmed as positive; 1 had a negative HIC value, indicating inhibition. Of the 14 previously PCR-negative specimens, 13 were confirmed as true negative; 1 had a negative HIC value, indicating inhibition. The assay was then used with 237 nasopharyngeal specimens from patients with pneumonia. Twenty-one of 237 (8.9%) were positive for C. pneumoniae, and 42 (17.7%) were found to inhibit the PCR. Specimens showing inhibitory activity were diluted 1:10 and were retested. Ten specimens were still inhibitory to the PCR and required further DNA purification. No additional positive samples were detected and 3 nasopharyngeal specimens remained inhibitory to PCR. Coamplification of a HIC DNA can help confirm true-negative PCR results by ruling out the presence of inhibitors of DNA amplification. PMID:9650936

The Protein Interaction Network of Bacteriophage Lambda with Its Host, Escherichia coli

PubMed Central

Blasche, Sonja; Wuchty, Stefan; Rajagopala, Seesandra V.

2013-01-01

Although most of the 73 open reading frames (ORFs) in bacteriophage λ have been investigated intensively, the function of many genes in host-phage interactions remains poorly understood. Using yeast two-hybrid screens of all lambda ORFs for interactions with its host Escherichia coli, we determined a raw data set of 631 host-phage interactions resulting in a set of 62 high-confidence interactions after multiple rounds of retesting. These links suggest novel regulatory interactions between the E. coli transcriptional network and lambda proteins. Targeted host proteins and genes required for lambda infection are enriched among highly connected proteins, suggesting that bacteriophages resemble interaction patterns of human viruses. Lambda tail proteins interact with both bacterial fimbrial proteins and E. coli proteins homologous to other phage proteins. Lambda appears to dramatically differ from other phages, such as T7, because of its unusually large number of modified and processed proteins, which reduces the number of host-virus interactions detectable by yeast two-hybrid screens. PMID:24049175

The genome and structural proteome of YuA, a new Pseudomonas aeruginosa phage resembling M6.

PubMed

Ceyssens, Pieter-Jan; Mesyanzhinov, Vadim; Sykilinda, Nina; Briers, Yves; Roucourt, Bart; Lavigne, Rob; Robben, Johan; Domashin, Artem; Miroshnikov, Konstantin; Volckaert, Guido; Hertveldt, Kirsten

2008-02-01

Pseudomonas aeruginosa phage YuA (Siphoviridae) was isolated from a pond near Moscow, Russia. It has an elongated head, encapsulating a circularly permuted genome of 58,663 bp, and a flexible, noncontractile tail, which is terminally and subterminally decorated with short fibers. The YuA genome is neither Mu- nor lambda-like and encodes 78 gene products that cluster in three major regions involved in (i) DNA metabolism and replication, (ii) host interaction, and (iii) phage particle formation and host lysis. At the protein level, YuA displays significant homology with phages M6, phiJL001, 73, B3, DMS3, and D3112. Eighteen YuA proteins were identified as part of the phage particle by mass spectrometry analysis. Five different bacterial promoters were experimentally identified using a promoter trap assay, three of which have a sigma54-specific binding site and regulate transcription in the genome region involved in phage particle formation and host lysis. The dependency of these promoters on the host sigma54 factor was confirmed by analysis of an rpoN mutant strain of P. aeruginosa PAO1. At the DNA level, YuA is 91% identical to the recently (July 2007) annotated phage M6 of the Lindberg typing set. Despite this level of DNA homology throughout the genome, both phages combined have 15 unique genes that do not occur in the other phage. The genome organization of both phages differs substantially from those of the other known Pseudomonas-infecting Siphoviridae, delineating them as a distinct genus within this family.

Genome of Enterobacteriophage Lula/phi80 and Insights into Its Ability To Spread in the Laboratory Environment

PubMed Central

Rotman, Ella; Kouzminova, Elena; Plunkett, Guy

2012-01-01

The novel temperate bacteriophage Lula, contaminating laboratory Escherichia coli strains, turned out to be the well-known lambdoid phage phi80. Our previous studies revealed that two characteristics of Lula/phi80 facilitate its spread in the laboratory environment: cryptic lysogen productivity and stealthy infectivity. To understand the genetics/genomics behind these traits, we sequenced and annotated the Lula/phi80 genome, encountering an E. coli-toxic gene revealed as a gap in the sequencing contig and analyzing a few genes in more detail. Lula/phi80's genome layout copies that of lambda, yet homology with other lambdoid phages is mostly limited to the capsid genes. Lula/phi80's DNA is resistant to cutting with several restriction enzymes, suggesting DNA modification, but deletion of the phage's damL gene, coding for DNA adenine methylase, did not make DNA cuttable. The damL mutation of Lula/phi80 also did not change the phage titer in lysogen cultures, whereas the host dam mutation did increase it almost 100-fold. Since the high phage titer in cultures of Lula/phi80 lysogens is apparently in response to endogenous DNA damage, we deleted the only Lula/phi80 SOS-controlled gene, dinL. We found that dinL mutant lysogens release fewer phage in response to endogenous DNA damage but are unchanged in their response to external DNA damage. The toxic gene of Lula/phi80, gamL, encodes an inhibitor of the host ATP-dependent exonucleases, RecBCD and SbcCD. Its own antidote, agt, apparently encoding a modifier protein, was found nearby. Interestingly, Lula/phi80 lysogens are recD and sbcCD phenocopies, so GamL and Agt are part of lysogenic conversion. PMID:23042999

The Probiotic Escherichia coli Strain Nissle 1917 Combats Lambdoid Bacteriophages stx and λ.

PubMed

Bury, Susanne; Soundararajan, Manonmani; Bharti, Richa; von Bünau, Rudolf; Förstner, Konrad U; Oelschlaeger, Tobias A

2018-01-01

Shiga toxin (Stx) producing E. coli (STEC) such as Enterohemorrhagic E. coli (EHEC) are the major cause of foodborne illness in humans. In vitro studies showed the probiotic Escherichia coli strain Nissle 1917 (EcN) to efficiently inhibit the production of Stx. Life threatening EHEC strains as for example the serotype O104:H4, responsible for the great outbreak in 2011 in Germany, evolutionary developed from certain E. coli strains which got infected by stx2 -encoding lambdoid phages turning the E. coli into lysogenic and subsequently Stx producing strains. Since antibiotics induce stx genes and Stx production, EHEC infected persons are not recommended to be treated with antibiotics. Therefore, EcN might be an alternative medication. However, because even commensal E. coli strains might be converted into Stx-producers after becoming host to a stx encoding prophage, we tested EcN for stx -phage genome integration. Our experiments revealed the resistance of EcN toward not only stx -phages but also against lambda-phages. This resistance was not based on the lack of or by mutated phage receptors. Rather it involved the expression of a phage repressor ( pr ) gene of a defective prophage in EcN which was able to partially protect E. coli K-12 strain MG1655 against stx and lambda phage infection. Furthermore, we observed EcN to inactivate phages and thereby to protect E. coli K-12 strains against infection by stx - as well as lambda-phages. Inactivation of lambda-phages was due to binding of lambda-phages to LamB of EcN whereas inactivation of stx -phages was caused by a thermostable protein of EcN. These properties together with its ability to inhibit Stx production make EcN a good candidate for the prevention of illness caused by EHEC and probably for the treatment of already infected people.

Fluctuation Pressure Assisted Ejection of DNA From Bacteriophage

NASA Astrophysics Data System (ADS)

Harrison, Michael J.

2011-03-01

The role of thermal pressure fluctuations excited within tightly packaged DNA while it is ejected from protein capsid shells is discussed in a model calculation. At equilibrium before ejection we assume the DNA is folded many times into a bundle of parallel segments that forms an equilibrium conformation at minimum free energy, which presses tightly against capsid walls. Using a canonical ensemble at temperature T we calculate internal pressure fluctuations against a slowly moving or static capsid mantle for an elastic continuum model of the folded DNA bundle. It is found that fluctuating pressures on the capsid from thermal excitation of longitudinal acoustic vibrations in the bundle whose wavelengths are exceeded by the bend persistence length may have root-mean-square values that are several tens of atmospheres for typically small phage dimensions. Comparisons are given with measured data on three mutants of lambda phage with different base pair lengths and total genome ejection pressures.

Simulation experiments of the effect of space environment on bacteriophage and DNA thin films

NASA Technical Reports Server (NTRS)

Fekete, A.; Ronto, Gy; Hegedus, M.; Modos, K.; Berces, A.; Kovacs, G.; Lammer, H.; Panitz, C.

2004-01-01

The main goal of PUR experiment (phage and uracil response) is to examine and quantify the effect of specific space conditions on nucleic acid models. To achieve this an improved method was elaborated for the preparation of DNA and bacteriophage thin films. The homogeneity of the films was controlled by UV spectroscopy and microscopy. To provide experimental evidence for the hypothesis that interplanetary transfer of the genetic material is possible, phage T7 and isolated T7 DNA thin films have been exposed to selected space conditions: intense UVC radiation (lambda=254 nm) and high vacuum (10(-4) Pa). The effects of DNA hydration, conformation and packing on UV radiation damage were examined. Characteristic changes in the absorption spectrum, in the electrophoretic pattern of DNA and the decrease of the amount of PCR products have been detected indicating the photodamage of isolated and intraphage DNA. c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.

Oligopeptide M13 Phage Display in Pathogen Research

PubMed Central

Kügler, Jonas; Zantow, Jonas; Meyer, Torsten; Hust, Michael

2013-01-01

Phage display has become an established, widely used method for selection of peptides, antibodies or alternative scaffolds. The use of phage display for the selection of antigens from genomic or cDNA libraries of pathogens which is an alternative to the classical way of identifying immunogenic proteins is not well-known. In recent years several new applications for oligopeptide phage display in disease related fields have been developed which has led to the identification of various new antigens. These novel identified immunogenic proteins provide new insights into host pathogen interactions and can be used for the development of new diagnostic tests and vaccines. In this review we focus on the M13 oligopeptide phage display system for pathogen research but will also give examples for lambda phage display and for applications in other disease related fields. In addition, a detailed technical work flow for the identification of immunogenic oligopeptides using the pHORF system is given. The described identification of immunogenic proteins of pathogens using oligopeptide phage display can be linked to antibody phage display resulting in a vaccine pipeline. PMID:24136040

Oligopeptide m13 phage display in pathogen research.

PubMed

Kügler, Jonas; Zantow, Jonas; Meyer, Torsten; Hust, Michael

2013-10-16

Phage display has become an established, widely used method for selection of peptides, antibodies or alternative scaffolds. The use of phage display for the selection of antigens from genomic or cDNA libraries of pathogens which is an alternative to the classical way of identifying immunogenic proteins is not well-known. In recent years several new applications for oligopeptide phage display in disease related fields have been developed which has led to the identification of various new antigens. These novel identified immunogenic proteins provide new insights into host pathogen interactions and can be used for the development of new diagnostic tests and vaccines. In this review we focus on the M13 oligopeptide phage display system for pathogen research but will also give examples for lambda phage display and for applications in other disease related fields. In addition, a detailed technical work flow for the identification of immunogenic oligopeptides using the pHORF system is given. The described identification of immunogenic proteins of pathogens using oligopeptide phage display can be linked to antibody phage display resulting in a vaccine pipeline.

In vivo gene delivery and expression by bacteriophage lambda vectors.

PubMed

Lankes, H A; Zanghi, C N; Santos, K; Capella, C; Duke, C M P; Dewhurst, S

2007-05-01

Bacteriophage vectors have potential as gene transfer and vaccine delivery vectors because of their low cost, safety and physical stability. However, little is known concerning phage-mediated gene transfer in mammalian hosts. We therefore performed experiments to examine phage-mediated gene transfer in vivo. Mice were inoculated with recombinant lambda phage containing a mammalian expression cassette encoding firefly luciferase (luc). Efficient, dose-dependent in vivo luc expression was detected, which peaked within 24 h of delivery and declined to undetectable levels within a week. Display of an integrin-binding peptide increased cellular internalization of phage in vitro and enhanced phage-mediated gene transfer in vivo. Finally, in vivo depletion of phagocytic cells using clodronate liposomes had only a minor effect on the efficiency of phage-mediated gene transfer. Unmodified lambda phage particles are capable of transducing mammalian cells in vivo, and may be taken up -- at least in part -- by nonphagocytic mechanisms. Surface modifications that enhance phage uptake result in more efficient in vivo gene transfer. These experiments shed light on the mechanisms involved in phage-mediated gene transfer in vivo, and suggest new approaches that may enhance the efficiency of this process.

Ordered shotgun sequencing of a 135 kb Xq25 YAC containing ANT2 and four possible genes, including three confirmed by EST matches.

PubMed Central

Chen, C N; Su, Y; Baybayan, P; Siruno, A; Nagaraja, R; Mazzarella, R; Schlessinger, D; Chen, E

1996-01-01

Ordered shotgun sequencing (OSS) has been successfully carried out with an Xq25 YAC substrate. yWXD703 DNA was subcloned into lambda phage and sequences of insert ends of the lambda subclones were used to generate a map to select a minimum tiling path of clones to be completely sequenced. The sequence of 135 038 nt contains the entire ANT2 cDNA as well as four other candidates suggested by computer-assisted analyses. One of the putative genes is homologous to a gene implicated in Graves' disease and it, ANT2 and two others are confirmed by EST matches. The results suggest that OSS can be applied to YACs in accord with earlier simulations and further indicate that the sequence of the YAC accurately reflects the sequence of uncloned human DNA. PMID:8918809

Assessment of the microscreen phage-induction assay for screening hazardous wastes (1989)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Houk, V.S.; DeMarini, D.M.

1989-01-01

The Microscreen phage-induction assay, which quantitatively measures the induction of prophage Lambda in Escherichia coli WP2s(Lambda), was used to test 14 crude (unfractionated) hazardous industrial-waste samples for genotoxic activity in the presence and absence of metabolic activation. Eleven of the 14 wastes induced prophage, and induction was observed at concentrations as low as 0.4 picograms per ml. Comparisons of the mutagenic activity of these waste samples in Salmonella and their ability to induce prophage Lambda indicate that the phage-induction assay was a more-sensitive indicator of genetic damage for this group of wastes. All but one of the wastes that weremore » mutagenic to Salmonella were detected by the phage-induction assay, and 5 wastes not mutagenic to Salmonella were genetically active in the phage assay. The enhanced ability of the phage-induction assay to detect genotoxic activity may be related to the constituents comprising these waste samples. Partial chemical characterizations of the wastes showed high concentrations of carcinogenic metals, solvents, and chlorinated compounds, most of which are detected poorly by the Salmonella assay.« less

Cyborg lectins: novel leguminous lectins with unique specificities.

PubMed

Yamamoto, K; Maruyama, I N; Osawa, T

2000-01-01

Bauhinia purpurea lectin (BPA) is one of the beta-galactose-binding leguminous lectins. Leguminous lectins contain a long metal-binding loop, part of which determines their carbohydrate-binding specificities. Random mutations were introduced into a portion of the cDNA coding BPA that corresponds to the carbohydrate-binding loop of the lectin. An library of the mutant lectin expressed on the surface of lambda foo phages was screened by the panning method. Several phage clones with an affinity for mannose or N-acetylglucosamine were isolated. These results indicate the possibility of making artificial lectins (so-called "cyborg lectins") with distinct and desired carbohydrate-binding specificities.

Architecture of the 99 bp DNA-six-protein regulatory complex of the lambda att site.

PubMed

Sun, Xingmin; Mierke, Dale F; Biswas, Tapan; Lee, Sang Yeol; Landy, Arthur; Radman-Livaja, Marta

2006-11-17

The highly directional and tightly regulated recombination reaction used to site-specifically excise the bacteriophage lambda chromosome out of its E. coli host chromosome requires the binding of six sequence-specific proteins to a 99 bp segment of the phage att site. To gain structural insights into this recombination pathway, we measured 27 FRET distances between eight points on the 99 bp regulatory DNA bound with all six proteins. Triangulation of these distances using a metric matrix distance-geometry algorithm provided coordinates for these eight points. The resulting path for the protein-bound regulatory DNA, which fits well with the genetics, biochemistry, and X-ray crystal structures describing the individual proteins and their interactions with DNA, provides a new structural perspective into the molecular mechanism and regulation of the recombination reaction and illustrates a design by which different families of higher-order complexes can be assembled from different numbers and combinations of the same few proteins.

Display of HIV-1 Envelope Protein on Lambda Phage Scaffold as a Vaccine Platform.

PubMed

Mattiacio, Jonelle L; Brewer, Matt; Dewhurst, Stephen

2017-01-01

The generation of a strong antibody response to target antigens is a major goal for vaccine development. Here we describe the display of the human immunodeficiency virus (HIV) envelope spike protein (Env) on a virus-like scaffold provided by the lambda phage capsid. Phage vectors, in general, have advantages over mammalian virus vectors due to their genetic tractability, inexpensive production, suitability for scale-up, as well as their physical stability, making them an attractive vaccine platform.

Purification of bacteriophage lambda repressor

PubMed Central

Gao, Ning; Shearwin, Keith; Mack, John; Finzi, Laura; Dunlap, David

2013-01-01

Bacteriophage lambda repressor controls the lysogeny/lytic growth switch after infection of E. coli by lambda phage. In order to study in detail the looping of DNA mediated by the protein, tag-free repressor and a loss-of-cooperativity mutant were expressed in E.coli and purified by (1) ammonium sulfate fractionation, (2) anion-exchange chromatography and (3) heparin affinity chromatography. This method employs more recently developed and readily available chromatography resins to produce highly pure protein in good yield. In tethered particle motion looping assays and atomic force microscopy “footprinting” assays, both the wild-type protein and a C-terminal His-tagged variant, purified using immobilized metal affinity chromatography, bound specifically to high affinity sites to mediate loop formation. In contrast the G147D loss-of-cooperativity mutant bound specifically but did not secure loops. PMID:23831434

Unusual Structure of the attB Site of the Site-Specific Recombination System of Lactobacillus delbrueckii Bacteriophage mv4

PubMed Central

Auvray, Frédéric; Coddeville, Michèle; Ordonez, Romy Catoira; Ritzenthaler, Paul

1999-01-01

The temperate phage mv4 integrates its genome into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus by site-specific recombination within the 3′ end of a tRNASer gene. Recombination is catalyzed by the phage-encoded integrase and occurs between the phage attP site and the bacterial attB site. In this study, we show that the mv4 integrase functions in vivo in Escherichia coli and we characterize the bacterial attB site with a site-specific recombination test involving compatible plasmids carrying the recombination sites. The importance of particular nucleotides within the attB sequence was determined by site-directed mutagenesis. The structure of the attB site was found to be simple but rather unusual. A 16-bp DNA fragment was sufficient for function. Unlike most genetic elements that integrate their DNA into tRNA genes, none of the dyad symmetry elements of the tRNASer gene were present within the minimal attB site. No inverted repeats were detected within this site either, in contrast to the lambda site-specific recombination model. PMID:10572145

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maaloum, M.; Muller, P.; Beker, A-F.

Almost two decades ago, measurements of force versus extension on isolated double-stranded DNA molecules revealed a force plateau. This unusual stretching phenomenon in DNA suggests that the long molecules may be extended from the usual B form into a new conformation. Different models have been proposed to describe the nature of DNA in its stretched form, S-DNA. Using atomic force microscopy combined with a molecular combing method, we identified the structure of {lambda}-phage DNA for different stretching values. We provide strong evidence for the existence of a first-order transition between B form and S form. Beyond a certain extension ofmore » the natural length, DNA molecules adopt a new double-helix conformation characterized by a diameter of 1.2 nm and a helical pitch of18 nm.« less

A gene cassette for adapting Escherichia coli strains as hosts for att-Int-mediated rearrangement and pL expression vectors.

PubMed

Balakrishnan, R; Bolten, B; Backman, K C

1994-01-28

A cassette of genes from bacteriophage lambda, when carried on a derivative of bacteriophage Mu, renders strains of Escherichia coli (and in principle other Mu-sensitive bacteria) capable of supporting lambda-based expression vectors, such as rearrangement vectors and pL vectors. The gene cassette contains a temperature-sensitive allele of the repressor gene, cIts857, and a shortened leftward operon comprising, oLpL, N, xis and int. Transfection and lysogenization of this cassette into various host bacteria is mediated by phage Mu functions. Examples of regulated expression of the gene encoding T4 DNA ligase are presented.

Binding of transcription termination protein nun to nascent RNA and template DNA.

PubMed

Watnick, R S; Gottesman, M E

1999-12-17

The amino-terminal arginine-rich motif of coliphage HK022 Nun binds phage lambda nascent transcript, whereas the carboxyl-terminal domain interacts with RNA polymerase (RNAP) and blocks transcription elongation. RNA binding is inhibited by zinc (Zn2+) and stimulated by Escherichia coli NusA. To study these interactions, the Nun carboxyl terminus was extended by a cysteine residue conjugated to a photochemical cross-linker. The carboxyl terminus contacted NusA and made Zn2+-dependent intramolecular contacts. When Nun was added to a paused transcription elongation complex, it cross-linked to the DNA template. Nun may arrest transcription by anchoring RNAP to DNA.

Subtraction of cap-trapped full-length cDNA libraries to select rare transcripts.

PubMed

Hirozane-Kishikawa, Tomoko; Shiraki, Toshiyuki; Waki, Kazunori; Nakamura, Mari; Arakawa, Takahiro; Kawai, Jun; Fagiolini, Michela; Hensch, Takao K; Hayashizaki, Yoshihide; Carninci, Piero

2003-09-01

The normalization and subtraction of highly expressed cDNAs from relatively large tissues before cloning dramatically enhanced the gene discovery by sequencing for the mouse full-length cDNA encyclopedia, but these methods have not been suitable for limited RNA materials. To normalize and subtract full-length cDNA libraries derived from limited quantities of total RNA, here we report a method to subtract plasmid libraries excised from size-unbiased amplified lambda phage cDNA libraries that avoids heavily biasing steps such as PCR and plasmid library amplification. The proportion of full-length cDNAs and the gene discovery rate are high, and library diversity can be validated by in silico randomization.

Assessment of the Microscreen phage-induction assay for screening hazardous wastes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Houk, V.S.; DeMarini, D.M.

1987-09-01

The Microscreen phage-induction assay, which quantitatively measures the induction of prophage lambda in Escherichia coli WP2s(lambda), was used to test 14 crude (unfractionated) hazardous industrial waste samples for genotoxic activity in the presence and absence of metabolic activation. Eleven of the 14 wastes induced prophage, and induction was observed at concentrations as low as 0.4 picograms per ml. Comparisons between the mutagenicity of these waste samples in Salmonella and their ability to induce prophage lambda indicate that the Microscreen phage-induction assay detected genotoxic activity in all but one of the wastes that were mutagenic in Salmonella. Moreover, the Microscreen assaymore » detected as genotoxic 5 additional wastes that were not detected in the Salmonella assay. The applicability of the Microscreen phage-induction assay for screening hazardous wastes for genotoxic activity is discussed along with some of the problems associated with screening highly toxic wastes containing toxic volatile compounds.« less

Use of the Microscreen phage-induction assay to assess the genotoxicity of 14 hazardous industrial wastes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Houk, V.S.; DeMarini, D.M.

1988-01-01

The Microscreen phage-induction assay, which quantitatively measures the induction of prophage lambda in Escherichia coli WP2s lambda, was used to test 14 crude (unfractionated) hazardous industrial-waste samples for genotoxic activity in the presence and absence of metabolic activation. Eleven of the 14 wastes induced prophage, and induction was observed at concentrations as low as 0.4 picograms per ml. Comparisons between the mutagenicity of these waste samples in Salmonella and their ability to induce prophage lambda indicate that the Microscreen phage-induction assay detected genotoxic activity in all but one of the wastes that were mutagenic in Salmonella. Moreover, the Microscreen assaymore » detected as genotoxic 5 additional wastes that were not detected in the Salmonella assay. The applicability of the Microscreen phage-induction assay for screening hazardous wastes for genotoxic activity is discussed along with some of the problems associated with screening highly toxic wastes containing toxic volatile compounds.« less

Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing; Progress report, June 1, 1990--May 31, 1993

DOE Office of Scientific and Technical Information (OSTI.GOV)

Richardson, C.C.

1993-12-31

This project focuses on the DNA polymerase (gene 5 protein) of phage T7 for use in DNA sequence analysis. Gene 5 protein interacts with accessory proteins to acquire properties essential for DNA replication. One goal is to understand these interactions in order to modify the proteins for use in DNA sequencing. E. coli thioredoxin, binds to gene 5 protein and clamps it to a primer-template. They have analyzed the binding of gene 5 protein-thioredoxin to primer-templates and have defined the optimal conditions to form an extremely stable complex with a dNTP in the polymerase catalytic site. The spatial proximity ofmore » these components has been determined using fluorescence emission anisotropy. The T7 DNA binding protein, the gene 2.5 protein, interacts with gene 5 protein and gene 4 protein to increase processivity and primer synthesis, respectively. Mutant gene 2.5 proteins have been isolated that do not interact with T7 DNA polymerase and can not support T7 growth. The nucleotide binding site of the T7 helicase has been identified and mutations affecting the site provide information on how the hydrolysis of NTPs fuel its unidirectional translocation. The sequence, GTC, has been shown to be necessary and sufficient for recognition by the T7 primase. The T7 gene 5.5 protein interacts with the E. coli nucleoid protein, H-NS, and also overcomes the phage {lambda} rex restriction system.« less

A Single Molecule Study of Two Bacteriophage Epigenetic Switches

NASA Astrophysics Data System (ADS)

Wang, Haowei

Epigenetic switches allow organisms to evolve into different states by activating/repressing different sets of genes without mutations of the underlying DNA sequence. The study of epigenetic switches is very important to understand the mechanism of human development, the origin of cancer, mental illness and fundamental processes such as gene regulation. The coliphage lambda epigenetic switch, which allows switching from lysogeny to lysis, has been studied for more than 50 years as a paradigm, and has recently received renewed attention. Atomic force microscopy (AFM) was used here to show that the lambda repressor oligomerizes on DNA, primarily as a dodecamer, to secure a DNA loop, which is the basis of the lambda switch. This study also provides support for the idea that specifically bound repressor stabilizes adjacent, non-specifically bound repressor molecules, which confers robustness to the switch. 186 is a member of a different coliphage family. One of the major differences between the two coliphage families is that lambda phages can be induced to switch from the lysogenic to the lytic state by UV radiation, but most coliphages of P2 family, to which 186 belongs, cannot. Interaction between coliphage 186 repressor and DNA is characterized by AFM and tethered particle motion (TPM). To expedite analysis of the AFM data, MatLab codes were written to automate the laborious, manual tracing procedures. The programs automatically recognize DNA segments and protein particles in an image, in order to measure the DNA length and position of bound particles as well as their height, diameter and volume. Application of these algorithms greatly improved the efficiency of AFM analysis. It was showed that 186 CI dimers form heptameric wheels, which induce DNA wrapping and different kinds of DNA looping producing various conformations of nucleoprotein complexes. Information about the dynamics of DNA wrapping and looping on 186 CI particles was also obtained by TPM.

Interaction between the phage HK022 Nun protein and the nut RNA of phage lambda.

PubMed

Chattopadhyay, S; Hung, S C; Stuart, A C; Palmer, A G; Garcia-Mena, J; Das, A; Gottesman, M E

1995-12-19

The nun gene product of prophage HK022 excludes phage lambda infection by blocking the expression of genes downstream from the lambda nut sequence. The Nun protein functions both by competing with lambda N transcription-antitermination protein and by actively inducing transcription termination on the lambda chromosome. We demonstrate that Nun binds directly to a stem-loop structure within nut RNA, boxB, which is also the target for the N antiterminator. The two proteins show comparable affinities for boxB and they compete with each other. Their interactions with boxB are similar, as shown by RNase protection experiments, NMR spectroscopy, and analysis of boxB mutants. Each protein binds the 5' strand of the boxB stem and the adjacent loop. The stem does not melt upon the binding of Nun or N, as the 3' strand remains sensitive to a double-strand-specific RNase. The binding of RNA partially protects Nun from proteolysis and changes its NMR spectra. Evidently, although Nun and N bind to the same surface of boxB RNA, their respective complexes interact differently with RNA polymerase, inducing transcription termination or antitermination, respectively.

Domestication of Lambda Phage Genes into a Putative Third Type of Replicative Helicase Matchmaker

PubMed Central

Brézellec, Pierre; Petit, Marie-Agnès; Pasek, Sophie; Vallet-Gely, Isabelle; Possoz, Christophe

2017-01-01

Abstract At the onset of the initiation of chromosome replication, bacterial replicative helicases are recruited and loaded on the DnaA-oriC nucleoprotein platform, assisted by proteins like DnaC/DnaI or DciA. Two orders of bacteria appear, however, to lack either of these factors, raising the question of the essentiality of these factors in bacteria. Through a phylogenomic approach, we identified a pair of genes that could have substituted for dciA. The two domesticated genes are specific of the dnaC/dnaI- and dciA-lacking organisms and apparently domesticated from lambdoid phage genes. They derive from λO and λP and were renamed dopC and dopE, respectively. DopE is expected to bring the replicative helicase to the bacterial origin of replication, while DopC might assist DopE in this function. The confirmation of the implication of DopCE in the handling of the replicative helicase at the onset of replication in these organisms would generalize to all bacteria and therefore to all living organisms the need for specific factors dedicated to this function. PMID:28854626

Chemical and Molecular Biological Aspects of Alkylhydrazine-Induced Carcinogenesis in Human Cells in Vitro. Revised

DTIC Science & Technology

1984-04-01

weights using the following phage DNA markers : lambda, T2 and T7. The number of alkaline labile sites (breaks due to apurinic sites and phosphotriesters...exhibit cell invasiveness in chick embryo skin, human malignancy specific cell- surface antigenic determinants (19), and produce tumors in pre...they eluted from the column, were identified by co-chromatographed markers , (Chart 2). When randomly proliferating cells were treated with either 1,1-DMH

Analysis of capsid portal protein and terminase functional domains: interaction sites required for DNA packaging in bacteriophage T4.

PubMed

Lin, H; Rao, V B; Black, L W

1999-06-04

Bacteriophage DNA packaging results from an ATP-driven translocation of concatemeric DNA into the prohead by the phage terminase complexed with the portal vertex dodecamer of the prohead. Functional domains of the bacteriophage T4 terminase and portal gene 20 product (gp20) were determined by mutant analysis and sequence localization within the structural genes. Interaction regions of the portal vertex and large terminase subunit (gp17) were determined by genetic (terminase-portal intergenic suppressor mutations), biochemical (column retention of gp17 and inhibition of in vitro DNA packaging by gp20 peptides), and immunological (co-immunoprecipitation of polymerized gp20 peptide and gp17) studies. The specificity of the interaction was tested by means of a phage T4 HOC (highly antigenicoutercapsid protein) display system in which wild-type, cs20, and scrambled portal peptide sequences were displayed on the HOC protein of phage T4. Binding affinities of these recombinant phages as determined by the retention of these phages by a His-tag immobilized gp17 column, and by co-immunoprecipitation with purified terminase supported the specific nature of the portal protein and terminase interaction sites. In further support of specificity, a gp20 peptide corresponding to a portion of the identified site inhibited packaging whereas the scrambled sequence peptide did not block DNA packaging in vitro. The portal interaction site is localized to 28 residues in the central portion of the linear sequence of gp20 (524 residues). As judged by two pairs of intergenic portal-terminase suppressor mutations, two separate regions of the terminase large subunit gp17 (central and COOH-terminal) interact through hydrophobic contacts at the portal site. Although the terminase apparently interacts with this gp20 portal peptide, polyclonal antibody against the portal peptide appears unable to access it in the native structure, suggesting intimate association of gp20 and gp17 possibly internalizes terminase regions within the portal in the packasome complex. Both similarities and differences are seen in comparison to analogous sites which have been identified in phages T3 and lambda. Copyright 1999 Academic Press.

Family values in the age of genomics: comparative analyses of temperate bacteriophage HK022.

PubMed

Weisberg, R A; Gottesmann, M E; Hendrix, R W; Little, J W

1999-01-01

HK022 is a temperate coliphage related to phage lambda. Its chromosome has been completely sequenced, and several aspects of its life cycle have been intensively studied. In the overall arrangement, expression, and function of most of its genes, HK022 broadly resembles lambda and other members of the lambda family. Upon closer view, significant differences emerge. The differences reveal alternative strategies used by related phages to cope with similar problems and illuminate previously unknown regulatory and structural motifs. HK022 prophages protect lysogens from superinfection by producing a sequence-specific RNA binding protein that prematurely terminates nascent transcripts of infecting phage. It uses a novel RNA-based mechanism to antiterminate its own early transcription. The HK022 protein shell is strengthened by a complex pattern of covalent subunit interlinking to form a unitary structure that resembles chain-mail armour. Its integrase and repressor proteins are similar to those of lambda, but the differences provide insights into the evolution of biological specificity and the elements needed for construction of a stable genetic switch.

Divergence and Mosaicism among Virulent Soil Phages of the Burkholderia cepacia Complex‡

PubMed Central

Summer, Elizabeth J.; Gonzalez, Carlos F.; Bomer, Morgan; Carlile, Thomas; Embry, Addie; Kucherka, Amalie M.; Lee, Jonte; Mebane, Leslie; Morrison, William C.; Mark, Louise; King, Maria D.; LiPuma, John J.; Vidaver, Anne K.; Young, Ry

2006-01-01

We have determined the genomic sequences of four virulent myophages, Bcep1, Bcep43, BcepB1A, and Bcep781, whose hosts are soil isolates of the Burkholderia cepacia complex. Despite temporal and spatial separations between initial isolations, three of the phages (Bcep1, Bcep43, and Bcep781, designated the Bcep781 group) exhibit 87% to 99% sequence identity to one another and most coding region differences are due to synonymous nucleotide substitutions, a hallmark of neutral genetic drift. Phage BcepB1A has a very different genome organization but is clearly a mosaic with respect to many of the genes of the Bcep781 group, as is a defective prophage element in Photorhabdus luminescens. Functions were assigned to 27 out of 71 predicted genes of Bcep1 despite extreme sequence divergence. Using a lambda repressor fusion technique, 10 Bcep781-encoded proteins were identified for their ability to support homotypic interactions. While head and tail morphogenesis genes have retained canonical gene order despite extreme sequence divergence, genes involved in DNA metabolism and host lysis are not organized as in other phages. This unusual genome arrangement may contribute to the ability of the Bcep781-like phages to maintain a unified genomic type. However, the Bcep781 group phages can also engage in lateral gene transfer events with otherwise unrelated phages, a process that contributes to the broader-scale genomic mosaicism prevalent among the tailed phages. PMID:16352842

Genome, Integration, and Transduction of a Novel Temperate Phage of Helicobacter pylori

PubMed Central

Luo, Cheng-Hung; Chiou, Pei-Yu; Yang, Chiou-Ying

2012-01-01

Helicobacter pylori is a common human pathogen that has been identified to be carcinogenic. This study isolated the temperate bacteriophage 1961P from the lysate of a clinical strain of H. pylori isolated in Taiwan. The bacteriophage has an icosahedral head and a short tail, typical of the Podoviridae family. Its double-stranded DNA genome is 26,836 bp long and has 33 open reading frames. Only 9 of the predicted proteins have homologs of known functions, while the remaining 24 are only similar to unknown proteins encoded by Helicobacter prophages and remnants. Analysis of sequences proximal to the phage-host junctions suggests that 1961P may integrate into the host chromosome via a mechanism similar to that of bacteriophage lambda. In addition, 1961P is capable of generalized transduction. To the best of our knowledge, this is the first report of the isolation, characterization, genome analysis, integration, and transduction of a Helicobacter pylori phage. PMID:22696647

Analysis of autoimmune bone marrow by antibody-phage display: somatic mutations and third complementarity-determining region arginines in anti-DNA gamma and kappa V genes.

PubMed

Seal, S N; Hoet, R M; Raats, J M; Radic, M Z

2000-09-01

To examine anti-double-stranded DNA (anti-dsDNA) IgG autoantibodies from the bone marrow of individuals with systemic lupus erythematosus (SLE). A library of single-chain variable fragments (scFv) was constructed from SLE bone marrow complementary DNA of gamma, kappa, and lambda isotype by cloning into the pHENIX phagemid vector. The library was screened with dsDNA in solution, and 2 anti-DNA phage, DNA1 and DNA4, were isolated and their Ig V genes sequenced. Soluble scFv corresponding to DNA1 and DNA4, and their heavy (H)- and light (L)-chain recombinants, were prepared, purified, and analyzed for binding to DNA by enzyme-linked immunosorbent assay. DNA1 and DNA4 used different Ig H-chain (3-30 and 5-51, respectively) and L-chain (DPK15 and DPK22, respectively) V genes. The ratios of replacement mutations to silent mutations in DNA1 and DNA4 suggest that their V genes were selected for improved antigen binding in vivo. The recombinant between DNA4VH and DNA1VL showed the highest relative affinity for both single-stranded DNA and dsDNA. These 2 Ig subunits contained third complementarity-determining region arginines and had acquired the majority of replacement mutations. Anti-dsDNA IgG autoantibodies from the bone marrow of SLE patients exploit diverse V genes and cationic V-D-J and V-J junctions for DNA binding, and accumulate replacement mutations that enhance binding.

Phage Lambda P Protein: Trans-Activation, Inhibition Phenotypes and their Suppression

PubMed Central

Hayes, Sidney; Erker, Craig; Horbay, Monique A.; Marciniuk, Kristen; Wang, Wen; Hayes, Connie

2013-01-01

The initiation of bacteriophage λ replication depends upon interactions between the oriλ DNA site, phage proteins O and P, and E. coli host replication proteins. P exhibits a high affinity for DnaB, the major replicative helicase for unwinding double stranded DNA. The concept of P-lethality relates to the hypothesis that P can sequester DnaB and in turn prevent cellular replication initiation from oriC. Alternatively, it was suggested that P-lethality does not involve an interaction between P and DnaB, but is targeted to DnaA. P-lethality is assessed by examining host cells for transformation by ColE1-type plasmids that can express P, and the absence of transformants is attributed to a lethal effect of P expression. The plasmid we employed enabled conditional expression of P, where under permissive conditions, cells were efficiently transformed. We observed that ColE1 replication and plasmid establishment upon transformation is extremely sensitive to P, and distinguish this effect from P-lethality directed to cells. We show that alleles of dnaB protect the variant cells from P expression. P-dependent cellular filamentation arose in ΔrecA or lexA[Ind-] cells, defective for SOS induction. Replication propagation and restart could represent additional targets for P interference of E. coli replication, beyond the oriC-dependent initiation step. PMID:23389467

Organization of genes for transcription and translation in the rif region of the Escherichia coli chromosome. [uv radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamamoto, M.; Nomura, M.

1979-01-01

The lambda rif/sup d/18 transducing phage is known to carry several genes for components of transcriptional and translational machineries; these genes are clustered in the rif region at 88 min on the Escherichia coli genetic map. They include a set of genes for rRNA's (rrnB), a gene for spacer tRNA, tRNA/sub 2//sup Glu/(tgtB), one of the two genes for EF-TU (tufB), genes for four ribosomal proteins (rplK, A, J, and L), genes for the ..beta.. and ..beta..' subunits of RNA polymerase (rpoB and rpoC), and genes for three tRNA's (tyrU, gluT, and thrT). An additional tRNA gene (subsequently identified asmore » thrU by Landy and his co-workers) and a gene for a protein (protein U) with unknown functions were found to be carried by lambda rif/sup d/18. We analyzed the organization of these genes by using various deletion and hybrid phages derived from lambda rif/sup d/18 and lambda rif/sup d/12, a phage related to lambda rif/sup d/18. The expression of various genes was examined in uv-irradiated cells infected with these transducing phages. Two main conclusions were obtained. First, the four tRNA genes are not cotranscribed with the genes in rrnB, even though these tRNA genes are located close to the distal end of rrnB. Second, the four ribosomal protein genes are organized into two separate transcriptional units; rplK and A are in one unit and rplJ and L are in the second unit.« less

Molecular Biology and Biotechnology of Bacteriophage

NASA Astrophysics Data System (ADS)

Onodera, Kazukiyo

The development of the molecular biology of bacteriophage such as T4, lambda and filamentous phages was described and the process that the fundamental knowledge obtained in this field has subsequently led us to the technology of phage display was introduced.

Long-range correlations and charge transport properties of DNA sequences

NASA Astrophysics Data System (ADS)

Liu, Xiao-liang; Ren, Yi; Xie, Qiong-tao; Deng, Chao-sheng; Xu, Hui

2010-04-01

By using Hurst's analysis and transfer approach, the rescaled range functions and Hurst exponents of human chromosome 22 and enterobacteria phage lambda DNA sequences are investigated and the transmission coefficients, Landauer resistances and Lyapunov coefficients of finite segments based on above genomic DNA sequences are calculated. In a comparison with quasiperiodic and random artificial DNA sequences, we find that λ-DNA exhibits anticorrelation behavior characterized by a Hurst exponent 0.5

New restriction enzymes discovered from Escherichia coli clinical strains using a plasmid transformation method

PubMed Central

Kasarjian, Julie K. A.; Iida, Masatake; Ryu, Junichi

2003-01-01

The presence of restriction enzymes in bacterial cells has been predicted by either classical phage restriction-modification (R-M) tests, direct in vitro enzyme assays or more recently from bacterial genome sequence analysis. We have applied phage R-M test principles to the transformation of plasmid DNA and established a plasmid R-M test. To validate this test, six plasmids that contain BamHI fragments of phage lambda DNA were constructed and transformed into Escherichia coli strains containing known R-M systems including: type I (EcoBI, EcoAI, Eco124I), type II (HindIII) and type III (EcoP1I). Plasmid DNA with a single recognition site showed a reduction of relative efficiency of transformation (EOT = 10–1–10–2). When multiple recognition sites were present, greater reductions in EOT values were observed. Once established in the cell, the plasmids were subjected to modification (EOT = 1.0). We applied this test to screen E.coli clinical strains and detected the presence of restriction enzymes in 93% (14/15) of cells. Using additional subclones and the computer program, RM Search, we identified four new restriction enzymes, Eco377I, Eco585I, Eco646I and Eco777I, along with their recognition sequences, GGA(8N)ATGC, GCC(6N)TGCG, CCA(7N)CTTC, and GGA(6N)TATC, respectively. Eco1158I, an isoschizomer of EcoBI, was also found in this study. PMID:12595571

T7 RNA polymerase-driven inducible cell lysis for DNA transfer from Escherichia coli to Bacillus subtilis.

PubMed

Juhas, Mario; Ajioka, James W

2017-11-01

The majority of the good DNA editing techniques have been developed in Escherichia coli; however, Bacillus subtilis is better host for a plethora of synthetic biology and biotechnology applications. Reliable and efficient systems for the transfer of synthetic DNA between E. coli and B. subtilis are therefore of the highest importance. Using synthetic biology approaches, such as streamlined lambda Red recombineering and Gibson Isothermal Assembly, we integrated genetic circuits pT7L123, Repr-ts-1 and pLT7pol encoding the lysis genes of bacteriophages MS2, ΦX174 and lambda, the thermosensitive repressor and the T7 RNA polymerase into the E. coli chromosome. In this system, T7 RNA polymerase regulated by the thermosensitive repressor drives the expression of the phage lysis genes. We showed that T7 RNA polymerase significantly increases efficiency of cell lysis and transfer of the plasmid and bacterial artificial chromosome-encoded DNA from the lysed E. coli into B. subtilis. The T7 RNA polymerase-driven inducible cell lysis system is suitable for the efficient cell lysis and transfer of the DNA engineered in E. coli to other naturally competent hosts, such as B. subtilis. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Initiation of lambda DNA replication. The Escherichia coli small heat shock proteins, DnaJ and GrpE, increase DnaK's affinity for the lambda P protein.

PubMed

Osipiuk, J; Georgopoulos, C; Zylicz, M

1993-03-05

It is known that the initiation of bacteriophage lambda replication requires the orderly assembly of the lambda O.lambda P.DnaB helicase protein preprimosomal complex at the ori lambda DNA site. The DnaK, DnaJ, and GrpE heat shock proteins act together to destabilize the lambda P.DnaB complex, thus freeing DnaB and allowing it to unwind lambda DNA near the ori lambda site. The first step of this disassembly reaction is the binding of DnaK to the lambda P protein. In this report, we examined the influence of the DnaJ and GrpE proteins on the stability of the lambda P.DnaK complex. We present evidence for the existence of the following protein-protein complexes: lambda P.DnaK, lambda P.DnaJ, DnaJ.DnaK, DnaK.GrpE, and lambda P.DnaK.GrpE. Our results suggest that the presence of GrpE alone destabilizes the lambda P.DnaK complex, whereas the presence of DnaJ alone stabilizes the lambda P.DnaK complex. Using immunoprecipitation, we show that in the presence of GrpE, DnaK exhibits a higher affinity for the lambda P.DnaJ complex than it does alone. Using cross-linking with glutaraldehyde, we show that oligomeric forms of DnaK exhibit a higher affinity for lambda P than monomeric DnaK. However, in the presence of GrpE, monomeric DnaK can efficiently bind lambda P protein. These findings help explain our previous results, namely that in the GrpE-dependent lambda DNA replication system, the DnaK protein requirement can be reduced up to 10-fold.

Use of the microscreen phage-induction assay to assess the genotoxicity of 14 hazardous industrial wastes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Houk, V.S.; DeMarini, D.M.

1988-01-01

The Microscreen phage-induction assay, which quantitatively measures the induction of prophage lambda in Escherichia coli WP2s(lambda), was used to test 14 crude (unfractionated) hazardous industrial waste samples for genotoxic activity in the presence and absence of metabolic activation. Eleven of the 14 wastes induced prophage, and induction was observed at concentrations as low as 0.4 pg per ml. Comparisons between the ability of these waste samples to induce prophage and their mutagenicity in the Salmonella reverse mutation assay indicate that the phage-induction assay detected genotoxic activity in all but one of the wastes that were mutagenic in Salmonella. Moreover, themore » Microscreen assay detected as genotoxic five additional wastes that were not detected in the Salmonella assay. The applicability of the Microscreen phage-induction assay for screening hazardous wastes for genotoxic activity is discussed, as are some of the problems associated with screening highly toxic wastes containing toxic volatile compounds.« less

Single-Molecule Denaturation Mapping of DNA in Nanofluidic Channels

NASA Astrophysics Data System (ADS)

Reisner, Walter; Larsen, Niels; Silahtaroglu, Asli; Kristensen, Anders; Tommerup, Niels; Tegenfeldt, Jonas O.; Flyvbjerg, Henrik

2010-03-01

Nanochannel based DNA stretching can serve as a platform for a new optical mapping technique based on measuring the pattern of partial melting along the extended molecules. We partially melt DNA extended in nanofluidic channels via a combination of local heating and added chemical denaturants. The melted molecules, imaged via a standard fluorescence videomicroscopy setup, exhibit a nonuniform fluorescence profile corresponding to a series of local dips and peaks in the intensity trace along the stretched molecule. We show that this barcode is consistent with the presence of locally melted regions along the molecule and can be explained by calculations of sequence-dependent melting probability. Specifically, we obtain experimental melting profiles for T4, T7, lambda-phage and bacterial artificial chromosome DNA (from human chromosome 12) and compare these profiles to theory. In addition, we demonstrate that the BAC melting profile can be used to align the BAC to its correct position on chromosome 12.

Protein chainmail variants in dsDNA viruses

PubMed Central

Zhou, Z. Hong; Chiou, Joshua

2017-01-01

First discovered in bacteriophage HK97, biological chainmail is a highly stable system formed by concatenated protein rings. Each subunit of the ring contains the HK97-like fold, which is characterized by its submarine-like shape with a 5-stranded β sheet in the axial (A) domain, spine helix in the peripheral (P) domain, and an extended (E) loop. HK97 capsid consists of covalently-linked copies of just one HK97-like fold protein and represents the most effective strategy to form highly stable chainmail needed for dsDNA genome encapsidation. Recently, near-atomic resolution structures enabled by cryo electron microscopy (cryoEM) have revealed a range of other, more complex variants of this strategy for constructing dsDNA viruses. The first strategy, exemplified by P22-like phages, is the attachment of an insertional (I) domain to the core 5-stranded β sheet of the HK97-like fold. The atomic models of the Bordetella phage BPP-1 showcases an alternative topology of the classic HK97 topology of the HK97-like fold, as well as the second strategy for constructing stable capsids, where an auxiliary jellyroll protein dimer serves to cement the non-covalent chainmail formed by capsid protein subunits. The third strategy, found in lambda-like phages, uses auxiliary protein trimers to stabilize the underlying non-covalent chainmail near the 3-fold axis. Herpesviruses represent highly complex viruses that use a combination of these strategies, resulting in four-level hierarchical organization including a non-covalent chainmail formed by the HK97-like fold domain found in the floor region. A thorough understanding of these structures should help unlock the enigma of the emergence and evolution of dsDNA viruses and inform bioengineering efforts based on these viruses. PMID:29177192

Mutations of the phage lambda nutL region that prevent the action of Nun, a site-specific transcription termination factor.

PubMed Central

Baron, J; Weisberg, R A

1992-01-01

Phage HK022 encodes a protein, Nun, that promotes transcription termination within the pL and pR operons of its relative, phage lambda. The lambda sequences required for termination had previously been shown to overlap the nut sites, which are essential for transcription antitermination during normal lambda growth. To further specify the Nun target and to determine its relation to the nut sites, we constructed deletion and base substitution mutations of the lambda nutL region and measured Nun-dependent reduction of the expression of a downstream reporter gene. The shortest construct that retained full Nun responsiveness was a 42-bp segment that included both boxA and boxB, sequences that have been implicated in lambda antitermination. Deletion of boxA reduced Nun termination, and deletion of both sequences eliminated Nun termination. Base substitutions in boxA and the proximal portion of boxB impaired Nun termination, while base substitutions between boxA and boxB, in the distal portion of boxB, and immediately downstream from boxB had no appreciable effect. The termination defect of all of the base substitution mutations was relieved by increasing the level of Nun protein; in contrast, the deletions and a multiple-base substitution did not regain full Nun responsiveness at elevated Nun concentrations. We also asked if these mutant nut regions retained their ability to interact with N, the lambda-encoded antitermination protein. A qualitative assay showed that mutations within boxA or boxB reduced interaction, while mutations outside boxA and boxB did not. These data show that (i) the recognition sites for N and Nun overlap to a very considerable extent but are probably not identical and (ii) a high concentration of Nun promotes its interaction with mutant nut sites, a behavior also reported to be characteristic of N. PMID:1532174

Packaging of a unit-length viral genome: the role of nucleotides and the gpD decoration protein in stable nucleocapsid assembly in bacteriophage lambda.

PubMed

Yang, Qin; Maluf, Nasib Karl; Catalano, Carlos Enrique

2008-11-28

The developmental pathways for a variety of eukaryotic and prokaryotic double-stranded DNA viruses include packaging of viral DNA into a preformed procapsid structure, catalyzed by terminase enzymes and fueled by ATP hydrolysis. In most instances, a capsid expansion process accompanies DNA packaging, which significantly increases the volume of the capsid to accommodate the full-length viral genome. "Decoration" proteins add to the surface of the expanded capsid lattice, and the terminase motors tightly package DNA, generating up to approximately 20 atm of internal capsid pressure. Herein we describe biochemical studies on genome packaging using bacteriophage lambda as a model system. Kinetic analysis suggests that the packaging motor possesses at least four ATPase catalytic sites that act cooperatively to effect DNA translocation, and that the motor is highly processive. While not required for DNA translocation into the capsid, the phage lambda capsid decoration protein gpD is essential for the packaging of the penultimate 8-10 kb (15-20%) of the viral genome; virtually no DNA is packaged in the absence of gpD when large DNA substrates are used, most likely due to a loss of capsid structural integrity. Finally, we show that ATP hydrolysis is required to retain the genome in a packaged state subsequent to condensation within the capsid. Presumably, the packaging motor continues to "idle" at the genome end and to maintain a positive pressure towards the packaged state. Surprisingly, ADP, guanosine triphosphate, and the nonhydrolyzable ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) similarly stabilize the packaged viral genome despite the fact that they fail to support genome packaging. In contrast, the poorly hydrolyzed ATP analog ATP-gammaS only partially stabilizes the nucleocapsid, and a DNA is released in "quantized" steps. We interpret the ensemble of data to indicate that (i) the viral procapsid possesses a degree of plasticity that is required to accommodate the packaging of large DNA substrates; (ii) the gpD decoration protein is required to stabilize the fully expanded capsid; and (iii) nucleotides regulate high-affinity DNA binding interactions that are required to maintain DNA in the packaged state.

Combining Comprehensive Analysis of Off-Site Lambda Phage Integration with a CRISPR-Based Means of Characterizing Downstream Physiology.

PubMed

Tanouchi, Yu; Covert, Markus W

2017-09-19

During its lysogenic life cycle, the phage genome is integrated into the host chromosome by site-specific recombination. In this report, we analyze lambda phage integration into noncanonical sites using next-generation sequencing and show that it generates significant genetic diversity by targeting over 300 unique sites in the host Escherichia coli genome. Moreover, these integration events can have important phenotypic consequences for the host, including changes in cell motility and increased antibiotic resistance. Importantly, the new technologies that we developed to enable this study-sequencing secondary sites using next-generation sequencing and then selecting relevant lysogens using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-based selection-are broadly applicable to other phage-bacterium systems. IMPORTANCE Bacteriophages play an important role in bacterial evolution through lysogeny, where the phage genome is integrated into the host chromosome. While phage integration generally occurs at a specific site in the host chromosome, it is also known to occur at other, so-called secondary sites. In this study, we developed a new experimental technology to comprehensively study secondary integration sites and discovered that phage can integrate into over 300 unique sites in the host genome, resulting in significant genetic diversity in bacteria. We further developed an assay to examine the phenotypic consequence of such diverse integration events and found that phage integration can cause changes in evolutionarily relevant traits such as bacterial motility and increases in antibiotic resistance. Importantly, our method is readily applicable to other phage-bacterium systems. Copyright © 2017 Tanouchi and Covert.

The recognition and modification sites for the bacterial type I restriction systems KpnAI, StySEAI, StySENI and StySGI

PubMed Central

Kasarjian, Julie K. A.; Hidaka, Masumi; Horiuchi, Takashi; Iida, Masatake; Ryu, Junichi

2004-01-01

Using an in vivo plasmid transformation method, we have determined the DNA sequences recognized by the KpnAI, StySEAI, StySENI and StySGI R-M systems from Klebsiella oxytoca strain M5a1, Salmonella eastbourne, Salmonella enteritidis and Salmonella gelsenkirchen, respectively. These type I restriction-modification systems were originally identified using traditional phage assay, and described here is the plasmid transformation test and computer program used to determine their DNA recognition sequences. For this test, we constructed two sets of plasmids, pL and pE, that contain phage lambda and Escherichia coli K-12 chromosomal DNA fragments, respectively. Further, using the methylation sensitivities of various known type II restriction enzymes, we identified the target adenines for methylation (listed in bold italics below as A or T in case of the complementary strand). The recognition sequence and methylation sites are GAA(6N)TGCC (KpnAI), ACA(6N)TYCA (StySEAI), CGA(6N)TACC (StySENI) and TAAC(7N)RTCG (StySGI). These DNA recognition sequences all have a typical type I bipartite pattern and represent three novel specificities and one isoschizomer (StySENI). For confirmation, oligonucleotides containing each of the predicted sequences were synthesized, cloned into plasmid pMECA and transformed into each strain, resulting in a large reduction in efficiency of transformation (EOT). PMID:15199175

Identification and analysis of recombineering functions from Gram-negative and Gram-positive bacteria and their phages

PubMed Central

Datta, Simanti; Costantino, Nina; Zhou, Xiaomei; Court, Donald L.

2008-01-01

We report the identification and functional analysis of nine genes from Gram-positive and Gram-negative bacteria and their phages that are similar to lambda (λ) bet or Escherichia coli recT. Beta and RecT are single-strand DNA annealing proteins, referred to here as recombinases. Each of the nine other genes when expressed in E. coli carries out oligonucleotide-mediated recombination. To our knowledge, this is the first study showing single-strand recombinase activity from diverse bacteria. Similar to bet and recT, most of these other recombinases were found to be associated with putative exonuclease genes. Beta and RecT in conjunction with their cognate exonucleases carry out recombination of linear double-strand DNA. Among four of these foreign recombinase/exonuclease pairs tested for recombination with double-strand DNA, three had activity, albeit barely detectable. Thus, although these recombinases can function in E. coli to catalyze oligonucleotide recombination, the double-strand DNA recombination activities with their exonuclease partners were inefficient. This study also demonstrated that Gam, by inhibiting host RecBCD nuclease activity, helps to improve the efficiency of λ Red-mediated recombination with linear double-strand DNA, but Gam is not absolutely essential. Thus, in other bacterial species where Gam analogs have not been identified, double-strand DNA recombination may still work in the absence of a Gam-like function. We anticipate that at least some of the recombineering systems studied here will potentiate oligonucleotide and double-strand DNA-mediated recombineering in their native or related bacteria. PMID:18230724

Role of the Escherichia coli grpE heat shock protein in the initiation of bacteriophage lambda DNA replication.

PubMed

Osipiuk, J; Zylicz, M

1991-01-01

Initiation of replication of lambda DNA requires assembly of the proper nucleoprotein complex consisting of the lambda origin of replication-lambda O-lambda P-dnaB proteins. The dnaJ, dnaK and grpE heat shock proteins destabilize the lambda P-dnaB interaction in this complex permitting dnaB helicase to unwind lambda DNA near ori lambda sequence. First step of this disassembling reaction is the binding of dnaK protein to lambda P protein. In this report we examined the influence of dnaJ and grpE proteins on stability of the lambda P-dnaK complex. Our results show that grpE alone dissociates this complex, but both grpE and dnaJ together do not. These results suggest that, in the presence of grpE protein, dnaK protein has a higher affinity for lambda P protein complexed with dnaJ protein than in the situation where grpE protein is not used.

Plasmid DNA production combining antibiotic-free selection, inducible high yield fermentation, and novel autolytic purification.

PubMed

Carnes, Aaron E; Hodgson, Clague P; Luke, Jeremy M; Vincent, Justin M; Williams, James A

2009-10-15

DNA vaccines and gene medicines, derived from bacterial plasmids, are emerging as an important new class of pharmaceuticals. However, the challenges of performing cell lysis processes for plasmid DNA purification at an industrial scale are well known. To address downstream purification challenges, we have developed autolytic Escherichia coli host strains that express endolysin (phage lambdaR) in the cytoplasm. Expression of the endolysin is induced during fermentation by a heat inducible promoter. The endolysin remains in the cytoplasm, where it is separated from its peptidoglycan substrate in the cell wall; hence the cells remain alive and intact and can be harvested by the usual methods. The plasmid DNA is then recovered by autolytic extraction under slightly acidic, low salt buffer conditions and treatment with a low concentration of non-ionic detergent. Under these conditions the E. coli genomic DNA remains associated with the insoluble cell debris and is removed by a solid-liquid separation. Here, we report fermentation, lysis methods, and plasmid purification using autolytic hosts.

The effects of temperature and magnetic flux on electron transport through a four-channel DNA model

NASA Astrophysics Data System (ADS)

Lee, Sunhee; Hedin, Eric; Joe, Yong

2010-03-01

The temperature dependence of the conductivity of lambda phage DNA has been measured by Tran et al [1] experimentally, where the conductivity displayed strong (weak) temperature dependence above (below) a threshold temperature. In order to understand the temperature effects of electron transport theoretically, we study a two-dimensional and four-channel DNA model using a tight-binding (TB) Hamiltonian. The thermal effects within a TB model are incorporated into the hopping integral and the relative twist angle from its equilibrium value between base-pairs. Since these thermal structural fluctuations localize the electronic wave functions in DNA, we examine a temperature-dependent localization length, a temperature-driven transmission, and current-voltage characteristics in this system. In addition, we incorporate magnetic field effects into the analysis of the transmission through DNA in order to modulate the quantum interference between the electron paths that comprise the 4-channel structure. [1] P. Tran, B. Alavi, and G. Gruner, PRL 85, 1564 (2000).

[Construction and characterization of a cDNA library from human liver tissue of cirrhosis].

PubMed

Chen, Xiao-hong; Chen, Zhi; Chen, Feng; Zhu, Hai-hong; Zhou, Hong-juan; Yao, Hang-ping

2005-03-01

To construct a cDNA library from human liver tissue of cirrhosis. The total RNA from human liver tissue of cirrhosis was extracted using Trizol method, and the mRNA was purified using mRNA purification kit. SMART technique and CDSIII/3' primer were used for first-strand cDNA synthesis. Long distance PCR was then used to synthesize the double-strand cDNA that was then digested by proteinase K and Sfi I, and was fractionated by CHOMA SPIN-400 column. The cDNA fragments longer than 0.4 kb were collected and ligated to lambdaTripl Ex2 vector. Then lambda-phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries was strictly checked by conventional titer determination. Eleven plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. The titers of unamplifed and amplified libraries were 1.03 x 10(6) pfu/ml and 1.36 x 10(9) pfu/ml respectively. The percentages of recombinants from both libraries were 97.24 % in unamplified library and 99.02 % in amplified library. The lengths of the inserts were 1.02 kb in average (36.36 % 1 approximately equals 2 kb and 63.64 % 0.5 approximately equals 1.0 kb). A high quality cDNA library from human liver tissue of cirrhosis was constructed successfully, which can be used for screening and cloning new special genes associated with the occurrence of cirrhosis.

ArgO145, a Stx2a prophage of a bovine O145:H- STEC strain, is closely related to phages of virulent human strains.

PubMed

Krüger, A; Burgán, J; Friedrich, A W; Rossen, J W A; Lucchesi, P M A

2018-06-01

Shiga toxins (Stx) are the main virulence factor of a pathogroup of Escherichia coli strains that cause severe human diseases. These toxins are encoded in prophages (Stx prophages), and generally their expression depends on prophage induction. Several studies have reported high diversity among both Stx prophages and Stx. In particular, the toxin subtype Stx2a is associated with high virulence and HUS. Here, we report the genome of ArgO145, an inducible Stx2a prophage identified in a bovine O145:H- strain which produced high levels of Shiga toxin and Stx phage particles. The ArgO145 genome shared lambda phage organization, with recombination, regulation, replication, lysis, and head and tail structural gene regions, although some lambda genes encoding regulatory proteins could not be identified. Remarkably, some Stx2a phages of strains isolated from patients in other countries showed high similarity to ArgO145. Copyright © 2018 Elsevier B.V. All rights reserved.

Understanding the enormous diversity of bacteriophages: the tailed phages that infect the bacterial family Enterobacteriaceae.

PubMed

Grose, Julianne H; Casjens, Sherwood R

2014-11-01

Bacteriophages are the predominant biological entity on the planet. The recent explosion of sequence information has made estimates of their diversity possible. We describe the genomic comparison of 337 fully sequenced tailed phages isolated on 18 genera and 31 species of bacteria in the Enterobacteriaceae. These phages were largely unambiguously grouped into 56 diverse clusters (32 lytic and 24 temperate) that have syntenic similarity over >50% of the genomes within each cluster, but substantially less sequence similarity between clusters. Most clusters naturally break into sets of more closely related subclusters, 78% of which are correlated with their host genera. The largest groups of related phages are superclusters united by genome synteny to lambda (81 phages) and T7 (51 phages). This study forms a robust framework for understanding diversity and evolutionary relationships of existing tailed phages, for relating newly discovered phages and for determining host/phage relationships.

Understanding the enormous diversity of bacteriophages: the tailed phages that infect the bacterial family Enterobacteriaceae

PubMed Central

Grose, Julianne H.; Casjens, Sherwood R.

2014-01-01

Bacteriophages are the predominant biological entity on the planet. The recent explosion of sequence information has made estimates of their diversity possible. We describe the genomic comparison of 337 fully sequenced tailed phages isolated on 18 genera and 31 species of bacteria in the Enterobacteriaceae. These phages were largely unambiguously grouped into 56 diverse clusters (32 lytic and 24 temperate) that have syntenic similarity over >50% of the genomes within each cluster, but substantially less sequence similarity between clusters. Most clusters naturally break into sets of more closely related subclusters, 78% of which are correlated with their host genera. The largest groups of related phages are superclusters united by genome synteny to lambda (81 phages) and T7 (51 phages). This study forms a robust framework for understanding diversity and evolutionary relationships of existing tailed phages, for relating newly discovered phages and for determining host/phage relationships. PMID:25240328

UVB-induced mutagenesis in hairless {lambda}lacZ-transgenic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frijhoff, A.F.W.; Rebel, H.; Mientjes, E.J.

UVB-induced mutagenesis was studied in hairless 40.6 transgenic mice (Muta{trademark}Mouse), which contain the {lambda}gt1OlacZ shuttle vector as a target for mutagenesis. Mice were exposed at the dorsal side to either single doses of 200, 500, 800, or 1000 J/m{sup 2} UVB or to two successive irradiations of either 200 and 800 J/m{sup 2} UVB, with intervals of 1,3, or 5 days, or to 800 and 200 J/m{sup 2} UVB with a 5-day interval. At 23 days after the last exposure, lacZ mutant frequencies (MF) were determined in the epidermis. The lacZ MF increased linearly with increasing dose of UVB. Themore » mutagenic effect of two successive irradiations appeared to be additive. The UV-induced mutation spectrum was dominated by G:C{r_arrow}A:T transitions at dipyrimidine sites. DNA-sequence analysis of spontaneously mutated phages showed a diverse spectrum consisting of insertions, deletions and G:C {r_arrow} A:T transitions at CpG sites. the results indicate that the hairless {lambda}lacZ-transgenic mouse is a suitable in vivo model for studying UVB-induced mutations. 29 refs., 5 tabs.« less

Genetic characterization of ØVC8 lytic phage for Vibrio cholerae O1.

PubMed

Solís-Sánchez, Alejandro; Hernández-Chiñas, Ulises; Navarro-Ocaña, Armando; De la Mora, Javier; Xicohtencatl-Cortes, Juan; Eslava-Campos, Carlos

2016-03-22

Epidemics and pandemics of cholera, a diarrheal disease, are attributed to Vibrio cholera serogroups O1 and O139. In recent years, specific lytic phages of V. cholera have been proposed to be important factors in the cyclic occurrence of cholera in endemic areas. However, the role and potential participation of lytic phages during long interepidemic periods of cholera in non-endemic regions have not yet been described. The purpose of this study was to isolate and characterize specific lytic phages of V. cholera O1 strains. Sixteen phages were isolated from wastewater samples collected at the Endhó Dam in Hidalgo State, Mexico, concentrated with PEG/NaCl, and purified by density gradient. The lytic activity of the purified phages was tested using different V. cholerae O1 and O139 strains. Phage morphology was visualized by transmission electron microscopy (TEM), and phage genome sequencing was performed using the Genome Analyzer IIx System. Genome assembly and bioinformatics analysis were performed using a set of high-throughput programs. Phage structural proteins were analyzed by mass spectrometry. Sixteen phages with lytic and lysogenic activity were isolated; only phage ØVC8 showed specific lytic activity against V. cholerae O1 strains. TEM images of ØVC8 revealed a phage with a short tail and an isometric head. The ØVC8 genome comprises linear double-stranded DNA of 39,422 bp with 50.8 % G + C. Of the 48 annotated ORFs, 16 exhibit homology with sequences of known function and several conserved domains. Bioinformatics analysis showed multiple conserved domains, including an Ig domain, suggesting that ØVC8 might adhere to different mucus substrates such as the human intestinal epithelium. The results suggest that ØVC8 genome utilize the "single-stranded cohesive ends" packaging strategy of the lambda-like group. The two structural proteins sequenced and analyzed are proteins of known function. ØVC8 is a lytic phage with specific activity against V. cholerae O1 strains and is grouped as a member of the VP2-like phage subfamily. The encoding of an Ig domain by ØVC8 makes this phage a good candidate for use in phage therapy and an alternative tool for monitoring V. cholerae populations.

Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries.

PubMed

Terrón-González, L; Medina, C; Limón-Mortés, M C; Santero, E

2013-01-01

The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic gene expression, while the other approach uses the lambda phage transcription anti-termination protein N to limit transcription termination. A metagenomic library was constructed and functionally screened to identify genes conferring carbenicillin resistance to E. coli. The use of these enhanced expression systems resulted in a 6-fold increase in the frequency of carbenicillin resistant clones. Subcloning and sequence analysis showed that, besides β-lactamases, efflux pumps are not only able contribute to carbenicillin resistance but may in fact be sufficient by themselves to convey carbenicillin resistance.

Diversity and distribution of single-stranded DNA phages in the North Atlantic Ocean

PubMed Central

Tucker, Kimberly P; Parsons, Rachel; Symonds, Erin M; Breitbart, Mya

2011-01-01

Knowledge of marine phages is highly biased toward double-stranded DNA (dsDNA) phages; however, recent metagenomic surveys have also identified single-stranded DNA (ssDNA) phages in the oceans. Here, we describe two complete ssDNA phage genomes that were reconstructed from a viral metagenome from 80 m depth at the Bermuda Atlantic Time-series Study (BATS) site in the northwestern Sargasso Sea and examine their spatial and temporal distributions. Both genomes (SARssφ1 and SARssφ2) exhibited similarity to known phages of the Microviridae family in terms of size, GC content, genome organization and protein sequence. PCR amplification of the replication initiation protein (Rep) gene revealed narrow and distinct depth distributions for the newly described ssDNA phages within the upper 200 m of the water column at the BATS site. Comparison of Rep gene sequences obtained from the BATS site over time revealed changes in the diversity of ssDNA phages over monthly time scales, although some nearly identical sequences were recovered from samples collected 4 years apart. Examination of ssDNA phage diversity along transects through the North Atlantic Ocean revealed a positive correlation between genetic distance and geographic distance between sampling sites. Together, the data suggest fundamental differences between the distribution of these ssDNA phages and the distribution of known marine dsDNA phages, possibly because of differences in host range, host distribution, virion stability, or viral evolution mechanisms and rates. Future work needs to elucidate the host ranges for oceanic ssDNA phages and determine their ecological roles in the marine ecosystem. PMID:21124487

Discovery of a protein phosphatase activity encoded in the genome of bacteriophage lambda. Probable identity with open reading frame 221.

PubMed

Cohen, P T; Cohen, P

1989-06-15

Infection of Escherichia coli with phage lambda gt10 resulted in the appearance of a protein phosphatase with activity towards 32P-labelled casein. Activity reached a maximum near the point of cell lysis and declined thereafter. The phosphatase was stimulated 30-fold by Mn2+, while Mg2+ and Ca2+ were much less effective. Activity was unaffected by inhibitors 1 and 2, okadaic acid, calmodulin and trifluoperazine, distinguishing it from the major serine/threonine-specific protein phosphatases of eukaryotic cells. The lambda phosphatase was also capable of dephosphorylating other substrates in the presence of Mn2+, although activity towards 32P-labelled phosphorylase was 10-fold lower, and activity towards phosphorylase kinase and glycogen synthase 25 50-fold lower than with casein. No casein phosphatase activity was present in either uninfected cells, or in E. coli infected with phage lambda gt11. Since lambda gt11 lacks part of the open reading frame (orf) 221, previously shown to encode a protein with sequence similarity to protein phosphatase-1 and protein phosphatase-2A of mammalian cells [Cohen, Collins, Coulson, Berndt & da Cruz e Silva (1988) Gene 69, 131-134], the results indicate that ORF221 is the protein phosphatase detected in cells infected with lambda gt10. Comparison of the sequence of ORF221 with other mammalian protein phosphatases defines three highly conserved regions which are likely to be essential for function. The first of these is deleted in lambda gt11.

Bypassing bacterial infection in phage display by sequencing DNA released from phage particles.

PubMed

Villequey, Camille; Kong, Xu-Dong; Heinis, Christian

2017-11-01

Phage display relies on a bacterial infection step in which the phage particles are replicated to perform multiple affinity selection rounds and to enable the identification of isolated clones by DNA sequencing. While this process is efficient for wild-type phage, the bacterial infection rate of phage with mutant or chemically modified coat proteins can be low. For example, a phage mutant with a disulfide-free p3 coat protein, used for the selection of bicyclic peptides, has a more than 100-fold reduced infection rate compared to the wild-type. A potential strategy for bypassing the bacterial infection step is to directly sequence DNA extracted from phage particles after a single round of phage panning using high-throughput sequencing. In this work, we have quantified the fraction of phage clones that can be identified by directly sequencing DNA from phage particles. The results show that the DNA of essentially all of the phage particles can be 'decoded', and that the sequence coverage for mutants equals that of amplified DNA extracted from cells infected with wild-type phage. This procedure is particularly attractive for selections with phage that have a compromised infection capacity, and it may allow phage display to be performed with particles that are not infective at all. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Characterization of Natronobacterium magadii phage phi Ch1, a unique archaeal phage containing DNA and RNA.

PubMed

Witte, A; Baranyi, U; Klein, R; Sulzner, M; Luo, C; Wanner, G; Krüger, D H; Lubitz, W

1997-02-01

A novel archaeal bacteriophage, phi Ch1, was isolated from a haloalkalophilic archaeon Natronobacterium magadii upon spontaneous lysis. The phage-cured strain N. magadii(L13) was used to demonstrate infectivity of phage phi Ch1. The turbid-plaque morphology and the fact that N. magadii cells isolated from plaques were able to produce phage indicated that phi Ch1 is a temperate phage. The phage morphology resembles other members of Myoviridae-infecting Halobacterium species. In solution below 2M NaCl, the phage lost its morphological stability and infectivity. One- and two-dimensional SDS-PAGE of phage particles revealed at least four major and five minor proteins with molecular masses ranging from 15 to 80 kDa and acidic isoelectric points. Southern blot analysis of chromosomal DNA of a lysogenic N. magadii strain showed that phi Ch1 exists as a chromosomally integrated prophage. The phage particles contain both double-stranded, linear DNA (approx. 55 kbp) as well as several RNA species (80-700 nucleotides). Hybridization of labelled RNA fragments to total DNA from N. magadii and phi Ch1 showed that the virion-associated RNA is host encoded. Part of the phage DNA population is modified and restriction analysis revealed evidence for adenine methylation. Phage phi Ch1 is the first virus described for the genus natronobacterium, and the first phage containing DNA and RNA in mature phage particles.

Induction of lambda prophage by 213 nm laser radiation: a quantitative comparison with 193 nm excimer radiation using image analysis.

PubMed

Matchette, L S; Grossman, L W; Hahn, D W; Cooney, C

1996-03-01

We compared the DNA damage produced by radiation from two UV laser wavelengths, 213 nm and 193 nm, with that produced by noncoherent 254 nm radiation. Following irradiation of Escherichia coli BR339, a bacteriophage lambda lysogen containing the lacZ gene, pro-phage induction was measured by assaying for beta-galactosidase. Because of the limited penetration by UV laser wavelengths an agar overlay of the lysogen was used as the irradiation target. Irradiation of 254 nm was performed in buffer suspension followed by transfer of 5 microL spots onto assay plants. Computer image analysis was used to monitor the rate of product formation, observed as an increase in optical density of the irradiated zones on assay plates. We found that the rate of product formation was a more reproducible unit of comparison than the optical density present at the end of the reaction. Although the rate of product formation was not linearly related to enzyme concentration, the data could be fit to a simple logarithmic function. Using this method, we concluded that the DNA damaging ability of 213 nm radiation was 10 times more efficient than 193 nm radiation and about 100 times less efficient than 254 nm noncoherent radiation.

Molecular cloning and characterization of Hymenolepis diminuta alpha-tubulin gene.

PubMed

Mohajer-Maghari, Behrokh; Amini-Bavil-Olyaee, Samad; Webb, Rodney A; Coe, Imogen R

2007-02-01

To isolate a full-length alpha-tubulin cDNA from an eucestode, Hymenolepis diminuta, a lambda phage cDNA library was constructed. The alpha-tubulin gene was cloned, sequenced and characterized. The H. diminuta alpha-tubulin consisted of 450 amino acids. This protein contained putative sites for all posttranslational modifications as detyrosination/tyrosination at the carboxyl-terminal of protien, phosphorylation at residues R79 and K336, glycylation/glutamylation at residue G445 and acetylation at residue K40. Comparisons of H. diminuta alpha-tubulin with all full-length alpha-tubulin proteins revealed that H. diminuta alpha-tubulin possesses 10 distinctive residues, which are not found in any other alpha-tubulins. Phylogenetic analysis showed that H. diminuta alpha-tubulin has grouped in a separated branch adjacent eucestode and trematodes branch with 92% bootstrap value (1000 replicates). In conclusion, this is the first report of H. diminuta cDNA library construction, cloning and characterization of H. diminuta alpha-tubulin gene.

A homolog of Escherichia coli RecA in mitochondria of the cellular slime mold Dictyostelium discoideum.

PubMed

Hasegawa, Yasuna; Wakabayashi, Masayuki; Nakamura, Shogo; Kodaira, Ken-ichi; Shinohara, Hiroaki; Yasukawa, Hiro

2004-05-04

The cellular slime mold Dictyostelium discoideum expresses a gene encoding a 452-amino-acid polypeptide that is 47% identical to Escherichia coli RecA. A recA-deficient E. coli, JE6651, was transformed by pYSN1, which was designed to express the truncated form of the D. discoideum gene, and used in suppression assays. The viability of the transformant, JE6651(pYSN1), increased following UV irradiation or mitomycin C treatment. Phage lambda (red(-) gam(-)), which required RecA activity for DNA packaging, formed plaques on a lawn of JE6651(pYSN1). These results indicate that the gene product has a DNA recombination activity. Fluorescence of D. discoideum protein fused with GFP was detected in mitochondria. The gene disruption mutant was hypersensitive to UV-light (254nm), mitomycin C and H(2)O(2), indicating that D. discoideum recA is important for survival following exposure to DNA damaging agents.

Cloning and determination of the transcription termination site of ribosomal RNA gene of the mouse.

PubMed Central

Kominami, R; Mishima, Y; Urano, Y; Sakai, M; Muramatsu, M

1982-01-01

A Eco RI 6.6 kb DNA fragment containing the 3'-end of 28S ribosomal RNA gene of the mouse was detected by Southern blot hybridization, and cloned in a lambda-phage vector. The site of transcription termination and the processed 3'-end of 28S RNA were determined on the cloned fragment and the surrounding nucleotide sequence determined. The 3'-terminal nucleotides of mouse 28S RNA are similar to those of yeast, Drosophila and Xenopus although the homology was lost drastically beyond the 3'-end of 28S RNA. 45S precursor RNA terminated at 30 nucleotides downstream from the 3'-end of 28S RNA gene. A structure of a dyad symmetry with a loop was found immediately prior to the termination site of 45S RNA. The rDNA termination site thus shares some common features with termination sites recognized by other RNA polymerases. Images PMID:6281727

Improvement and Optimization of Two Engineered Phage Resistance Mechanisms in Lactococcus lactis

PubMed Central

McGrath, Stephen; Fitzgerald, Gerald F.; van Sinderen, Douwe

2001-01-01

Homologous replication module genes were identified for four P335 type phages. DNA sequence analysis revealed that all four phages exhibited more than 90% DNA homology for at least two genes, designated rep2009 and orf17. One of these genes, rep2009, codes for a putative replisome organizer protein and contains an assumed origin of phage DNA replication (ori2009), which was identical for all four phages. DNA fragments representing the ori2009 sequence confer a phage-encoded resistance (Per) phenotype on lactococcal hosts when they are supplied on a high-copy-number vector. Furthermore, cloning multiple copies of the ori2009 sequence was found to increase the effectiveness of the Per phenotype conferred. A number of antisense plasmids targeting specific genes of the replication module were constructed. Two separate plasmids targeting rep2009 and orf17 were found to efficiently inhibit proliferation of all four phages by interfering with intracellular phage DNA replication. These results represent two highly effective strategies for inhibiting bacteriophage proliferation, and they also identify a novel gene, orf17, which appears to be important for phage DNA replication. Furthermore, these results indicate that although the actual mechanisms of DNA replication are very similar, if not identical, for all four phages, expression of the replication genes is significantly different in each case. PMID:11157223

Genetic Variation of Lactobacillus delbrueckii subsp. lactis Bacteriophages Isolated from Cheese Processing Plants in Finland

PubMed Central

Forsman, Päivi; Alatossava, Tapani

1991-01-01

The genomes of four Lactobacillus delbrueckii subsp. lactis bacteriophages were characterized by restriction endonuclease mapping, Southern hybridization, and heteroduplex analysis. The phages were isolated from different cheese processing plants in Finland between 1950 and 1972. All four phages had a small isometric head and a long noncontractile tail. Two different types of genome (double-stranded DNA) organization existed among the different phages, the pac type and the cos type, corresponding to alternative types of phage DNA packaging. Three phages belonged to the pac type, and a fourth was a cos-type phage. The pac-type phages were genetically closely related. In the genomes of the pac-type phages, three putative insertion/deletions (0.7 to 0.8 kb, 1.0 kb, and 1.5 kb) and one other region (0.9 kb) containing clustered base substitutions were discovered and localized. At the phenotype level, three main differences were observed among the pac-type phages. These concerned two minor structural proteins and the efficiency of phage DNA packaging. The genomes of the pac-type phages showed only weak homology with that of the cos-type phage. Phage-related DNA, probably a defective prophage, was located in the chromosome of the host strain sensitive to the cos-type phage. This DNA exhibited homology under stringent conditions to the pac-type phages. Images PMID:16348513

Genes and Structural Proteins of the Phage Syn5 of the Marine Cyanobacteria Synechococcus

DTIC Science & Technology

2005-09-01

typhimurium phage P22, a podoviridae, was shown to possess extensive genomic similarity to coliphage lambda, a siphoviridae (Botstein and Herskowitz...are found among cyanobacteria in the surface waters during the winter. Temperature influences the number of infectious particles produced during lytic...grids and stained with 1% uranyl acetate for 15 minutes, washed three times in double-distilled water , stained in 1% lead citrate for 4 minutes, and

Lambda Red Mediated Gap Repair Utilizes a Novel Replicative Intermediate in Escherichia coli

PubMed Central

Reddy, Thimma R.; Fevat, Léna M. S.; Munson, Sarah E.; Stewart, A. Francis; Cowley, Shaun M.

2015-01-01

The lambda phage Red recombination system can mediate efficient homologous recombination in Escherichia coli, which is the basis of the DNA engineering technique termed recombineering. Red mediated insertion of DNA requires DNA replication, involves a single-stranded DNA intermediate and is more efficient on the lagging strand of the replication fork. Lagging strand recombination has also been postulated to explain the Red mediated repair of gapped plasmids by an Okazaki fragment gap filling model. Here, we demonstrate that gap repair involves a different strand independent mechanism. Gap repair assays examining the strand asymmetry of recombination did not show a lagging strand bias. Directly testing an ssDNA plasmid showed lagging strand recombination is possible but dsDNA plasmids did not employ this mechanism. Insertional recombination combined with gap repair also did not demonstrate preferential lagging strand bias, supporting a different gap repair mechanism. The predominant recombination route involved concerted insertion and subcloning though other routes also operated at lower frequencies. Simultaneous insertion of DNA resulted in modification of both strands and was unaffected by mutations to DNA polymerase I, responsible for Okazaki fragment maturation. The lower efficiency of an alternate Red mediated ends-in recombination pathway and the apparent lack of a Holliday junction intermediate suggested that gap repair does not involve a different Red recombination pathway. Our results may be explained by a novel replicative intermediate in gap repair that does not involve a replication fork. We exploited these observations by developing a new recombineering application based on concerted insertion and gap repair, termed SPI (subcloning plus insertion). SPI selected against empty vector background and selected for correct gap repair recombinants. We used SPI to simultaneously insert up to four different gene cassettes in a single recombineering reaction. Consequently, our findings have important implications for the understanding of E. coli replication and Red recombination. PMID:25803509

Engineering T7 bacteriophage as a potential DNA vaccine targeting delivery vector.

PubMed

Xu, Hai; Bao, Xi; Wang, Yiwei; Xu, Yue; Deng, Bihua; Lu, Yu; Hou, Jibo

2018-03-20

DNA delivery with bacteriophage by surface-displayed mammalian cell penetrating peptides has been reported. Although, various phages have been used to facilitate DNA transfer by surface displaying the protein transduction domain of human immunodeficiency virus type 1 Tat protein (Tat peptide), no similar study has been conducted using T7 phage. In this study, we engineeredT7 phage as a DNA targeting delivery vector to facilitate cellular internalization. We constructed recombinant T7 phages that displayed Tat peptide on their surface and carried eukaryotic expression box (EEB) as a part of their genomes (T7-EEB-Tat). We demonstrated that T7 phage harboring foreign gene insertion had packaged into infective progeny phage particles. Moreover, when mammalian cells that were briefly exposed to T7-EEB-Tat, expressed a significant higher level of the marker gene with the control cells infected with the wide type phage without displaying Tat peptides. These data suggested that the potential of T7 phage as an effective delivery vector for DNA vaccine transfer.

Extending the Host Range of Bacteriophage Particles for DNA Transduction.

PubMed

Yosef, Ido; Goren, Moran G; Globus, Rea; Molshanski-Mor, Shahar; Qimron, Udi

2017-06-01

A major limitation in using bacteriophage-based applications is their narrow host range. Approaches for extending the host range have focused primarily on lytic phages in hosts supporting their propagation rather than approaches for extending the ability of DNA transduction into phage-restrictive hosts. To extend the host range of T7 phage for DNA transduction, we have designed hybrid particles displaying various phage tail/tail fiber proteins. These modular particles were programmed to package and transduce DNA into hosts that restrict T7 phage propagation. We have also developed an innovative generalizable platform that considerably enhances DNA transfer into new hosts by artificially selecting tails that efficiently transduce DNA. In addition, we have demonstrated that the hybrid particles transduce desired DNA into desired hosts. This study thus critically extends and improves the ability of the particles to transduce DNA into novel phage-restrictive hosts, providing a platform for myriad applications that require this ability. Copyright © 2017 Elsevier Inc. All rights reserved.

tif-1 mutation alters polynucleotide recognition by the recA protein of Escherichia coli.

PubMed Central

McEntee, K; Weinstock, G M

1981-01-01

The requirements for polynucleotide-dependent hydrolysis of ATP and for proteolytic cleavage of phage lambda repressor have been examined for both the wild-type (recA+ protein) and the tif-1 mutant form [tif(recA) protein] of the recA gene product. The recA+ and tif(recA) proteins catalyze both reactions in the presence of long single-stranded DNAs or certain deoxyhomopolymers. However, short oligonucleotides [(dT)12, (dA)14] stimulate neither the protease nor the ATPase activities of the recA+ protein. In contrast, these short oligonucleotides activate tif(recA) protein to cleave lambda repressor without stimulating its ATPase activity. Moreover, both the ATPase and protease activities of the tif(recA) protein are stimulated by poly(rU) and poly(rC) whereas the recA+ protein does not respond to these ribopolymers. We have purified the recA protein from a strain in which the tif mutation is intragenically suppressed. This mutant protein (recA629) is inactive in the presence of (dT)12, (dA)14, poly(rU), and poly(rC) for lambda repressor cleavage and ATP hydrolysis. These results argue that the tif-1 mutation (or mutations) alters the DNA binding site of the recA protein. We suggest that in vivo the tif(recA) protein is activated for cleaving repressors of SOS genes by complex formation with short single-stranded regions or gaps that normally occur near the growing fork of replicating chromosomes and are too short for activating the recA+ enzyme. This mechanism can account for the expression of SOS functions in the absence of DNA damage in tif mutant strains. Images PMID:7031642

Heat Induction of Prophage φ105 in Bacillus subtilis: Replication of the Bacterial and Bacteriophage Genomes

PubMed Central

Armentrout, Richard W.; Rutberg, Lars

1971-01-01

A temperature-inducible mutant of temperate Bacillus bacteriophage φ105 was isolated and used to lysogenize a thymine-requiring strain of Bacillus subtilis 168. Synthesis of phage and bacterial deoxyribonucleic acid (DNA) was studied by sucrose gradient centrifugation and density equilibrium centrifugation of DNA extracted from induced bacteria. The distribution of DNA in the gradients was measured by differential isotope and density labeling of DNA before and after induction and by measuring the biological activity of the DNA in genetic transformation, in rescue of phage markers, and in infectivity assays. At early times after induction, but after at least one round of replication, phage DNA remains associated with high-molecular-weight DNA, whereas, later in the infection, phage DNA is associated with material of decreasing molecular weight. Genetic linkage between phage and bacterial markers can be demonstrated in replicated DNA from induced cells. Prophage induction is shown to affect replication of the bacterial chromosome. The overall rate of replication of prelabeled bacterial DNA is identical in temperature-induced lysogenics and in “mock-induced” wild-type φ105 lysogenics. The rate of replication of the bacterial marker phe-1 (and also of nia-38), located close to the prophage in direction of the terminus of the bacterial chromosome, is increased in induced cells, however, relative to other bacterial markers tested. In temperature-inducible lysogenics, where the prophage also carries a ts mutation which blocks phage DNA synthesis, replication of both phage and bacterial DNA stops after about 50% of the phage DNA has replicated once. The results of these experiments suggest that the prophage is not initially excised in induced cells, but rather it is specifically replicated in situ together with adjacent parts of the bacterial chromosome. PMID:5002012

Mechanisms of inactivation of bacteriophage phiX174 and its DNA in aerosols by ozone and ozonized cyclohexene.

PubMed Central

de Mik, G.; de Groot, I.

1977-01-01

The mechanisms of inactivation of aerosolized bacteriophage phiX174 in atmospheres containing ozone, cyclohexene, or ozonized cyclohexene were studied by using 32P-labelled phage. The inactivation of the aerosolized phage in clear air or in air containing cyclohexene is due to damage of the protein coat since the deoxyribonucleic acid (DNA) extracted from the inactivated phage retains its biological activity. Inactivation of the phage in air containing ozonized cyclohexene is due both to protein and DNA damage. Sucrose gradient analysis shows that aerosolized inactivated phiX174 releases unbroken DNA. In contrast, the DNA from phage phiX174 inactivated by ozonized cyclohexene is broken. The inactivation of aerosolized phage phiX174-DNA was studied in the same atmospheres using 32P-labelled DNA. phiX174-DNA aerosolized in clear air or air containing cyclohexene at 75% r.h. is inactivated by a factor of 2 in 30 min. The inactivated DNA is broken. Ozone as well as ozonized cyclohexene inactivates KNA very fast causing breaks in the molecule. This is in contrast with the intact bacteriophage in which ozone does not produce breaks in the DNA. PMID:265342

Reversible Condensation of DNA using a Redox-Active Surfactant

PubMed Central

Hays, Melissa E.; Jewell, Christopher M.; Lynn, David M.; Abbott, Nicholas L.

2008-01-01

We report characterization of aqueous solutions of dilute Lambda phage DNA containing the redox-active surfactant (11-ferrocenylundecyl)trimethylammonium bromide (FTMA) as a function of the oxidation state of the FTMA. FTMA undergoes a reversible one-electron oxidation from a reduced state that forms micelles in aqueous solution to an oxidized state (containing the ferrocenium cation) that does not selfassociate in solution. This investigation sought to test the hypothesis that FTMA can be used to achieve reversible control over the conformation of DNA-surfactant complexes in solution. Whereas DNA adopts extended coil conformations in aqueous solutions, our measurements revealed that addition of reduced FTMA (2–5μM) to aqueous solutions of DNA (5 μM in nucleotide units) resulted in coexistence of extended coils and compact globules in solution. At higher concentrations of reduced FTMA (up to 30μM), the DNA was present as compact globules only. In contrast, oxidized FTMA had no measurable effect on the conformation of DNA, allowing DNA to maintain an extended coil state up to a concentration of 75μM oxidized FTMA. We further demonstrate that it is possible to chemically or electrochemically transform the oxidation state of FTMA in preformed complexes of FTMA and DNA, thus achieving in situ control over the conformations of the DNA in solution. These results provide guidance for the design of surfactant systems that permit active control of DNA-surfactant interactions. PMID:17428073

Lambda phage-based vaccine induces antitumor immunity in hepatocellular carcinoma.

PubMed

Iwagami, Yoshifumi; Casulli, Sarah; Nagaoka, Katsuya; Kim, Miran; Carlson, Rolf I; Ogawa, Kosuke; Lebowitz, Michael S; Fuller, Steve; Biswas, Biswajit; Stewart, Solomon; Dong, Xiaoqun; Ghanbari, Hossein; Wands, Jack R

2017-09-01

Hepatocellular carcinoma (HCC) is a difficult to treat tumor with a poor prognosis. Aspartate β-hydroxylase (ASPH) is a highly conserved enzyme overexpressed on the cell surface of both murine and human HCC cells. We evaluated therapeutic effects of nanoparticle lambda (λ) phage vaccine constructs against ASPH expressing murine liver tumors. Mice were immunized before and after subcutaneous implantation of a syngeneic BNL HCC cell line. Antitumor actively was assessed by generation of antigen specific cellular immune responses and the identification of tumor infiltrating lymphocytes. Prophylactic and therapeutic immunization significantly delayed HCC growth and progression. ASPH-antigen specific CD4+ and CD8+ lymphocytes were identified in the spleen of tumor bearing mice and cytotoxicity was directed against ASPH expressing BNL HCC cells. Furthermore, vaccination generated antigen specific Th1 and Th2 cytokine secretion by immune cells. There was widespread necrosis with infiltration of CD3+ and CD8+ T cells in HCC tumors of λ phage vaccinated mice compared to controls. Moreover, further confirmation of anti-tumor effects on ASPH expressing tumor cell growth were obtained in another murine syngeneic vaccine model with pulmonary metastases. These observations suggest that ASPH may serve as a highly antigenic target for immunotherapy.

Bacteriophage of Haemophilus influenzae III. Morphology, DNA Homology, and Immunity Properties of HP1c1, S2, and the Defective Bacteriophage from Strain Rd

PubMed Central

Boling, Maxon E.; Allison, David P.; Setlow, Jane K.

1973-01-01

The phages HP1c1 and S2 and a defective phage of Haemophilus influenzae have been compared. The morphology of the phages and the mol wt of their DNAs are similar, although the defective phage appears to have a different tail plate region. Electron microscope observation indicates that the defective phage does not attach to the cell surface, and its DNA appears to lack cohesive ends. The homology of the DNAs of the phages has been measured by hydridization. DNA from the defective phage shows little or no homology with the other phage DNAs. HP1c1 and S2 DNAs show a high level of homology. Each of these phages can form plaques on lawns of the lysogen of the other phage but at reduced plating efficiencies, suggesting that the two phages have related but not identical immunity systems. Images PMID:4540713

Amino Acid Control over Deoxyribonucleic Acid Synthesis in Escherichia coli Infected With T-Even Bacteriophage

PubMed Central

Donini, Pierluigi

1970-01-01

Starvation for a required amino acid of normal or RCstrEscherichia coli infected with T-even phages arrests further synthesis of phage deoxyribonucleic acid (DNA). This amino acid control over phage DNA synthesis does not occur in RCrelE. coli mutants. Heat inactivation of a temperature-sensitive aminoacyl-transfer ribonucleic acid (RNA) synthetase similarly causes an arrest of phage DNA synthesis in infected cells of RCstr phenotype but not in cells of RCrel phenotype. Inhibition of phage DNA synthesis in amino acid-starved RCstr host cells can be reversed by addition of chloramphenicol to the culture. Thus, the general features of amino acid control over T-even phage DNA synthesis are entirely analogous to those known for amino acid control over net RNA synthesis of uninfected bacteria. This analogy shows that the bacterial rel locus controls a wider range of macromolecular syntheses than had been previously thought. PMID:4914067

Investigation of interaction between magnetic silica particles and lambda phage DNA fragment.

PubMed

Smerkova, Kristyna; Dostalova, Simona; Vaculovicova, Marketa; Kynicky, Jindrich; Trnkova, Libuse; Kralik, Miroslav; Adam, Vojtech; Hubalek, Jaromir; Provaznik, Ivo; Kizek, Rene

2013-12-01

Nucleic acids belong to the most important molecules and therefore the understanding of their properties, function and behavior is crucial. Even though a range of analytical and biochemical methods have been developed for this purpose, one common step is essential for all of them - isolation of the nucleic acid from the from complex sample matrix. The use of magnetic particles for the separation of nucleic acids has many advantages over other isolation methods. In this study, an isolation procedure for extraction of DNA was optimized. Each step of the isolation process including washing, immobilization and elution was optimized and therefore the efficiency was increased from 1.7% to 28.7% and the total time was shortened from 75 to 30min comparing to the previously described method. Quantification of the particular parameter influence was performed by square-wave voltammetry using hanging drop mercury electrode. Further, we compared the optimized method with standard chloroform extraction and applied on isolation of DNA from Staphylococcus aureus and Escherichia coli. Copyright © 2013 Elsevier B.V. All rights reserved.

Effectiveness of the lactococcal abortive infection systems AbiA, AbiE, AbiF and AbiG against P335 type phages.

PubMed

Tangney, Mark; Fitzgerald, Gerald F

2002-04-23

Four lactococcal abortive infection mechanisms were introduced into strains which were sensitive hosts for P335 type phages and plaque assay experiments performed to assess their effect on five lactococcal bacteriophages from this family. Results indicate that AbiA inhibits all five P335 phages tested, while AbiG affects phiP335 itself and phiQ30 but not the other P335 species phages. AbiA was shown to retard phage Q30 DNA replication as previously reported for other phages. It was also demonstrated that AbiG, previously shown to act at a point after DNA replication in the cases of c2 type and 936 type phages, acts at the level of, or prior to phage Q30 DNA replication. AbiE and AbiF had no effect on the P335 type phages examined.

Fragmentation of the CRISPR-Cas Type I-B signature protein Cas8b.

PubMed

Richter, Hagen; Rompf, Judith; Wiegel, Julia; Rau, Kristina; Randau, Lennart

2017-11-01

CRISPR arrays are transcribed into long precursor RNA species, which are further processed into mature CRISPR RNAs (crRNAs). Cas proteins utilize these crRNAs, which contain spacer sequences that can be derived from mobile genetic elements, to mediate immunity during a reoccurring virus infection. Type I CRISPR-Cas systems are defined by the presence of different Cascade interference complexes containing large and small subunits that play major roles during target DNA selection. Here, we produce the protein and crRNA components of the Type I-B CRISPR-Cas complex of Clostridium thermocellum and Methanococcus maripaludis. The C. thermocellum Cascade complexes were reconstituted and analyzed via size-exclusion chromatography. Activity of the heterologous M. maripaludis CRISPR-Cas system was followed using phage lambda plaques assays. The reconstituted Type-I-B Cascade complex contains Cas7, Cas5, Cas6b and the large subunit Cas8b. Cas6b can be omitted from the reconstitution protocol. The large subunit Cas8b was found to be represented by two tightly associated protein fragments and a small C-terminal Cas8b segment was identified in recombinant complexes and C. thermocellum cell lysate. Production of Cas8b generates a small C-terminal fragment, which is suggested to fulfill the role of the missing small subunit. A heterologous, synthetic M. maripaludis Type I-B system is active in E. coli against phage lambda, highlighting a potential for genome editing using endogenous Type-I-B CRISPR-Cas machineries. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

Assignment of the human type I IMP dehydrogenase gene (IMPDH1) to chromosome 7q31.3-q32

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gu, Jing Jin; Kaiser-Rogers, K.; Rao, K.

Two phage {lambda} clones that contain 5{prime} portion of the human type I inosine 5{prime}-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) gene were isolated. Both polymerase chain reaction analysis of a panel of human-mouse and human-hamster cell somatic hybrids using primers specific for the type I IMPDH gene and fluorescence in situ hybridization with metaphase human chromosome using type I IMPDH genomic DNA as probes indicate that the type I IMPDH gene (symbol IMPDH1) is located on chromosome 7. Sequential GTG-banding was performed to assign the band location of the type I IMPDH gene to chromosome 7q31.3-q32. 16 refs., 1 fig., 1more » tab.« less

Biophysics and bioinformatics of transcription regulation in bacteria and bacteriophages

NASA Astrophysics Data System (ADS)

Djordjevic, Marko

2005-11-01

Due to rapid accumulation of biological data, bioinformatics has become a very important branch of biological research. In this thesis, we develop novel bioinformatic approaches and aid design of biological experiments by using ideas and methods from statistical physics. Identification of transcription factor binding sites within the regulatory segments of genomic DNA is an important step towards understanding of the regulatory circuits that control expression of genes. We propose a novel, biophysics based algorithm, for the supervised detection of transcription factor (TF) binding sites. The method classifies potential binding sites by explicitly estimating the sequence-specific binding energy and the chemical potential of a given TF. In contrast with the widely used information theory based weight matrix method, our approach correctly incorporates saturation in the transcription factor/DNA binding probability. This results in a significant reduction in the number of expected false positives, and in the explicit appearance---and determination---of a binding threshold. The new method was used to identify likely genomic binding sites for the Escherichia coli TFs, and to examine the relationship between TF binding specificity and degree of pleiotropy (number of regulatory targets). We next address how parameters of protein-DNA interactions can be obtained from data on protein binding to random oligos under controlled conditions (SELEX experiment data). We show that 'robust' generation of an appropriate data set is achieved by a suitable modification of the standard SELEX procedure, and propose a novel bioinformatic algorithm for analysis of such data. Finally, we use quantitative data analysis, bioinformatic methods and kinetic modeling to analyze gene expression strategies of bacterial viruses. We study bacteriophage Xp10 that infects rice pathogen Xanthomonas oryzae. Xp10 is an unusual bacteriophage, which has morphology and genome organization that most closely resembles temperate phages, such as lambda. It, however, encodes its own T7-like RNA polymerase (characteristic of virulent phages), whose role in gene expression was unclear. Our analysis resulted in quantitative understanding of the role of both host and phage RNA polymerase, and in the identification of the previously unknown promoter sequence for Xp10 RNA polymerase. More generally, an increasing number of phage genomes are being sequenced every year, and we expect that methods of quantitative data analysis that we introduced will provide an efficient way to study gene expression strategies of novel bacterial viruses.

New Deoxyribonucleic Acid Polymerase Induced by Bacillus subtilis Bacteriophage PBS2

PubMed Central

Price, Alan R.; Cook, Sandra J.

1972-01-01

The deoxyribonucleic acid (DNA) of Bacillus subtilis phage PBS2 has been confirmed to contain uracil instead of thymine. PBS2 phage infection of wild-type cells or DNA polymerase-deficient cells results in an increase in the specific activity of DNA polymerase. This induction of DNA polymerase activity is prevented by actinomycin D and chloramphenicol. In contrast to the major B. subtilis DNA polymerase, which prefers deoxythymidine triphosphate (dTTP) to deoxyuridine triphosphate (dUTP), the DNA polymerase in crude extracts of PBS2-infected cells is equally active whether dTTP or dUTP is employed. This phage-induced polymerase may be responsible for the synthesis of uracil-containing DNA during PBS2 phage infection. PMID:4623224

Rapid isolation of novel FK506 binding proteins from multiple organisms using gDNA and cDNA T7 phage display.

PubMed

Piggott, Andrew M; Kriegel, Alison M; Willows, Robert D; Karuso, Peter

2009-10-01

Reverse chemical proteomics using T7 phage display is a powerful technique for identifying cellular receptors of biologically active small molecules. However, to date this method has generally been limited to cDNA libraries constructed from mRNA isolated from eukaryotes. In this paper, we describe the construction of the first prokaryotic T7 phage display libraries from randomly digested Pseudomonas stutzeri and Vibrio fischeri gDNA, as well as a plant cDNA library from Arabidopsis thaliana. We also describe the use of T7 phage display to identify novel proteins from environmental DNA samples using biotinylated FK506 as a model affinity probe.

φX-174 Bacteriophage Structural Mutants Which Affect Deoxyribonucleic Acid Synthesis

PubMed Central

Siegel, Jeff E. D.; Hayashi, Masaki

1969-01-01

Seven cistrons in φX-174 were identified and one in particular was studied intensively: cistron A, which is assigned a protein in the mature phage. Amber mutants in this cistron synthesize a new deoxyribonucleic acid (DNA) form in addition to circular phage DNA upon infection of the restrictive host. This DNA is linear, non-infectious, and single-stranded; it is formed from the phage strand of replicative form φX-174 DNA. These mutants produce two different defective particles in the restrictive host. One particle contains circular phage DNA but is not infectious; the other contains the new DNA form and is similar to the 70S particles found in wild-type phage lysates. The mutant A gene product acts independently of normal A protein upon mixed infection of the restrictive host with an A mutant and a mutant from any other cistron or wild type. PMID:5823229

QUANTITATIVE STUDY OF ENDOLYSIN SYNTHESIS DURING REPRODUCTION OF LAMBDA PHAGES

PubMed Central

Groman, Neal B.; Suzuki, Grace

1963-01-01

Groman, Neal B. (University of Washington, Seattle) and Grace Suzuki. Quantitative study of endolysin synthesis during reproduction of lambda phages. J. Bacteriol. 86:187–194. 1963.—Endolysin is presumed to be a phage-induced enzyme participating in lysis through its destructive action on the host cell wall. A method for assaying endolysin is described, which was utilized in studying endolysin synthesis at 37 and 44 C by induced strains of K-12 (λ), K-12 (λtem), and K-12 (λ112). In all cases, endolysin was detected prior to the appearance of mature, intracellular phage and was detected earlier at 44 C than at 37 C. It was synthesized at a linear rate, as was phage, and both syntheses terminated at the same time. Surprisingly, endolysin also accumulated under conditions in which induced K-12 (λ112) exhibited lysis inhibition. Under these conditions, endolysin concentration per induced cell was 2 to 2.5 times that produced by normally lysing K-12 (λ). Since alterations introduced into the lytic process by temperature, mutation, or both correlate well with the timing and rate of endolysin synthesis, the data tend to support the concept that endolysin determines the kinetics of the process. However, the accumulation of endolysin during lysis inhibition suggests the need for alternative hypotheses. One hypothesis is that although endolysin action is the key to lysis some preliminary steps are required to release the enzyme so that it may contact its substrate in the cell wall. A second hypothesis is that basically the lytic process involves an alteration in the permeability barrier of the cell and that lytic enzymes such as endolysin have evolved as an auxillary but dispensable mechanism to this process. PMID:14058940

Dynamics of bacteriophage genome ejection in vitro and in vivo

NASA Astrophysics Data System (ADS)

Panja, Debabrata; Molineux, Ian J.

2010-12-01

Bacteriophages, phages for short, are viruses of bacteria. The majority of phages contain a double-stranded DNA genome packaged in a capsid at a density of ~500 mg ml-1. This high density requires substantial compression of the normal B-form helix, leading to the conjecture that DNA in mature phage virions is under significant pressure, and that pressure is used to eject the DNA during infection. A large number of theoretical, computer simulation and in vitro experimental studies surrounding this conjecture have revealed many—though often isolated and/or contradictory—aspects of packaged DNA. This prompts us to present a unified view of the statistical physics and thermodynamics of DNA packaged in phage capsids. We argue that the DNA in a mature phage is in a (meta)stable state, wherein electrostatic self-repulsion is balanced by curvature stress due to confinement in the capsid. We show that in addition to the osmotic pressure associated with the packaged DNA and its counterions, there are four different pressures within the capsid: pressure on the DNA, hydrostatic pressure, the pressure experienced by the capsid and the pressure associated with the chemical potential of DNA ejection. Significantly, we analyze the mechanism of force transmission in the packaged DNA and demonstrate that the pressure on DNA is not important for ejection. We derive equations showing a strong hydrostatic pressure difference across the capsid shell. We propose that when a phage is triggered to eject by interaction with its receptor in vitro, the (thermodynamic) incentive of water molecules to enter the phage capsid flushes the DNA out of the capsid. In vivo, the difference between the osmotic pressures in the bacterial cell cytoplasm and the culture medium similarly results in a water flow that drags the DNA out of the capsid and into the bacterial cell.

Stumbling across the Same Phage: Comparative Genomics of Widespread Temperate Phages Infecting the Fish Pathogen Vibrio anguillarum

PubMed Central

Kalatzis, Panos G.; Rørbo, Nanna; Castillo, Daniel; Mauritzen, Jesper Juel; Jørgensen, Jóhanna; Kokkari, Constantina; Zhang, Faxing; Katharios, Pantelis; Middelboe, Mathias

2017-01-01

Nineteen Vibrio anguillarum-specific temperate bacteriophages isolated across Europe and Chile from aquaculture and environmental sites were genome sequenced and analyzed for host range, morphology and life cycle characteristics. The phages were classified as Siphoviridae with genome sizes between 46,006 and 54,201 bp. All 19 phages showed high genetic similarity, and 13 phages were genetically identical. Apart from sporadically distributed single nucleotide polymorphisms (SNPs), genetic diversifications were located in three variable regions (VR1, VR2 and VR3) in six of the phage genomes. Identification of specific genes, such as N6-adenine methyltransferase and lambda like repressor, as well as the presence of a tRNAArg, suggested a both mutualistic and parasitic interaction between phages and hosts. During short term phage exposure experiments, 28% of a V. anguillarum host population was lysogenized by the temperate phages and a genomic analysis of a collection of 31 virulent V. anguillarum showed that the isolated phages were present as prophages in >50% of the strains covering large geographical distances. Further, phage sequences were widely distributed among CRISPR-Cas arrays of publicly available sequenced Vibrios. The observed distribution of these specific temperate Vibriophages across large geographical scales may be explained by efficient dispersal of phages and bacteria in the marine environment combined with a mutualistic interaction between temperate phages and their hosts which selects for co-existence rather than arms race dynamics. PMID:28531104

Linkage and homology analysis divides the eight genes for the small subunit of petunia ribulose 1,5-bisphosphate carboxylase into three gene families

PubMed Central

Dean, Caroline; van den Elzen, Peter; Tamaki, Stanley; Dunsmuir, Pamela; Bedbrook, John

1985-01-01

Twenty-six λ phage clones with homology to coding sequences of the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase have been isolated from an EMBL3 λ phage bank of Petunia (Mitchell) DNA. Restriction mapping of the phage inserts shows that the clones were obtained from five nonoverlapping regions of petunia DNA that carry seven SSU genes. Comparison of the HindIII genomic fragments of petunia DNA with the HindIII restriction fragments of the isolated phage indicates that petunia nuclear DNA encodes eight SSU genes, seven of which are present in the phage clones. Two incomplete genes, which contain only the 3′ end of an SSU gene, were also found in the phage clones. We demonstrate that the eight SSU genes of petunia can be divided into three gene families based on homology to three petunia cDNA clones. Two gene families contain single SSU genes and the third contains six genes, four of which are closely linked within petunia nuclear DNA. Images PMID:16593584

Characterization of Two Virulent Phages of Lactobacillus plantarum

PubMed Central

Briggiler Marcó, Mariángeles; Garneau, Josiane E.; Tremblay, Denise; Quiberoni, Andrea

2012-01-01

We characterized two Lactobacillus plantarum virulent siphophages, ATCC 8014-B1 (B1) and ATCC 8014-B2 (B2), previously isolated from corn silage and anaerobic sewage sludge, respectively. Phage B2 infected two of the eight L. plantarum strains tested, while phage B1 infected three. Phage adsorption was highly variable depending on the strain used. Phage defense systems were found in at least two L. plantarum strains, LMG9211 and WCSF1. The linear double-stranded DNA genome of the pac-type phage B1 had 38,002 bp, a G+C content of 47.6%, and 60 open reading frames (ORFs). Surprisingly, the phage B1 genome has 97% identity with that of Pediococcus damnosus phage clP1 and 77% identity with that of L. plantarum phage JL-1; these phages were isolated from sewage and cucumber fermentation, respectively. The double-stranded DNA (dsDNA) genome of the cos-type phage B2 had 80,618 bp, a G+C content of 36.9%, and 127 ORFs with similarities to those of Bacillus and Lactobacillus strains as well as phages. Some phage B2 genes were similar to ORFs from L. plantarum phage LP65 of the Myoviridae family. Additionally, 6 tRNAs were found in the phage B2 genome. Protein analysis revealed 13 (phage B1) and 9 (phage B2) structural proteins. To our knowledge, this is the first report describing such high identity between phage genomes infecting different genera of lactic acid bacteria. PMID:23042172

Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda: localized unwinding of duplex DNA by a six-protein reaction.

PubMed Central

Dodson, M; Echols, H; Wickner, S; Alfano, C; Mensa-Wilmot, K; Gomes, B; LeBowitz, J; Roberts, J D; McMacken, R

1986-01-01

The O protein of bacteriophage lambda localizes the initiation of DNA replication to a unique site on the lambda genome, ori lambda. By means of electron microscopy, we infer that the binding of O to ori lambda initiates a series of protein addition and transfer reactions that culminate in localized unwinding of the origin DNA, generating a prepriming structure for the initiation of DNA replication. We can define three stages of this prepriming reaction, the first two of which we have characterized previously. First, dimeric O protein binds to multiple DNA binding sites and self-associates to form a nucleoprotein structure, the O-some. Second, lambda P and host DnaB proteins interact with the O-some to generate a larger complex that includes additional DNA from an A + T-rich region adjacent to the O binding sites. Third, the addition of the DnaJ, DnaK, and Ssb proteins and ATP results in an origin-specific unwinding reaction, probably catalyzed by the helicase activity of DnaB. The unwinding reaction is unidirectional, proceeding "rightward" from the origin. The minimal DNA sequence competent for unwinding consists of two O binding sites and the adjacent A + T-rich region to the right of the binding sites. We conclude that the lambda O protein localizes and initiates a six-protein sequential reaction responsible for but preceding the precise initiation of DNA replication. Specialized nucleoprotein structures similar to the O-some may be a general feature of DNA transactions requiring extraordinary precision in localization and control. Images PMID:3020552

Membrane fusion during phage lysis.

PubMed

Rajaure, Manoj; Berry, Joel; Kongari, Rohit; Cahill, Jesse; Young, Ry

2015-04-28

In general, phages cause lysis of the bacterial host to effect release of the progeny virions. Until recently, it was thought that degradation of the peptidoglycan (PG) was necessary and sufficient for osmotic bursting of the cell. Recently, we have shown that in Gram-negative hosts, phage lysis also requires the disruption of the outer membrane (OM). This is accomplished by spanins, which are phage-encoded proteins that connect the cytoplasmic membrane (inner membrane, IM) and the OM. The mechanism by which the spanins destroy the OM is unknown. Here we show that the spanins of the paradigm coliphage lambda mediate efficient membrane fusion. This supports the notion that the last step of lysis is the fusion of the IM and OM. Moreover, data are provided indicating that spanin-mediated fusion is regulated by the meshwork of the PG, thus coupling fusion to murein degradation by the phage endolysin. Because endolysin function requires the formation of μm-scale holes by the phage holin, the lysis pathway is seen to require dramatic dynamics on the part of the OM and IM, as well as destruction of the PG.

Bacteriophage-associated gene transfer in pneumococcus: transduction or pseudotransduction?

PubMed Central

Porter, R D; Shoemaker, N B; Rampe, G; Guild, W R

1979-01-01

Lysates of pneumococcal phage PG24 transferred genes from one host to another in a process with many of the properties of generalized transduction, in that the host genes were packaged in DNase-resistant particles that closely resembled infectious phage in physical properties, adsorbed to the recipient cells like phage, and were inhibited by antisera to the phage and by trypsin. However, phage processes did not complete the transfer of host DNA as they did phage DNA. Instead, gene transfer required development of competence and entry of the host DNA by the endonuclease-dependent pathway used for transforming and transfecting DNA. This process often occurred on the assay plate hours after adsorption of the particles to the cells, and the transfer was DNase sensitive if challenged at this time. Phenotypic expression was therefore also delayed. The product of entry was like that in transformation, a single strand of DNA that integrates by formation of a hex-sensitive donor-recipient heteroduplex. Whether this gene transfer process is unique to this system or is only the first one described is not clear. The term "pseudotransduction" may be useful in calling attention to its unexpected features. The DNA of PG24 phage has anomalous physical properties reflecting unusual bases. Images PMID:33154

Identification and biosynthesis of thymidine hypermodifications in the genomic DNA of widespread bacterial viruses

PubMed Central

Lee, Yan-Jiun; Dai, Nan; Walsh, Shannon E.; Müller, Stephanie; Fraser, Morgan E.; Kauffman, Kathryn M.; Guan, Chudi; Weigele, Peter R.

2018-01-01

Certain viruses of bacteria (bacteriophages) enzymatically hypermodify their DNA to protect their genetic material from host restriction endonuclease-mediated cleavage. Historically, it has been known that virion DNAs from the Delftia phage ΦW-14 and the Bacillus phage SP10 contain the hypermodified pyrimidines α-putrescinylthymidine and α-glutamylthymidine, respectively. These bases derive from the modification of 5-hydroxymethyl-2′-deoxyuridine (5-hmdU) in newly replicated phage DNA via a pyrophosphorylated intermediate. Like ΦW-14 and SP10, the Pseudomonas phage M6 and the Salmonella phage ViI encode kinase homologs predicted to phosphorylate 5-hmdU DNA but have uncharacterized nucleotide content [Iyer et al. (2013) Nucleic Acids Res 41:7635–7655]. We report here the discovery and characterization of two bases, 5-(2-aminoethoxy)methyluridine (5-NeOmdU) and 5-(2-aminoethyl)uridine (5-NedU), in the virion DNA of ViI and M6 phages, respectively. Furthermore, we show that recombinant expression of five gene products encoded by phage ViI is sufficient to reconstitute the formation of 5-NeOmdU in vitro. These findings point to an unexplored diversity of DNA modifications and the underlying biochemistry of their formation. PMID:29555775

Structure of a bacterial virus DNA-injection protein complex reveals a decameric assembly with a constricted molecular channel

DOE PAGES

Zhao, Haiyan; Speir, Jeffrey A.; Matsui, Tsutomu; ...

2016-02-16

The multi-layered cell envelope structure of Gram-negative bacteria represents significant physical and chemical barriers for short-tailed phages to inject phage DNA into the host cytoplasm. Here we show that a DNA-injection protein of bacteriophage Sf6, gp12, forms a 465-kDa, decameric assembly in vitro. The electron microscopic structure of the gp12 assembly shows a ~150-Å, mushroom-like architecture consisting of a crown domain and a tube-like domain, which embraces a 25-Å-wide channel that could precisely accommodate dsDNA. The constricted channel suggests that gp12 mediates rapid, uni-directional injection of phage DNA into host cells by providing a molecular conduit for DNA translocation. Themore » assembly exhibits a 10-fold symmetry, which may be a common feature among DNA-injection proteins of P22-like phages and may suggest a symmetry mismatch with respect to the 6-fold symmetric phage tail. As a result, the gp12 monomer is highly flexible in solution, supporting a mechanism for translocation of the protein through the conduit of the phage tail toward the host cell envelope, where it assembles into a DNA-injection device.« less

Structure of a bacterial virus DNA-injection protein complex reveals a decameric assembly with a constricted molecular channel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Haiyan; Speir, Jeffrey A.; Matsui, Tsutomu

The multi-layered cell envelope structure of Gram-negative bacteria represents significant physical and chemical barriers for short-tailed phages to inject phage DNA into the host cytoplasm. Here we show that a DNA-injection protein of bacteriophage Sf6, gp12, forms a 465-kDa, decameric assembly in vitro. The electron microscopic structure of the gp12 assembly shows a ~150-Å, mushroom-like architecture consisting of a crown domain and a tube-like domain, which embraces a 25-Å-wide channel that could precisely accommodate dsDNA. The constricted channel suggests that gp12 mediates rapid, uni-directional injection of phage DNA into host cells by providing a molecular conduit for DNA translocation. Themore » assembly exhibits a 10-fold symmetry, which may be a common feature among DNA-injection proteins of P22-like phages and may suggest a symmetry mismatch with respect to the 6-fold symmetric phage tail. As a result, the gp12 monomer is highly flexible in solution, supporting a mechanism for translocation of the protein through the conduit of the phage tail toward the host cell envelope, where it assembles into a DNA-injection device.« less

Transcription attenuation is the major mechanism by which the leu operon of Salmonella typhimurium is controlled.

PubMed

Searles, L L; Wessler, S R; Calvo, J M

1983-01-25

Three mutations, each causing constitutive expression of the Salmonella typhimurium leu operon, were cloned into phage vector lambda gt4 on EcoRI DNA fragments carrying all of that operon except for part of the promoter-distal last gene. Sequence analysis of DNA from these phage demonstrated that each contains a single base change in the leu attenuator. Transcription of mutant DNA in vitro resulted in transcription beyond the usual site of termination. The level of beta-IPM dehydrogenase, the leuB enzyme, was elevated 40-fold in a strain carrying one of these mutations, and starvation of this strain for leucine had little effect on the amount of activity expressed. Using a strain with a wild-type promoter-leader region of the leu operon, the rates of synthesis and degradation of leu leader RNA and readthrough RNA (leu mRNA) were measured by DNA-RNA hybridizations with specific DNA probes. The rate of synthesis of the leu leader was about the same in cells grown with excess or with limiting leucine. On the other hand, the rate of synthesis of leu mRNA was 12-fold higher for cells grown in limiting leucine as opposed to excess leucine. The rate of degradation of these RNA species was the same under both conditions of growth. Thus, the variation in expression of the leu operon observed for cells grown in minimal medium is, for the most part, not caused by control over the frequency of initiation or by the differential stability of these RNA species. Rather, the variation is a direct result of the frequency of transcription termination at an attenuator site. These results taken together suggest that transcription attenuation is the major mechanism by which leucine regulates expression of the leu operon of S. typhimurium for cells growing in a minimal medium.

Sequence Analysis of Leuconostoc mesenteroides Bacteriophage Φ1-A4 Isolated from an Industrial Vegetable Fermentation▿

PubMed Central

Lu, Z.; Altermann, E.; Breidt, F.; Kozyavkin, S.

2010-01-01

Vegetable fermentations rely on the proper succession of a variety of lactic acid bacteria (LAB). Leuconostoc mesenteroides initiates fermentation. As fermentation proceeds, L. mesenteroides dies off and other LAB complete the fermentation. Phages infecting L. mesenteroides may significantly influence the die-off of L. mesenteroides. However, no L. mesenteroides phages have been previously genetically characterized. Knowledge of more phage genome sequences may provide new insights into phage genomics, phage evolution, and phage-host interactions. We have determined the complete genome sequence of L. mesenteroides phage Φ1-A4, isolated from an industrial sauerkraut fermentation. The phage possesses a linear, double-stranded DNA genome consisting of 29,508 bp with a G+C content of 36%. Fifty open reading frames (ORFs) were predicted. Putative functions were assigned to 26 ORFs (52%), including 5 ORFs of structural proteins. The phage genome was modularly organized, containing DNA replication, DNA-packaging, head and tail morphogenesis, cell lysis, and DNA regulation/modification modules. In silico analyses showed that Φ1-A4 is a unique lytic phage with a large-scale genome inversion (∼30% of the genome). The genome inversion encompassed the lysis module, part of the structural protein module, and a cos site. The endolysin gene was flanked by two holin genes. The tail morphogenesis module was interspersed with cell lysis genes and other genes with unknown functions. The predicted amino acid sequences of the phage proteins showed little similarity to other phages, but functional analyses showed that Φ1-A4 clusters with several Lactococcus phages. To our knowledge, Φ1-A4 is the first genetically characterized L. mesenteroides phage. PMID:20118355

Design and screening of M13 phage display cDNA libraries.

PubMed

Georgieva, Yuliya; Konthur, Zoltán

2011-02-17

The last decade has seen a steady increase in screening of cDNA expression product libraries displayed on the surface of filamentous bacteriophage. At the same time, the range of applications extended from the identification of novel allergens over disease markers to protein-protein interaction studies. However, the generation and selection of cDNA phage display libraries is subjected to intrinsic biological limitations due to their complex nature and heterogeneity, as well as technical difficulties regarding protein presentation on the phage surface. Here, we review the latest developments in this field, discuss a number of strategies and improvements anticipated to overcome these challenges making cDNA and open reading frame (ORF) libraries more readily accessible for phage display. Furthermore, future trends combining phage display with next generation sequencing (NGS) will be presented.

Romulus and Remus, two phage isolates representing a distinct clade within the Twortlikevirus genus, display suitable properties for phage therapy applications.

PubMed

Vandersteegen, Katrien; Kropinski, Andrew M; Nash, John H E; Noben, Jean-Paul; Hermans, Katleen; Lavigne, Rob

2013-03-01

The renewed interest in controlling Staphylococcus aureus infections using their natural enemies, bacteriophages, has led to the isolation of a limited number of virulent phages so far. These phages are all members of the Twortlikevirus, displaying little variance. We present two novel closely related (95.9% DNA homology) lytic myoviruses, Romulus and Remus, with double-stranded DNA (dsDNA) genomes of 131,333 bp and 134,643 bp, respectively. Despite their relatedness to Staphylococcus phages K, G1, ISP, and Twort and Listeria phages A511 and P100, Romulus and Remus can be proposed as isolates of a new species within the Twortlikevirus genus. A distinguishing feature for these phage genomes is the unique distribution of group I introns compared to that in other staphylococcal myoviruses. In addition, a hedgehog/intein domain was found within their DNA polymerase genes, and an insertion sequence-encoded transposase exhibits splicing behavior and produces a functional portal protein. From a phage therapy application perspective, Romulus and Remus infected approximately 70% of the tested S. aureus isolates and displayed promising lytic activity against these isolates. Furthermore, both phages showed a rapid initial adsorption and demonstrated biofilm-degrading capacity in a proof-of-concept experiment.

ORF phage display to identify cellular proteins with different functions.

PubMed

Li, Wei

2012-09-01

Open reading frame (ORF) phage display is a new branch of phage display aimed at improving its efficiency to identify cellular proteins with specific binding or functional activities. Despite the success of phage display with antibody libraries and random peptide libraries, phage display with cDNA libraries of cellular proteins identifies a high percentage of non-ORF clones encoding unnatural short peptides with minimal biological implications. This is mainly because of the uncontrollable reading frames of cellular proteins in conventional cDNA libraries. ORF phage display solves this problem by eliminating non-ORF clones to generate ORF cDNA libraries. Here I summarize the procedures of ORF phage display, discuss the factors influencing its efficiency, present examples of its versatile applications, and highlight evidence of its capability of identifying biologically relevant cellular proteins. ORF phage display coupled with different selection strategies is capable of delineating diverse functions of cellular proteins with unique advantages. Copyright © 2012 Elsevier Inc. All rights reserved.

Occurrence of small Hsd plasmids in Salmonella typhi, Shigella boydii, and Escherichia coli.

PubMed Central

Yoshida, Y; Mise, K

1986-01-01

The natural occurrence of small Hsd (host specificity for DNA) plasmids was demonstrated in restriction endonuclease-producing strains of Salmonella typhi, Shigella boydii, and Escherichia coli. The five Hsd plasmids isolated were between 5.0 and 12.2 kilobases long. The copy number of all the Hsd plasmids was high (more than 10 copies per cell). Introduction of these small plasmids into E. coli strain 0 drastically lowered the efficiency of plating of the lambda.0 phages (the efficiency of plating was less than 5 X 10(-5) PFU-1). High restriction endonuclease activities were detected in the Hsd plasmid-positive strains because of the elevated copy numbers of the hsdR+ gene. The advantages of using E. coli strains containing the small Hsd plasmids for purification of type II restriction endonucleases are discussed. Images PMID:3003023

Genomic Diversity of Phages Infecting Probiotic Strains of Lactobacillus paracasei

PubMed Central

Rousseau, Geneviève M.; Capra, María L.; Quiberoni, Andrea; Tremblay, Denise M.; Labrie, Simon J.

2015-01-01

Strains of the Lactobacillus casei group have been extensively studied because some are used as probiotics in foods. Conversely, their phages have received much less attention. We analyzed the complete genome sequences of five L. paracasei temperate phages: CL1, CL2, iLp84, iLp1308, and iA2. Only phage iA2 could not replicate in an indicator strain. The genome lengths ranged from 34,155 bp (iA2) to 39,474 bp (CL1). Phages iA2 and iLp1308 (34,176 bp) possess the smallest genomes reported, thus far, for phages of the L. casei group. The GC contents of the five phage genomes ranged from 44.8 to 45.6%. As observed with many other phages, their genomes were organized as follows: genes coding for DNA packaging, morphogenesis, lysis, lysogeny, and replication. Phages CL1, CL2, and iLp1308 are highly related to each other. Phage iLp84 was also related to these three phages, but the similarities were limited to gene products involved in DNA packaging and structural proteins. Genomic fragments of phages CL1, CL2, iLp1308, and iLp84 were found in several genomes of L. casei strains. Prophage iA2 is unrelated to these four phages, but almost all of its genome was found in at least four L. casei strains. Overall, these phages are distinct from previously characterized Lactobacillus phages. Our results highlight the diversity of L. casei phages and indicate frequent DNA exchanges between phages and their hosts. PMID:26475105

Capstan Friction Model for DNA Ejection from Bacteriophages

NASA Astrophysics Data System (ADS)

Ghosal, Sandip

2012-12-01

Bacteriophages infect cells by attaching to the outer membrane and injecting their DNA into the cell. The phage DNA is then transcribed by the cell’s transcription machinery. A number of physical mechanisms by which DNA can be translocated from the phage capsid into the cell have been identified. A fast ejection driven by the elastic and electrostatic potential energy of the compacted DNA within the viral capsid appears to be used by most phages, at least to initiate infection. In recent in vitro experiments, the speed of DNA translocation from a λ phage capsid has been measured as a function of ejected length over the entire duration of the event. Here, a mechanical model is proposed that is able to explain the observed dependence of exit velocity on ejected length, and that is also consistent with the accepted picture of the geometric arrangement of DNA within the viral capsid.

Organization and sequence of four flagellin-encoding genes of Edwardsiella icataluri

USDA-ARS?s Scientific Manuscript database

Edwardsiella ictaluri, the cause of enteric septicemia in channel catfish (Ictalurus punctatus), is motile by means of peritrichous flagella. We determined the complete flagellin gene sequences and their organization in E. ictaluri by sequencing genomic segments selected from a lambda-ZAP phage gen...

Discovery of cyanophage genomes which contain mitochondrial DNA polymerase.

PubMed

Chan, Yi-Wah; Mohr, Remus; Millard, Andrew D; Holmes, Antony B; Larkum, Anthony W; Whitworth, Anna L; Mann, Nicholas H; Scanlan, David J; Hess, Wolfgang R; Clokie, Martha R J

2011-08-01

DNA polymerase γ is a family A DNA polymerase responsible for the replication of mitochondrial DNA in eukaryotes. The origins of DNA polymerase γ have remained elusive because it is not present in any known bacterium, though it has been hypothesized that mitochondria may have inherited the enzyme by phage-mediated nonorthologous displacement. Here, we present an analysis of two full-length homologues of this gene, which were found in the genomes of two bacteriophages, which infect the chlorophyll-d containing cyanobacterium Acaryochloris marina. Phylogenetic analyses of these phage DNA polymerase γ proteins show that they branch deeply within the DNA polymerase γ clade and therefore share a common origin with their eukaryotic homologues. We also found homologues of these phage polymerases in the environmental Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis (CAMERA) database, which fell in the same clade. An analysis of the CAMERA assemblies containing the environmental homologues together with the filter fraction metadata indicated some of these assemblies may be of bacterial origin. We also show that the phage-encoded DNA polymerase γ is highly transcribed as the phage genomes are replicated. These findings provide data that may assist in reconstructing the evolution of mitochondria.

Molecular specialization of breast vasculature: A breast-homing phage-displayed peptide binds to aminopeptidase P in breast vasculature

NASA Astrophysics Data System (ADS)

Essler, Markus; Ruoslahti, Erkki

2002-02-01

In vivo phage display identifies peptides that selectively home to the vasculature of individual organs, tissues, and tumors. Here we report the identification of a cyclic nonapeptide, CPGPEGAGC, which homes to normal breast tissue with a 100-fold selectivity over nontargeted phage. The homing of the phage is inhibited by its cognate synthetic peptide. Phage localization in tissue sections showed that the breast-homing phage binds to the blood vessels in the breast, but not in other tissues. The phage also bound to the vasculature of hyperplastic and malignant lesions in transgenic breast cancer mice. Expression cloning with a phage-displayed cDNA library yielded a phage that specifically bound to the breast-homing peptide. The cDNA insert was homologous to a fragment of aminopeptidase P. The homing peptide bound aminopeptidase P from malignant breast tissue in affinity chromatography. Antibodies against aminopeptidase P inhibited the in vitro binding of the phage-displayed cDNA to the peptide and the in vivo homing of phage carrying the peptide. These results indicate that aminopeptidase P is the receptor for the breast-homing peptide. This peptide may be useful in designing drugs for the prevention and treatment of breast cancer.

DNA fusion product of phage P2 with plasmid pBR322 - A new phasmid

NASA Technical Reports Server (NTRS)

Nicoletti, M.; Bertani, G.

1983-01-01

The chromosome of the temperate bacteriophage P2 and that of the plasmid pBR322 have been joined in vitro after treatment with restriction endonuclease EcoRI. The fusion product - a phasmid - can behave as a plasmid, as a phage and as a prophage. It can replicate its DNA under the control of either the specific replication mechanism of the parent phage in a polA mutant or that of the parent plasmid in a rep mutant. Several interesting interactions between the two replication modes are indicated. In particular, phage particles may be produced even when the phage mode of DNA replication is blocked, and this throws new light on the involvement of the early gene A in the regulation of late gene expression in phage P2.

The interaction of E. coli integration host factor and lambda cos DNA: multiple complex formation and protein-induced bending.

PubMed Central

Kosturko, L D; Daub, E; Murialdo, H

1989-01-01

The interaction of E. coli's integration Host Factor (IHF) with fragments of lambda DNA containing the cos site has been studied by gel-mobility retardation and electron microscopy. The cos fragment used in the mobility assays is 398 bp and spans a region from 48,298 to 194 on the lambda chromosome. Several different complexes of IHF with this fragment can be distinguished by their differential mobility on polyacrylamide gels. Relative band intensities indicate that the formation of a complex between IHF and this DNA fragment has an equilibrium binding constant of the same magnitude as DNA fragments containing lambda's attP site. Gel-mobility retardation and electron microscopy have been employed to show that IHF sharply bends DNA near cos and to map the bending site. The protein-induced bend is near an intrinsic bend due to DNA sequence. The position of the bend suggests that IHF's role in lambda DNA packaging may be the enhancement of terminase binding/cos cutting by manipulating DNA structure. Images PMID:2521383

Cylinders vs. Spheres: Biofluid Shear Thinning in Driven Nanoparticle Transport

PubMed Central

Cribb, Jeremy A.; Meehan, Timothy D.; Shah, Sheel M.; Skinner, Kwan; Superfine, Richard

2011-01-01

Increasingly, the research community applies magnetophoresis to micro and nanoscale particles for drug delivery applications and the nanoscale rheological characterization of complex biological materials. Of particular interest is the design and transport of these magnetic particles through entangled polymeric fluids commonly found in biological systems. We report the magnetophoretic transport of spherical and rod-shaped particles through viscoelastic, entangled solutions using lambda-phage DNA (λ-DNA) as a model system. In order to understand and predict the observed phenomena, we fully characterize three fundamental components: the magnetic field and field gradient, the shape and magnetic properties of the probe particles, and the macroscopic rheology of the solution. Particle velocities obtained in Newtonian solutions correspond to macroscale rheology, with forces calculated via Stokes Law. In λ-DNA solutions, nanorod velocities are 100 times larger than predicted by measured zero-shear viscosity. These results are consistent with particles experiencing transport through a shear thinning fluid, indicating magnetically driven transport in shear thinning may be especially effective and favor narrow diameter, high aspect ratio particles. A complete framework for designing single-particle magnetic-based delivery systems results when we combine a quantified magnetic system with qualified particles embedded in a characterized viscoelastic medium. PMID:20571853

Effects of Relative Humidity and Spraying Medium on Ultraviolet (UV) Decontamination of Filters Loaded with Viral Aerosols

DTIC Science & Technology

2012-02-01

K (m2/J) at: Reference or source 68% RH 75% RH Adenovirus dsDNA 0.039 0.068 31 Vaccinia virus dsDNA 6.01 1.42 17 Phage T7 dsDNA 0.33 0.22 27 Phage ...viruses, need of only biosafety level 1 (BSL-1) containment, and ease of preparation and assay (2, 21). Nonen- veloped MS2 phage was used for the worst...Iodine disinfection of a model bacterio- phage , MS2, demonstrating apparent rebound. Water Res. 33:169 –179. 3. Burgener J. 2006. Position paper on

Pseudomonas aeruginosa phage PaP1 DNA polymerase is an A-family DNA polymerase demonstrating ssDNA and dsDNA 3'-5' exonuclease activity.

PubMed

Liu, Binyan; Gu, Shiling; Liang, Nengsong; Xiong, Mei; Xue, Qizhen; Lu, Shuguang; Hu, Fuquan; Zhang, Huidong

2016-08-01

Most phages contain DNA polymerases, which are essential for DNA replication and propagation in infected host bacteria. However, our knowledge on phage-encoded DNA polymerases remains limited. This study investigated the function of a novel DNA polymerase of PaP1, which is the lytic phage of Pseudomonas aeruginosa. PaP1 encodes its sole DNA polymerase called Gp90 that was predicted as an A-family DNA polymerase with polymerase and 3'-5' exonuclease activities. The sequence of Gp90 is homologous but not identical to that of other A-family DNA polymerases, such as T7 DNA polymerases (Pol) and DNA Pol I. The purified Gp90 demonstrated a polymerase activity. The processivity of Gp90 in DNA replication and its efficiency in single-dNTP incorporation are similar to those of T7 Pol with processive thioredoxin (T7 Pol/trx). Gp90 can degrade ssDNA and dsDNA in 3'-5' direction at a similar rate, which is considerably lower than that of T7 Pol/trx. The optimized conditions for polymerization were a temperature of 37 °C and a buffer consisting of 40 mM Tris-HCl (pH 8.0), 30 mM MgCl2, and 200 mM NaCl. These studies on DNA polymerase encoded by PaP1 help advance our knowledge on phage-encoded DNA polymerases and elucidate PaP1 propagation in infected P. aeruginosa.

Location of the unique integration site on an Escherichia coli chromosome by bacteriophage lambda DNA in vivo.

PubMed

Tal, Asaf; Arbel-Goren, Rinat; Costantino, Nina; Court, Donald L; Stavans, Joel

2014-05-20

The search for specific sequences on long genomes is a key process in many biological contexts. How can specific target sequences be located with high efficiency, within physiologically relevant times? We addressed this question for viral integration, a fundamental mechanism of horizontal gene transfer driving prokaryotic evolution, using the infection of Escherichia coli bacteria with bacteriophage λ and following the establishment of a lysogenic state. Following the targeting process in individual live E. coli cells in real time revealed that λ DNA remains confined near the entry point of a cell following infection. The encounter between the 15-bp-long target sequence on the chromosome and the recombination site on the viral genome is facilitated by the directed motion of bacterial DNA generated during chromosome replication, in conjunction with constrained diffusion of phage DNA. Moving the native bacterial integration site to different locations on the genome and measuring the integration frequency in these strains reveals that the frequencies of the native site and a site symmetric to it relative to the origin are similar, whereas both are significantly higher than when the integration site is moved near the terminus, consistent with the replication-driven mechanism we propose. This novel search mechanism is yet another example of the exquisite coevolution of λ with its host.

Burkholderia mallei Cluster 1 Type VI Secretion Mutants Exhibit Growth and Actin Polymerization Defects in RAW 264.7 Murine Macrophages

DTIC Science & Technology

2010-01-01

mallei virAG; Kmr 45 pBHR4-GFP Broad-host-range vector containing gfp from pQBI T7 GFP (Quantum Biotech); Gmr 45 pBHR1-TG pBHR1 containing gfp...Bonanno, J. M. Sauder, S. Pukatzki, S. K. Burley, S. C. Almo, and J. J. Mekalanos. 2009. Type VI secretion apparatus and phage tail-associated protein...14251. 37. Pell, L. G., V. Kanelis, L. W. Donaldson, P. L. Howell, and A. R. Davidson. 2009. The phage lambda major tail protein structure reveals a

Energetics of genome ejection from phage revealed by isothermal titration calorimetry

NASA Astrophysics Data System (ADS)

Jeembaeva, Meerim; Jonsson, Bengt; Castelnovo, Martin; Evilevitch, Alex

2009-03-01

It has been experimentally shown that ejection of double-stranded DNA from phage is driven by internal pressure reaching tens of atmospheres. This internal pressure is partially responsible for delivery of DNA into the host cell. While several theoretical models and simulations nicely describe the experimental data of internal forces either resisting active packaging or equivalently favoring spontaneous ejection, there are no direct energy measurements available that would help to verify how quantitative these theories are. We performed direct measurements of the enthalpy responsible for DNA ejection from phage λ, using Isothermal Titration Calorimetry. The phage capsids were ``opened'' in vitro by titrating λ into a solution with LamB receptor and the enthalpy of DNA ejection process was measured. In his way, enthalpy stored in λ was determined as a function of packaged DNA length comparing wild-type phage λ (48.5 kb) with a shorter λ-DNA length mutant (37.7 kb). The temperature dependence of the ejection enthalpy was also investigated. The values obtained were in good agreement with existing models and provide a better understanding of ds- DNA packaging and release mechanisms in motor-packaged viruses (e.g., tailed bacteriophages, Herpes Simplex, and adenoviruses).

A novel helper phage for HaloTag-mediated co-display of enzyme and substrate on phage.

PubMed

Delespaul, Wouter; Peeters, Yves; Herdewijn, Piet; Robben, Johan

2015-05-01

Phage display is an established technique for the molecular evolution of peptides and proteins. For the selection of enzymes based on catalytic activity however, simultaneous coupling of an enzyme and its substrate to the phage surface is required. To facilitate this process of co-display, we developed a new helper phage displaying HaloTag, a modified haloalkane dehalogenase that binds specifically and covalently to functionalized haloalkane ligands. The display of functional HaloTag was demonstrated by capture on streptavidin-coated magnetic beads, after coupling a biotinylated haloalkane ligand, or after on-phage extension of a DNA oligonucleotide primer with a biotinylated nucleotide by phi29 DNA polymerase. We also achieved co-display of HaloTag and phi29 DNA polymerase, thereby opening perspectives for the molecular evolution of this enzyme (and others) towards new substrate specificities. Copyright © 2015 Elsevier Inc. All rights reserved.

Role of C-terminal residues in oligomerization and stability of lambda CII: implications for lysis-lysogeny decision of the phage.

PubMed

Datta, Ajit Bikram; Roy, Siddhartha; Parrack, Pradeep

2005-01-14

A crucial element in the lysis-lysogeny decision of the temperate coliphage lambda is the phage protein CII, which has several interesting properties. It promotes lysogeny through activation of three phage promoters p(E), p(I) and p(aQ), recognizing a direct repeat sequence TTGCN6TTGC at each. The three-dimensional structure of CII, a homo-tetramer of 97 residue subunits, is unknown. It is an unstable protein in vivo, being rapidly degraded by the host protease HflB (FtsH). This instability is essential for the function of CII in the lysis-lysogeny switch. From NMR and limited proteolysis we show that about 15 C-terminal residues of CII are highly flexible, and may act as a target for proteolysis in vivo. From in vitro transcription, isothermal calorimetry and gel chromatography of CII (1-97) and its truncated fragments CIIA (4-81/82) and CIIB (4-69), we find that residues 70-81/82 are essential for (a) tetramer formation, (b) operator binding and (c) transcription activation. Presumably, tetramerization is necessary for the latter functions. Based on these results, we propose a model for CII structure, in which protein-protein contacts for dimer and tetramer formation are different. The implications of tetrameric organization, essential for CII activity, on the recognition of the direct repeat sequence is discussed.

Comparative genomics of 9 novel Paenibacillus larvae bacteriophages

PubMed Central

Stamereilers, Casey; LeBlanc, Lucy; Yost, Diane; Amy, Penny S.; Tsourkas, Philippos K.

2016-01-01

ABSTRACT American Foulbrood Disease, caused by the bacterium Paenibacillus larvae, is one of the most destructive diseases of the honeybee, Apis mellifera. Our group recently published the sequences of 9 new phages with the ability to infect and lyse P. larvae. Here, we characterize the genomes of these P. larvae phages, compare them to each other and to other sequenced P. larvae phages, and putatively identify protein function. The phage genomes are 38–45 kb in size and contain 68–86 genes, most of which appear to be unique to P. larvae phages. We classify P. larvae phages into 2 main clusters and one singleton based on nucleotide sequence identity. Three of the new phages show sequence similarity to other sequenced P. larvae phages, while the remaining 6 do not. We identified functions for roughly half of the P. larvae phage proteins, including structural, assembly, host lysis, DNA replication/metabolism, regulatory, and host-related functions. Structural and assembly proteins are highly conserved among our phages and are located at the start of the genome. DNA replication/metabolism, regulatory, and host-related proteins are located in the middle and end of the genome, and are not conserved, with many of these genes found in some of our phages but not others. All nine phages code for a conserved N-acetylmuramoyl-L-alanine amidase. Comparative analysis showed the phages use the “cohesive ends with 3′ overhang” DNA packaging strategy. This work is the first in-depth study of P. larvae phage genomics, and serves as a marker for future work in this area. PMID:27738559

Regulation of infection efficiency in a globally abundant marine Bacteriodetes virus

DOE PAGES

Howard-Varona, Cristina; Roux, Simon; Dore, Hugo; ...

2016-05-17

Microbes impact human health and disease, industrial processes and natural ecosystems, but do so under the influence of viruses. Problematically, knowledge of viral infection efficiencies and outcomes (e.g. lysis, lysogeny) derives from few model systems that over-represent efficient, lytic infections and under-represent virus-host natural diversity. Here we sought to understand how infection efficiency is regulated in an environmental Bacteroidetes virus that represents a globally abundant viral group and has drastically different infection efficiencies when infecting two nearly identical bacterial strains. To this end, we quantified bacterial virus (phage) and host DNA, transcripts and phage particles throughout the infection of bothmore » bacterial hosts. While the phage transcriptome was similar during both infections, host transcriptional differences appeared to have altered infection efficiency. Specifically, host transcriptomes suggested that the phage failed to repress early host expression in the inefficient nfection, thereby allowing the host to respond against infection by delaying phage DNA replication and protein translation. Further measurements showed that phage DNA and particle production were delayed (by >30 minutes) and reduced (by >50%) in the inefficient versus efficient infection as the host over-expressed DNA degradation genes and under-expressed translation genes, respectively. Together these results suggest that multiple levels of regulation can impact infection efficiencies as failure to repress host transcription allowed the host to defend against both phage DNA and protein production. Given that this phage type is ubiquitous and abundant in the global oceans and that variably efficient viral infections are likely common in any ecosystem with varying phage-host abundances and physiological states, these data provide a critically needed foundation for understanding and modeling viral infection efficiency in nature.« less

Regulation of infection efficiency in a globally abundant marine Bacteriodetes virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Howard-Varona, Cristina; Roux, Simon; Dore, Hugo

Microbes impact human health and disease, industrial processes and natural ecosystems, but do so under the influence of viruses. Problematically, knowledge of viral infection efficiencies and outcomes (e.g. lysis, lysogeny) derives from few model systems that over-represent efficient, lytic infections and under-represent virus-host natural diversity. Here we sought to understand how infection efficiency is regulated in an environmental Bacteroidetes virus that represents a globally abundant viral group and has drastically different infection efficiencies when infecting two nearly identical bacterial strains. To this end, we quantified bacterial virus (phage) and host DNA, transcripts and phage particles throughout the infection of bothmore » bacterial hosts. While the phage transcriptome was similar during both infections, host transcriptional differences appeared to have altered infection efficiency. Specifically, host transcriptomes suggested that the phage failed to repress early host expression in the inefficient nfection, thereby allowing the host to respond against infection by delaying phage DNA replication and protein translation. Further measurements showed that phage DNA and particle production were delayed (by >30 minutes) and reduced (by >50%) in the inefficient versus efficient infection as the host over-expressed DNA degradation genes and under-expressed translation genes, respectively. Together these results suggest that multiple levels of regulation can impact infection efficiencies as failure to repress host transcription allowed the host to defend against both phage DNA and protein production. Given that this phage type is ubiquitous and abundant in the global oceans and that variably efficient viral infections are likely common in any ecosystem with varying phage-host abundances and physiological states, these data provide a critically needed foundation for understanding and modeling viral infection efficiency in nature.« less

Acinetobacter phage genome is similar to Sphinx 2.36, the circular DNA copurified with TSE infected particles.

PubMed

Longkumer, Toshisangba; Kamireddy, Swetha; Muthyala, Venkateswar Reddy; Akbarpasha, Shaikh; Pitchika, Gopi Krishna; Kodetham, Gopinath; Ayaluru, Murali; Siddavattam, Dayananda

2013-01-01

While analyzing plasmids of Acinetobacter sp. DS002 we have detected a circular DNA molecule pTS236, which upon further investigation is identified as the genome of a phage. The phage genome has shown sequence similarity to the recently discovered Sphinx 2.36 DNA sequence co-purified with the Transmissible Spongiform Encephalopathy (TSE) particles isolated from infected brain samples collected from diverse geographical regions. As in Sphinx 2.36, the phage genome also codes for three proteins. One of them codes for RepA and is shown to be involved in replication of pTS236 through rolling circle (RC) mode. The other two translationally coupled ORFs, orf106 and orf96, code for coat proteins of the phage. Although an orf96 homologue was not previously reported in Sphinx 2.36, a closer examination of DNA sequence of Sphinx 2.36 revealed its presence downstream of orf106 homologue. TEM images and infection assays revealed existence of phage AbDs1 in Acinetobacter sp. DS002.

Acinetobacter phage genome is similar to Sphinx 2.36, the circular DNA copurified with TSE infected particles

PubMed Central

Longkumer, Toshisangba; Kamireddy, Swetha; Muthyala, Venkateswar Reddy; Akbarpasha, Shaikh; Pitchika, Gopi Krishna; Kodetham, Gopinath; Ayaluru, Murali; Siddavattam, Dayananda

2013-01-01

While analyzing plasmids of Acinetobacter sp. DS002 we have detected a circular DNA molecule pTS236, which upon further investigation is identified as the genome of a phage. The phage genome has shown sequence similarity to the recently discovered Sphinx 2.36 DNA sequence co-purified with the Transmissible Spongiform Encephalopathy (TSE) particles isolated from infected brain samples collected from diverse geographical regions. As in Sphinx 2.36, the phage genome also codes for three proteins. One of them codes for RepA and is shown to be involved in replication of pTS236 through rolling circle (RC) mode. The other two translationally coupled ORFs, orf106 and orf96, code for coat proteins of the phage. Although an orf96 homologue was not previously reported in Sphinx 2.36, a closer examination of DNA sequence of Sphinx 2.36 revealed its presence downstream of orf106 homologue. TEM images and infection assays revealed existence of phage AbDs1 in Acinetobacter sp. DS002. PMID:23867905

Sequence and structural characterization of great salt lake bacteriophage CW02, a member of the T7-like supergroup.

PubMed

Shen, Peter S; Domek, Matthew J; Sanz-García, Eduardo; Makaju, Aman; Taylor, Ryan M; Hoggan, Ryan; Culumber, Michele D; Oberg, Craig J; Breakwell, Donald P; Prince, John T; Belnap, David M

2012-08-01

Halophage CW02 infects a Salinivibrio costicola-like bacterium, SA50, isolated from the Great Salt Lake. Following isolation, cultivation, and purification, CW02 was characterized by DNA sequencing, mass spectrometry, and electron microscopy. A conserved module of structural genes places CW02 in the T7 supergroup, members of which are found in diverse aquatic environments, including marine and freshwater ecosystems. CW02 has morphological similarities to viruses of the Podoviridae family. The structure of CW02, solved by cryogenic electron microscopy and three-dimensional reconstruction, enabled the fitting of a portion of the bacteriophage HK97 capsid protein into CW02 capsid density, thereby providing additional evidence that capsid proteins of tailed double-stranded DNA phages have a conserved fold. The CW02 capsid consists of bacteriophage lambda gpD-like densities that likely contribute to particle stability. Turret-like densities were found on icosahedral vertices and may represent a unique adaptation similar to what has been seen in other extremophilic viruses that infect archaea, such as Sulfolobus turreted icosahedral virus and halophage SH1.

A comparison of the survival of F+RNA and F+DNA coliphages in lake water microcosms.

PubMed

Long, Sharon C; Sobsey, Mark D

2004-03-01

The survival of seven F+RNA phages (MS2 Group I ATCC type strain, two Group I environmental isolates, a Group II environmental isolate, a Group III environmental isolate, and two Group IV environmental isolates) and six F+DNA phages (M13, fd, f1, and ZJ/2 ATCC type strains, and two environmental isolates) were examined in microcosms using a surface drinking water source. Phages were spiked into replicate aliquots of a source water at about 20,000 pfu/ml. Replicate spikes were incubated at 4 and 20 degrees C and monitored for 110 days. At 4 degrees C, Groups I and II F+ RNA phages were detectable through 110 days, with reductions of about 1 and 3 log10, respectively. The Group III F+RNA phage demonstrated 5 log10 reduction after 3 weeks, and the Group IV F+RNA phages were reduced to detection limits (5 log10 reduction) within 10 days. Of the F+DNA phages, all four type strains were detectable with about 2.5 log10 reduction after 110 days at 4 degrees C. The F+DNA environmental isolates were detectable with about a 4 log10 reduction after 110 days at 4 degrees C. All phages demonstrated faster decay at 20 degrees C. These results suggest that differences in F+ phage survival may influence their prevalence in environmental waters and the ability to attribute their prevalence to specific human and animal sources of faecal contamination.

Comparison of five bacteriophages as models for viral aerosol studies.

PubMed

Turgeon, Nathalie; Toulouse, Marie-Josée; Martel, Bruno; Moineau, Sylvain; Duchaine, Caroline

2014-07-01

Bacteriophages are perceived to be good models for the study of airborne viruses because they are safe to use, some of them display structural features similar to those of human and animal viruses, and they are relatively easy to produce in large quantities. Yet, only a few studies have investigated them as models. It has previously been demonstrated that aerosolization, environmental conditions, and sampling conditions affect viral infectivity, but viral infectivity is virus dependent. Thus, several virus models are likely needed to study their general behavior in aerosols. The aim of this study was to compare the effects of aerosolization and sampling on the infectivity of five tail-less bacteriophages and two pathogenic viruses: MS2 (a single-stranded RNA [ssRNA] phage of the Leviviridae family), Φ6 (a segmented double-stranded RNA [dsRNA] phage of the Cystoviridae family), ΦX174 (a single-stranded DNA [ssDNA] phage of the Microviridae family), PM2 (a double-stranded DNA [dsDNA] phage of the Corticoviridae family), PR772 (a dsDNA phage of the Tectiviridae family), human influenza A virus H1N1 (an ssRNA virus of the Orthomyxoviridae family), and the poultry virus Newcastle disease virus (NDV; an ssRNA virus of the Paramyxoviridae family). Three nebulizers and two nebulization salt buffers (with or without organic fluid) were tested, as were two aerosol sampling devices, a liquid cyclone (SKC BioSampler) and a dry cyclone (National Institute for Occupational Safety and Health two-stage cyclone bioaerosol sampler). The presence of viruses in collected air samples was detected by culture and quantitative PCR (qPCR). Our results showed that these selected five phages behave differently when aerosolized and sampled. RNA phage MS2 and ssDNA phage ΦX174 were the most resistant to aerosolization and sampling. The presence of organic fluid in the nebulization buffer protected phages PR772 and Φ6 throughout the aerosolization and sampling with dry cyclones. In this experimental setup, the behavior of the influenza virus resembled that of phages PR772 and Φ6, while the behavior of NDV was closer to that of phages MS2 and ΦX174. These results provide critical information for the selection of appropriate phage models to mimic the behavior of specific human and animal viruses in aerosols. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Comparison of Five Bacteriophages as Models for Viral Aerosol Studies

PubMed Central

Turgeon, Nathalie; Toulouse, Marie-Josée; Martel, Bruno; Moineau, Sylvain

2014-01-01

Bacteriophages are perceived to be good models for the study of airborne viruses because they are safe to use, some of them display structural features similar to those of human and animal viruses, and they are relatively easy to produce in large quantities. Yet, only a few studies have investigated them as models. It has previously been demonstrated that aerosolization, environmental conditions, and sampling conditions affect viral infectivity, but viral infectivity is virus dependent. Thus, several virus models are likely needed to study their general behavior in aerosols. The aim of this study was to compare the effects of aerosolization and sampling on the infectivity of five tail-less bacteriophages and two pathogenic viruses: MS2 (a single-stranded RNA [ssRNA] phage of the Leviviridae family), Φ6 (a segmented double-stranded RNA [dsRNA] phage of the Cystoviridae family), ΦX174 (a single-stranded DNA [ssDNA] phage of the Microviridae family), PM2 (a double-stranded DNA [dsDNA] phage of the Corticoviridae family), PR772 (a dsDNA phage of the Tectiviridae family), human influenza A virus H1N1 (an ssRNA virus of the Orthomyxoviridae family), and the poultry virus Newcastle disease virus (NDV; an ssRNA virus of the Paramyxoviridae family). Three nebulizers and two nebulization salt buffers (with or without organic fluid) were tested, as were two aerosol sampling devices, a liquid cyclone (SKC BioSampler) and a dry cyclone (National Institute for Occupational Safety and Health two-stage cyclone bioaerosol sampler). The presence of viruses in collected air samples was detected by culture and quantitative PCR (qPCR). Our results showed that these selected five phages behave differently when aerosolized and sampled. RNA phage MS2 and ssDNA phage ΦX174 were the most resistant to aerosolization and sampling. The presence of organic fluid in the nebulization buffer protected phages PR772 and Φ6 throughout the aerosolization and sampling with dry cyclones. In this experimental setup, the behavior of the influenza virus resembled that of phages PR772 and Φ6, while the behavior of NDV was closer to that of phages MS2 and ΦX174. These results provide critical information for the selection of appropriate phage models to mimic the behavior of specific human and animal viruses in aerosols. PMID:24795379

Peginterferon lambda for the treatment of HBeAg-positive chronic hepatitis B: A randomized phase 2b study (LIRA-B).

PubMed

Chan, Henry L Y; Ahn, Sang Hoon; Chang, Ting-Tsung; Peng, Cheng-Yuan; Wong, David; Coffin, Carla S; Lim, Seng Gee; Chen, Pei-Jer; Janssen, Harry L A; Marcellin, Patrick; Serfaty, Lawrence; Zeuzem, Stefan; Cohen, David; Critelli, Linda; Xu, Dong; Wind-Rotolo, Megan; Cooney, Elizabeth

2016-05-01

Peginterferon lambda-1a (lambda) is a Type-III interferon, which, like alfa interferons, has antiviral activity in vitro against hepatitis B virus (HBV) and hepatitis C virus (HCV); however, lambda has a more limited extra-hepatic receptor distribution. This phase 2b study (LIRA-B) evaluated lambda in patients with chronic HBV infection. Adult HBeAg+ interferon-naive patients were randomized (1:1) to weekly lambda (180 μg) or peginterferon alfa-2a (alfa) for 48 weeks. The primary efficacy endpoint was HBeAg seroconversion at week 24 post-treatment; lambda non-inferiority was demonstrated if the 80% confidence interval (80% CI) lower bound was >-15%. Baseline characteristics were balanced across groups (lambda N=80; alfa N=83). Early on-treatment declines in HBV-DNA and qHBsAg through week 24 were greater with lambda. HBeAg seroconversion rates were comparable for lambda and alfa at week 48 (17.5% vs. 16.9%, respectively); however lambda non-inferiority was not met at week 24 post-treatment (13.8% vs. 30.1%, respectively; lambda vs. alfa 80% CI lower bound -24%). Results for other key secondary endpoints (virologic, serologic, biochemical) and post hoc combined endpoints (HBV-DNA <2000 IU/ml plus HBeAg seroconversion or ALT normalization) mostly favored alfa. Overall adverse events (AE), serious AE, and AE-discontinuation rates were comparable between arms but AE-spectra differed (more cytopenias, flu-like, and musculoskeletal symptoms observed with alfa, more ALT flares and bilirubin elevations seen with lambda). Most on-treatment flares occurred early (weeks 4-12), associated with HBV-DNA decline; all post-treatment flares were preceded by HBV-DNA rise. On-treatment, lambda showed greater early effects on HBV-DNA and qHBsAg, and comparable serologic/virologic responses at end-of-treatment. However, post-treatment, alfa-associated HBeAg seroconversion rates were higher, and key secondary results mostly favored alfa. ClinicalTrials.gov number: NCT01204762. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Physiological Properties and Genome Structure of the Hyperthermophilic Filamentous Phage φOH3 Which Infects Thermus thermophilus HB8.

PubMed

Nagayoshi, Yuko; Kumagae, Kenta; Mori, Kazuki; Tashiro, Kosuke; Nakamura, Ayano; Fujino, Yasuhiro; Hiromasa, Yasuaki; Iwamoto, Takeo; Kuhara, Satoru; Ohshima, Toshihisa; Doi, Katsumi

2016-01-01

A filamentous bacteriophage, φOH3, was isolated from hot spring sediment in Obama hot spring in Japan with the hyperthermophilic bacterium Thermus thermophilus HB8 as its host. Phage φOH3, which was classified into the Inoviridae family, consists of a flexible filamentous particle 830 nm long and 8 nm wide. φOH3 was stable at temperatures ranging from 70 to 90°C and at pHs ranging from 6 to 9. A one-step growth curve of the phage showed a 60-min latent period beginning immediately postinfection, followed by intracellular virus particle production during the subsequent 40 min. The released virion number of φOH3 was 109. During the latent period, both single stranded DNA (ssDNA) and the replicative form (RF) of phage DNA were multiplied from min 40 onward. During the release period, the copy numbers of both ssDNA and RF DNA increased sharply. The size of the φOH3 genome is 5688 bp, and eight putative open reading frames (ORFs) were annotated. These ORFs were encoded on the plus strand of RF DNA and showed no significant homology with any known phage genes, except ORF 5, which showed 60% identity with the gene VIII product of the Thermus filamentous phage PH75. All the ORFs were similar to predicted genes annotated in the Thermus aquaticus Y51MC23 and Meiothermus timidus DSM 17022 genomes at the amino acid sequence level. This is the first report of the whole genome structure and DNA multiplication of a filamentous T. thermophilus phage within its host cell.

Assignment of the gene encoding the 5-HT{sub 1E} serotonin receptor (S31) (locus HTR1E) to human chromosome 6q14-q15

DOE Office of Scientific and Technical Information (OSTI.GOV)

Levy, F.O.; Tasken, K.; Solberg, R.

1994-08-01

The human gene for the 5-HT{sub 1E} serotonin receptor was recently cloned, but no chromosomal assignment has yet been given to this gene (locus HTR1E). In this work, we demonstrate by two independent polymerase chain reactions on a panel of human-hamster somatic cell hybrid genomic DNA that the 5-HT{sub 1E} serotonin receptor gene is localized on human chromosome 6. Furthermore, by means of in situ hybridization to human metaphase chromosomes, using the cloned 5-HT{sub 1E} receptor gene (phage clone {lambda}-S31) as a probe, we demonstrate that this gene is localized to the q14-q15 region on chromosome 6. Screening of genomicmore » DNA from 15 unrelated Caucasian individuals, using as a probe the open reading frame of the cloned 5-HT{sub 1E} receptor gene, did not reveal any restriction fragment length polymorphisms with the enzymes BamHI, BanII, BglII, EcoRI, HincII, HindIII, HinfI, MspI, PstI, and PvuII. Since the 5-HT{sub 1E} receptor is found mainly in the cerebral cortex and abnormal function of the serotonergic system has been implicated in a variety of neurologic and psychiatric diseases, the precise chromosomal assignment of the 5-HT{sub 1E} receptor gene is the crucial first step toward the evaluation of this locus as a candidate for mutations in such syndromes. 28 refs., 2 figs., 2 tabs.« less

Role of Escherichia coli dnaA gene and its integrative suppression in M13 Coliphage DNA synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mitra, S.; Stallions, D.R.

An F/sup +/ derivative of Escherichia coli E508 thermosensitive in dnaA function (involved in DNA synthesis initiation), its revertant and an Hfr derivative of E508(ts) in which the temperature-sensitive phenotype is suppressed by integrative suppression have been compared for their ability to support M13 phage DNA synthesis at the nonpermissive temperature. Upon infection at the nonpermissive temperature, both the revertant and the Hfr strain support normal phage replication while the temperature-sensitive mutant does not. However, when infection is carried out at a permissive temperature and the temperature is shifted up after infection, phage synthesis occurs in the temperature-sensitive mutant also,more » but in lesser quantity than in the revertant strain. Analysis of intracellular labeled phage DNA indicates: (a) parental replicative form DNA synthesis is not dependent on dnaA function; (b) progeny replicative form DNA synthesis is strongly inhibited in the temperature-sensitive dnaA mutant at the nonpermissive temperature; (c) progeny single-strand DNA synthesis does not absolutely require dnaA function; (d) progeny single-strand DNA is present in the circular form. The implication of the host DNA replication in M13 DNA synthesis is discussed.« less

The Small Viral Membrane-Associated Protein P32 Is Involved in Bacteriophage PRD1 DNA Entry

PubMed Central

Grahn, A. Marika; Daugelavičius, Rimantas; Bamford, Dennis H.

2002-01-01

The lipid-containing bacteriophage PRD1 infects a variety of gram-negative cells by injecting its linear double-stranded DNA genome into the host cell cytoplasm, while the protein capsid is left outside. The virus membrane and several structural proteins are involved in phage DNA entry. In this work we identified a new infectivity protein of PRD1. Disruption of gene XXXII resulted in a mutant phenotype defective in phage reproduction. The absence of the protein P32 did not compromise the particle assembly but led to a defect in phage DNA injection. In P32-deficient particles the phage membrane is unable to undergo a structural transformation from a spherical to a tubular form. Since P32− particles are able to increase the permeability of the host cell envelope to a degree comparable to that found with wild-type particles, we suggest that the tail-tube formation is needed to eject the DNA from the phage particle rather than to reach the host cell interior. PMID:11967303

Gene 1.7 of bacteriophage T7 confers sensitivity of phage growth to dideoxythymidine.

PubMed

Tran, Ngoc Q; Rezende, Lisa F; Qimron, Udi; Richardson, Charles C; Tabor, Stanley

2008-07-08

Bacteriophage T7 DNA polymerase efficiently incorporates dideoxynucleotides into DNA, resulting in chain termination. Dideoxythymidine (ddT) present in the medium at levels not toxic to Escherichia coli inhibits phage T7. We isolated 95 T7 phage mutants that were resistant to ddT. All contained a mutation in T7 gene 1.7, a nonessential gene of unknown function. When gene 1.7 was expressed from a plasmid, T7 phage resistant to ddT still arose; analysis of 36 of these mutants revealed that all had a single mutation in gene 5, which encodes T7 DNA polymerase. This mutation changes tyrosine-526 to phenylalanine, which is known to increase dramatically the ability of T7 DNA polymerase to discriminate against dideoxynucleotides. DNA synthesis in cells infected with wild-type T7 phage was inhibited by ddT, suggesting that it resulted in chain termination of DNA synthesis in the presence of gene 1.7 protein. Overexpression of gene 1.7 from a plasmid rendered E. coli cells sensitive to ddT, indicating that no other T7 proteins are required to confer sensitivity to ddT.

Natural selection underlies apparent stress-induced mutagenesis in a bacteriophage infection model.

PubMed

Yosef, Ido; Edgar, Rotem; Levy, Asaf; Amitai, Gil; Sorek, Rotem; Munitz, Ariel; Qimron, Udi

2016-04-18

The emergence of mutations following growth-limiting conditions underlies bacterial drug resistance, viral escape from the immune system and fundamental evolution-driven events. Intriguingly, whether mutations are induced by growth limitation conditions or are randomly generated during growth and then selected by growth limitation conditions remains an open question(1). Here, we show that bacteriophage T7 undergoes apparent stress-induced mutagenesis when selected for improved recognition of its host's receptor. In our unique experimental set-up, the growth limitation condition is physically and temporally separated from mutagenesis: growth limitation occurs while phage DNA is outside the host, and spontaneous mutations occur during phage DNA replication inside the host. We show that the selected beneficial mutations are not pre-existing and that the initial slow phage growth is enabled by the phage particle's low-efficiency DNA injection into the host. Thus, the phage particle allows phage populations to initially extend their host range without mutagenesis by virtue of residual recognition of the host receptor. Mutations appear during non-selective intracellular replication, and the frequency of mutant phages increases by natural selection acting on free phages, which are not capable of mutagenesis.

Specificity of diffusion channels produced by lambda phage receptor protein of Escherichia coli.

PubMed Central

Luckey, M; Nikaido, H

1980-01-01

The lamB protein, the receptor for phage lambda, was purified from the outer membrane of Escherichia coli K-12 by extraction with Triton X-100 and EDTA, chromatography on DEAE-Sephacel in Triton X-100, exchange of Triton for cholate by gel filtration, and chromatography on Sephacryl S-200 in cholate, NaCl, and EDTA. The purified protein appeared to exist as several oligomeric species. In an equilibrium retention assay with reconstituted vesicles containing phospholipids and lipopolysaccharide, the lamB protein conferred permeability for disaccharides. In a liposome swelling assay designed to measure rates of diffusion, the lamB protein conferred permeability to phospholipid liposomes for a variety of substrates. The rates obtained indicate the permeation facilitated by the lamB protein is specific, discriminating among substrates by both size and configuration. For example, maltose diffused into liposomes 40 times faster than sucrose, about 8 times faster than cellobiose, and about 12 times faster than maltoheptaose. The results suggest that the lamB protein forms a transmembrane channel containing a site (or sites) that loosely interacts with the solutes. Images PMID:6444720

Thermoresistant revertants of an Escherichia coli strain carrying tif-1 and ruv mutations: non-suppressibility of ruv by sfi.

PubMed Central

Otsuji, N; Iyehara-Ogawa, H

1979-01-01

Spontaneous thermoresistant revertants were isolated from Tif1 Ruv- and Tif1 Ruv+ strains of Escherichia coli K-12. They were divided into five groups; backmutants to tif+ and recA structural gene mutants accounted for at least two of these groups. Mutations with an unconditional RecA- phenyotype were detected at a higher frequency in the Tif1 Ruv- strains (65%) than in the Tif1 Ruv+ strains (25%). A third group consisted of revertants exhibiting a RecA- phenotype at low temperature. Revertants with normal recombination ability and UV resistance, but with a thermosensitive defect in propagating lambda bio11 phage, were also isolated (group 4). The alleles responsible for this property were cotransducible with the srl gene, suggesting that they are located at the recA locus. Other revertants, which might carry lex, LEXB, or zab mutations, were UV sensitive and were able to propagate lambda bio11 phage (group 5). The sfi mutation, which suppresses filamentation in the Tif1 and UV-sensitive Lon- strains, does not restore UV resistance of the Ruv- mutant. PMID:374356

Characterising the biology of novel lytic bacteriophages infecting multidrug resistant Klebsiella pneumoniae.

PubMed

Kęsik-Szeloch, Agata; Drulis-Kawa, Zuzanna; Weber-Dąbrowska, Beata; Kassner, Jerzy; Majkowska-Skrobek, Grażyna; Augustyniak, Daria; Lusiak-Szelachowska, Marzanna; Zaczek, Maciej; Górski, Andrzej; Kropinski, Andrew M

2013-03-28

Members of the genus Klebsiella are among the leading microbial pathogens associated with nosocomial infection. The increased incidence of antimicrobial resistance in these species has propelled the need for alternate/combination therapeutic regimens to aid clinical treatment. Bacteriophage therapy forms one of these alternate strategies. Electron microscopy, burst size, host range, sensitivity of phage particles to temperature, chloroform, pH, and restriction digestion of phage DNA were used to characterize Klebsiella phages. Of the 32 isolated phages eight belonged to the family Myoviridae, eight to the Siphoviridae whilst the remaining 16 belonged to the Podoviridae. The host range of these phages was characterised against 254 clinical Enterobacteriaceae strains including multidrug resistant Klebsiella isolates producing extended-spectrum beta-lactamases (ESBLs). Based on their lytic potential, six of the phages were further characterised for burst size, physicochemical properties and sensitivity to restriction endonuclease digestion. In addition, five were fully sequenced. Multiple phage-encoded host resistance mechanisms were identified. The Siphoviridae phage genomes (KP16 and KP36) contained low numbers of host restriction sites similar to the strategy found in T7-like phages (KP32). In addition, phage KP36 encoded its own DNA adenine methyltransferase. The φKMV-like KP34 phage was sensitive to all endonucleases used in this study. Dam methylation of KP34 DNA was detected although this was in the absence of an identifiable phage encoded methyltransferase. The Myoviridae phages KP15 and KP27 both carried Dam and Dcm methyltransferase genes and other anti-restriction mechanisms elucidated in previous studies. No other anti-restriction mechanisms were found, e.g. atypical nucleotides (hmC or glucosyl hmC), although Myoviridae phage KP27 encodes an unknown anti-restriction mechanism that needs further investigation.

Characterization of proviruses cloned from mink cell focus-forming virus-infected cellular DNA.

PubMed Central

Khan, A S; Repaske, R; Garon, C F; Chan, H W; Rowe, W P; Martin, M A

1982-01-01

Two proviruses were cloned from EcoRI-digested DNA extracted from mink cells chronically infected with AKR mink cell focus-forming (MCF) 247 murine leukemia virus (MuLV), using a lambda phage host vector system. One cloned MuLV DNA fragment (designated MCF 1) contained sequences extending 6.8 kilobases from an EcoRI restriction site in the 5' long terminal repeat (LTR) to an EcoRI site located in the envelope (env) region and was indistinguishable by restriction endonuclease mapping for 5.1 kilobases (except for the EcoRI site in the LTR) from the 5' end of AKR ecotropic proviral DNA. The DNA segment extending from 5.1 to 6.8 kilobases contained several restriction sites that were not present in the AKR ecotropic provirus. A 0.5-kilobase DNA segment located at the 3' end of MCF 1 DNA contained sequences which hybridized to a xenotropic env-specific DNA probe but not to labeled ecotropic env-specific DNA. This dual character of MCF 1 proviral DNA was also confirmed by analyzing heteroduplex molecules by electron microscopy. The second cloned proviral DNA (designated MCF 2) was a 6.9-kilobase EcoRI DNA fragment which contained LTR sequences at each end and a 2.0-kilobase deletion encompassing most of the env region. The MCF 2 proviral DNA proved to be a useful reagent for detecting LTRs electron microscopically due to the presence of nonoverlapping, terminally located LTR sequences which effected its circularization with DNAs containing homologous LTR sequences. Nucleotide sequence analysis demonstrated the presence of a 104-base-pair direct repeat in the LTR of MCF 2 DNA. In contrast, only a single copy of the reiterated component of the direct repeat was present in MCF 1 DNA. Images PMID:6281459

[Vitamin para-aminobenzoic acid inhibits development of SOS function in tif-1 mutants of Escherichia coli at nonpermissive temperatures].

PubMed

Vasil'eva, S V; Gorb, T E; Rapoport, I A

1983-12-01

The development of "SOS" inducible functions in lysogenic and non-lysogenic strains of Escherichia coli tif-1 sfiA11 (lambda) at nonpermissive temperature of 42 degrees C was strongly suppressed by para-aminobenzoic acid (PABA). The rate of prophage lambda induction decreased 400 times, as compared to the control level; the efficiency of W-reactivation of UV-irradiated phage lambda decreased 37.5 to 16%. PABA also inhibited to some extent (1.5 times) the process of inducible recombination on the RecF pathway. The processes of spontaneous lambda induction and W-reactivation, as well as spontaneous recombination on RecBC and RecF pathways, were not influenced by PABA. The above data are in accordance with previous studies of PABA action when the manifestation of "SOS" functions was induced by chemical mutagens. The action of PABA has been tentatively interpreted on the basis of negative control of "SOS" repair pathway.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Caberoy, Nora B.; Zhou, Yixiong; Alvarado, Gabriela

To efficiently elucidate the biological roles of phosphatidylserine (PS), we developed open-reading-frame (ORF) phage display to identify PS-binding proteins. The procedure of phage panning was optimized with a phage clone expressing MFG-E8, a well-known PS-binding protein. Three rounds of phage panning with ORF phage display cDNA library resulted in {approx}300-fold enrichment in PS-binding activity. A total of 17 PS-binding phage clones were identified. Unlike phage display with conventional cDNA libraries, all 17 PS-binding clones were ORFs encoding 13 real proteins. Sequence analysis revealed that all identified PS-specific phage clones had dimeric basic amino acid residues. GST fusion proteins were expressedmore » for 3 PS-binding proteins and verified for their binding activity to PS liposomes, but not phosphatidylcholine liposomes. These results elucidated previously unknown PS-binding proteins and demonstrated that ORF phage display is a versatile technology capable of efficiently identifying binding proteins for non-protein molecules like PS.« less

Bacteriophage Infecting the Myxobacterium Chondrococcus columnaris

PubMed Central

Kingsbury, David T.; Ordal, Erling J.

1966-01-01

Kingsbury, David T. (University of Washington, Seattle), and Erling J. Ordal. Bacteriophage infecting the myxobacterium Chondrococcus columnaris. J. Bacteriol. 91:1327–1332. 1966.—During a series of screening experiments, seven bacteriophages which infect the pathogenic myxobacterium Chondrococcus columnaris were isolated. Of these, one was chosen for detailed study. This phage has a wide host range among strains of C. columnaris, but does not infect other myxobacterial species tested. Morphologically, this phage resembles coliphage T2, though it is smaller. It has a head diameter of 600 A, a tail length of 1,000 A, and a tail width of 200 A. The head is attached to the tail by a well-defined neck. The turbid plaques produced by this phage are similar in appearance to those produced by coliphage λ, and average 1 mm in diameter. The phage has a latent period of 100 min, a rise period of an additional 90 min, and a burst size of 23. Calcium ions at a concentration of 0.004 m are required for adsorption. This requirement cannot be met by substitution of magnesium ions. A purified preparation of 2 × 1012 phage particles was extracted with phenol, and the nucleic acid was identified as deoxyribonucleic acid (DNA). Base ratios of the phage DNA and the DNA of two propagating strains were similar. Streptomycin at a concentration of 70 μg/ml inhibits phage infection at an early stage, probably by inhibiting injection of the phage DNA. Images PMID:5929758

A novel lineage of myoviruses infecting cyanobacteria is widespread in the oceans.

PubMed

Sabehi, Gazalah; Shaulov, Lihi; Silver, David H; Yanai, Itai; Harel, Amnon; Lindell, Debbie

2012-02-07

Viruses infecting bacteria (phages) are thought to greatly impact microbial population dynamics as well as the genome diversity and evolution of their hosts. Here we report on the discovery of a novel lineage of tailed dsDNA phages belonging to the family Myoviridae and describe its first representative, S-TIM5, that infects the ubiquitous marine cyanobacterium, Synechococcus. The genome of this phage encodes an entirely unique set of structural proteins not found in any currently known phage, indicating that it uses lineage-specific genes for virion morphogenesis and represents a previously unknown lineage of myoviruses. Furthermore, among its distinctive collection of replication and DNA metabolism genes, it carries a mitochondrial-like DNA polymerase gene, providing strong evidence for the bacteriophage origin of the mitochondrial DNA polymerase. S-TIM5 also encodes an array of bacterial-like metabolism genes commonly found in phages infecting cyanobacteria including photosynthesis, carbon metabolism and phosphorus acquisition genes. This suggests a common gene pool and gene swapping of cyanophage-specific genes among different phage lineages despite distinct sets of structural and replication genes. All cytosines following purine nucleotides are methylated in the S-TIM5 genome, constituting a unique methylation pattern that likely protects the genome from nuclease degradation. This phage is abundant in the Red Sea and S-TIM5 gene homologs are widespread in the oceans. This unusual phage type is thus likely to be an important player in the oceans, impacting the population dynamics and evolution of their primary producing cyanobacterial hosts.

Experimental and numerical studies of tethered DNA dynamics in shear flow

NASA Astrophysics Data System (ADS)

Lueth, Christopher A.

Polymer physics has a rich tradition spanning nearly two centuries. In the 1830s, Henri Braconnot and coworkers were perhaps the first to work on what is today known as polymer science when they derived semi-synthetic materials from naturally occurring cellulose. However, the true nature of polymers, as long chain molecules, had not been proposed until 1910 by Pickles. It was not until the 1950's when polymer models were developed using statistical mechanics. Recently, the field has been revitalized by the ability to study individual polymer molecules for the first time. The development of DNA single molecule fluorescence microscopy coupled with ever increasing computational power has opened the door to molecular level understanding of polymer physics, resolving old disputes and uncovering new interesting phenomena. In this work, we use a combination of theoretical predictions and lambda-phage DNA single molecule fluorescence microscopy to study the behavior of polymers tethered to surfaces. Brownian dynamics simulations of a number of coarse-grained polymer models---dynamic and equilibrium Kratky-Porod chains as well as bead-spring chains---were completed and compared with analytical and experimental results. First, an expression is developed for the entropic exclusion force experienced by a tethered polymer chain. We propose that, for a freely jointed chain, a modification to the free entropic force of kBT/y is needed in the direction normal to the surface. Analogously, we propose that for a wormlike chain, a modification of 2kBT/y is needed, due to the finite curvature of the model. Then, the reliability of discretized bead spring simulations containing this modified entropic force are analyzed using Kratky-Porod simulations and are found to reproduce most statistics, except for those very near the surface, such as end-wall contact. Next, experiments of tethered lambda-phage DNA in shear flow are presented for the first time in the flow-gradient plane. The tethering surface chemistry proved to be arduous work, but with the aide of contact angle and ellipsometry measurements, success was achieved. Extension behavior was shown to agree well with bead spring simulations, but deviations were discovered for weak flows in the extensional fluctuations, mean distance from the wall, and orientation angle. Cyclic dynamics---where the polymer continuously diffuses away from the wall, subsequently undergoes stretch in the flow direction, is then "entropically pulled back" towards the wall, and finally recoils---was observed and quantified through correlation and power spectral densities. Again, quantitative agreement was observed between experiments and bead spring simulations. The onset of cyclic dynamics was found to occur at Wi ≈ 3 and was found to decline with increasing Wi up to ≈ 200. Finally, a reliable procedure was developed utilizing Dip Pen Nanolithography to controllably tether DNA to gold surfaces. Preliminary experiments were performed and successful tethering was achieved, an important first step toward creating DNA scaffolds for molecular wires. However, further research is needed to fully develop the process and successfully double tether DNA molecules between gold electrodes.

Engineering M13 for phage display.

PubMed

Sidhu, S S

2001-09-01

Phage display is achieved by fusing polypeptide libraries to phage coat proteins. The resulting phage particles display the polypeptides on their surfaces and they also contain the encoding DNA. Library members with particular functions can be isolated with simple selections and polypeptide sequences can be decoded from the encapsulated DNA. The technology's success depends on the efficiency with which polypeptides can be displayed on the phage surface, and significant progress has been made in engineering M13 bacteriophage coat proteins as improved phage display platforms. Functional display has been achieved with all five M13 coat proteins, with both N- and C-terminal fusions. Also, coat protein mutants have been designed and selected to improve the efficiency of heterologous protein display, and in the extreme case, completely artificial coat proteins have been evolved specifically as display platforms. These studies demonstrate that the M13 phage coat is extremely malleable, and this property can be used to engineer the phage particle specifically for phage display. These improvements expand the utility of phage display as a powerful tool in modern biotechnology.

Quantification of M13 and T7 bacteriophages by TaqMan and SYBR green qPCR.

PubMed

Peng, Xiujuan; Nguyen, Alex; Ghosh, Debadyuti

2018-02-01

TaqMan and SYBR Green quantitative PCR (qPCR) methods were developed as DNA-based approaches to reproducibly enumerate M13 and T7 phages from phage display selection experiments individually and simultaneously. The genome copies of M13 and T7 phages were quantified by TaqMan or SYBR Green qPCR referenced against M13 and T7 DNA standard curves of known concentrations. TaqMan qPCR was capable of quantifying M13 and T7 phage DNA simultaneously with a detection range of 2.75*10 1 -2.75*10 8 genome copies(gc)/μL and 2.66*10 1 -2.66*10 8 genome copies(gc)/μL respectively. TaqMan qPCR demonstrated an efficient amplification efficiency (E s ) of 0.97 and 0.90 for M13 and T7 phage DNA, respectively. SYBR Green qPCR was ten-fold more sensitive than TaqMan qPCR, able to quantify 2.75-2.75*10 7 gc/μL and 2.66*10 1 -2.66*10 7 gc/μL of M13 and T7 phage DNA, with an amplification efficiency E s of 1.06 and 0.78, respectively. Due to its superior sensitivity, SYBR Green qPCR was used to enumerate M13 and T7 phage display clones selected against a cell line, and quantified titers demonstrated accuracy comparable to titers from traditional double-layer plaque assay. Compared to enzyme linked immunosorbent assay, both qPCR methods exhibited increased detection sensitivity and reproducibility. These qPCR methods are reproducible, sensitive, and time-saving to determine their titers and to quantify a large number of phage samples individually or simultaneously, thus avoiding the need for time-intensive double-layer plaque assay. These findings highlight the attractiveness of qPCR for phage enumeration for applications ranging from selection to next-generation sequencing (NGS). Copyright © 2017 Elsevier B.V. All rights reserved.

Structure and Function of the Splice Variants of TMPRSS2-ERG, a Prevalent Genomic Alteration in Prostate Cancer

DTIC Science & Technology

2009-09-01

binding ETS domain) and five type II (without ETS domain). Fusion-positive type I– and type II–containing phages were amplified with T3 and T7 primers...will be performed to identify the authentic 3’ UTRs from the mRNA pool from CaP patient specimens. Using phage excision strategy, we will use to... phage DNA sequences plasmids (cDNA) clones were generated by using phage excision strategy. Figure 1. ERG splice variants in prostate cancer

Concentration methods for high-resolution THz spectroscopy of nucleic-acid biomolecules and crystals

NASA Astrophysics Data System (ADS)

Brown, E. R.; Zhang, W.; Mendoza, E. A.; Kuznetsova, Y.; Brueck, S. R. J.; Rahman, M.; Norton, M. L.

2012-03-01

Biomolecules can exhibit low-lying vibrational modes in the THz region which are detectable in transmission given a strong molecular dipole moment and optical depth, and a spectrometer of adequate sensitivity. The nucleic acids are particularly interesting because of applications such as label-free gene assay, bio-agent detection, etc. However for nucleic acids, sample preparation and THz coupling are of paramount importance because of the strong absorption by liquid water and the small concentration of molecules present in physiological solutions. Concentration methods become necessary to make the THz vibrational modes detectable, either by concentrating the nucleic-acid sample itself in a small volume but large area, or by concentrating the THz radiation down to the volume of the sample. This paper summarizes one type of the first method: nanofluidic channel arrays for biological nucleic acids; and two types of the second method: (1) a circular-waveguide pinhole, and (2) a circular-waveguide, conical-horn coupling structure, both for DNA crystals. The first method has been demonstrated on a very short artificial nucleic acid [small-interfering (si) RNA (17-to-25 bp)] and a much longer, biological molecule [Lambda-phage DNA (48.5 kbp)]. The second method has been demonstrated on small (~100 micron) single crystals of DNA grown by the sitting-drop method.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lagrimini, L.M.

Since this manuscript was submitted we have conducted a more thorough physiological analysis of water relations in wild-type and peroxidase overproducing plants. These experiments include pressure bomb, plasmolysis, and membrane integrity analysis. We are also in the process of analyzing other phenotypes in peroxidase overproducer plants such as excessive browning of tissue, the rapid death of tissue in culture, and poor germination of seed. Transformed plants of Nicotiana tabacum and Nicotiana sylvestris were obtained which have peroxidase activity 3--7 fold lower than wild-type plants. This was done by introducing a chimeric gene composed of the CaMV 35S promoter and themore » 5' half of the tobacco anionic peroxidase cDNA in the antisense RNA configuration. A manuscript which describes this work is being written, and will be submitted for publication in January 1990. The anionic peroxidase gene has been cloned by hybridization to the cloned cDNA. The entire gene is contained on an 8.7kb fragment within a lambda phage clone. Several smaller DNA fragments have been subcloned, and some have been sequenced. One exon within the coding sequence has been sequenced, along with the partial sequence of two introns. Further sequencing is being carried-out to identify the promoter, which will be later joined to a reporter gene. 6 figs.« less

Development of Shuttle Vectors for Halobacteria

DTIC Science & Technology

1989-02-01

the time of application, we had completed a study of transfection, using Halobacterium halobiun and its phage ,DH. We showed (Cline and Doolittle... phage particles), producing plaques titratable on H. halobium lawns. Uptake of DNA by both species appears to be about equally efficient, but DNA from H...halobium-grown phage is restricted in H. volcanii, which shows a 104 - 105 reduction in transfection efficiency (400 plaques per pg) but no reduction

Incorporation of Deoxyribonucleic Acid Precursors by T4 Deoxyribonucleic Acid-Protein Complexes Retained on Glass Fiber Filters

PubMed Central

Miller, Robert C.; Kozinski, Andrzej W.

1970-01-01

Bacteriophage T4 deoxyribonucleic acid (DNA)-protein complexes were retained preferentially on glass fiber filters. DNA polymerase activity in the complex was detected through the incorporation of 3H-labeled DNA precursors. The primer-product DNA hybridized with both phage and Escherichia coli DNA. Density labeling experiments showed that about 30% of incorporated 3H-deoxyadenosine triphosphate was found in DNA which hybridized with phage DNA; this DNA was found to be covalently attached to the primer DNA. PMID:5497903

Bacteriophages of wastewater foaming-associated filamentous Gordonia reduce host levels in raw activated sludge.

PubMed

Liu, Mei; Gill, Jason J; Young, Ry; Summer, Elizabeth J

2015-09-09

Filamentous bacteria are a normal and necessary component of the activated sludge wastewater treatment process, but the overgrowth of filamentous bacteria results in foaming and bulking associated disruptions. Bacteriophages, or phages, were investigated for their potential to reduce the titer of foaming bacteria in a mixed-microbial activated sludge matrix. Foaming-associated filamentous bacteria were isolated from activated sludge of a commercial wastewater treatment plan and identified as Gordonia species by 16S rDNA sequencing. Four representative phages were isolated that target G. malaquae and two un-named Gordonia species isolates. Electron microscopy revealed the phages to be siphophages with long tails. Three of the phages--GordTnk2, Gmala1, and GordDuk1--had very similar ~76 kb genomes, with >93% DNA identity. These genomes shared limited synteny with Rhodococcus equi phage ReqiDocB7 and Gordonia phage GTE7. In contrast, the genome of phage Gsput1 was smaller (43 kb) and was not similar enough to any known phage to be placed within an established phage type. Application of these four phages at MOIs of 5-15 significantly reduced Gordonia host levels in a wastewater sludge model by approximately 10-fold as compared to non-phage treated reactors. Phage control was observed for nine days after treatment.

[Isolation and characterization of siphovirus phages infecting bovine Streptococcus agalactiae].

PubMed

Bai, Qinqin; Yang, Yongchun; Lu, Chengping

2016-02-04

To isolate and identify Streptococcus agalactiae phages and screen candidate phages to control infection caused by bovine S. agalactiae. We used two methods for isolation of S. agalactiae phages, namely (1) isolation of phages from milk and environmental samples, and (2) isolation of phages via induction of lysogens with Mitomycin C. Double-layer agar culture method was used to purify phages. Then the newly obtained phages, with S. agalactiae phage JX01 isolated from mastitis milk, were comparatively analyzed in the following aspects: morphology of phages by transmission electron microscopy, host range of phages to 55 S. agalactiae strains and other Streptococcus strains, phages DNA using EcoR I, Xba I, Pst I and Sal I, the optical multiplicity of infection, absorption curve and one step growth curve, and the stability of phages at different storage conditions. The comparative analysis of the 3 novel phages LYGO9, HZ04 and pA11 (induced from S. agalctiae bovine clinical isolate HAJL2011070601) with JX01 showed that the 4 phages were classified as the member of Siphovirdae family. EcoR I, Sal I, Xba I and Pst I separately digested the 4 phages DNA provided 4, 3, 3 and 2 profiles, respectively. This suggested that they were different strains. All the 4 phages specifically infected bovine S. agalactiae isolates. LYGO9, pA11, JX01 and HZ04 could lyse 12, 13, 20 and 23 of 42 tested bovine S. agalctiae isolates, respectively. This clearly indicated that these 4 phages are closely related. The 3 new phages which specifically lyse bovine S. agalactiae isolates are siphovirus phages. Phage LYGO9 was shown having a short latent period and a larger burst size.

DNA bending-induced phase transition of encapsidated genome in phage λ

PubMed Central

Lander, Gabriel C.; Johnson, John E.; Rau, Donald C.; Potter, Clinton S.; Carragher, Bridget; Evilevitch, Alex

2013-01-01

The DNA structure in phage capsids is determined by DNA–DNA interactions and bending energy. The effects of repulsive interactions on DNA interaxial distance were previously investigated, but not the effect of DNA bending on its structure in viral capsids. By varying packaged DNA length and through addition of spermine ions, we transform the interaction energy from net repulsive to net attractive. This allowed us to isolate the effect of bending on the resulting DNA structure. We used single particle cryo-electron microscopy reconstruction analysis to determine the interstrand spacing of double-stranded DNA encapsidated in phage λ capsids. The data reveal that stress and packing defects, both resulting from DNA bending in the capsid, are able to induce a long-range phase transition in the encapsidated DNA genome from a hexagonal to a cholesteric packing structure. This structural observation suggests significant changes in genome fluidity as a result of a phase transition affecting the rates of viral DNA ejection and packaging. PMID:23449219

Cloning structural genes for Treponema pallidum immunogens and characterisation of recombinant treponemal surface protein, P2 (P2 star).

PubMed Central

Peterson, K M; Baseman, J B; Alderete, J F

1987-01-01

A genomic library consisting of partially digested 10 to 20 kilobase pair fragments of Treponema pallidum deoxyribonucleic acid (DNA) was constructed using bacteriophage lambda EMBL-3 as the vector. Positive clones expressing T pallidum antigens were detected with sera from experimentally infected rabbits. Treponemal proteins ranging in molecular weight from 37,000 daltons to 120,000 daltons were identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting of phage lysate proteins. One recombinant phage was examined further and contained an insert encoding a prominent treponemal 37,000 dalton protein. The recombinant protein was not recognised by antiserum directed against a fibronectin binding treponemal adhesion that contained the same electrophoretic mobility. Neither did antibody to the recombinant 37,000 dalton protein react with any treponemal proteins purified by fibronectin affinity chromatography. The recombinant protein in Escherichia coli lysates was labelled P2 (P2 star) to differentiate it from the comigrating adhesin protein called P2. Native P2 protein was present on T pallidum surfaces as shown by radioimmunoprecipitation assays with extrinsically labelled organisms. A cross reactive molecule like P2 was not synthesised by the avirulent spirochaete, T phagedenis biotype Reiter, which indicated that P2 is a protein specific to virulent T pallidum organisms. Finally, only sera of patients with primary syphilis possessed appreciable concentrations of antibody to recombinant P2 protein. Images PMID:3315959

Direct Isolation of Purines and Pyrimidines from Nucleic Acids Using Sublimation

NASA Technical Reports Server (NTRS)

Glavin, Daniel P.; Schubert, Michael; Bada, Jeffrey L.

2003-01-01

A sublimation technique was developed to isolate purines and pyrimidines directly from lambda-deoxyribonucleic acid (lambda-DNA) and Escherichia coli cells. The sublimation of adenine, cytosine, guanine, and thymine from lambda-DNA was tested under reduced pressure (approx. 0.5 Torr) at temperatures of >150 C. With the exception of guanine, approximately 60 -75% of each base was sublimed directly from the lambda-DNA and recovered on a coldfinger of the sublimation apparatus after heating to 450 C. Several nucleobases including adenine, cytosine, thymine, and uracil were also recovered from E. coli bacteria after heating the cells to the same temperature, although some thermal decomposition of the bases also occurred. These results demonstrate the feasibility of using sublimation to isolate purines and pyrimidines from native E. coli DNA and RNA without any chemical treatment of the cells.

Detection of hepatitis B virus core antigen by phage display mediated TaqMan real-time immuno-PCR.

PubMed

Monjezi, Razieh; Tan, Sheau Wei; Tey, Beng Ti; Sieo, Chin Chin; Tan, Wen Siang

2013-01-01

The core antigen (HBcAg) of hepatitis B virus (HBV) is one of the markers for the identification of the viral infection. The main purpose of this study was to develop a TaqMan real-time detection assay based on the concept of phage display mediated immuno-PCR (PD-IPCR) for the detection of HBcAg. PD-IPCR combines the advantages of immuno-PCR (IPCR) and phage display technology. IPCR integrates the versatility of enzyme-linked immunosorbent assay (ELISA) with the sensitivity and signal generation power of PCR. Whereas, phage display technology exploits the physical association between the displayed peptide and the encoding DNA within the same phage particle. In this study, a constrained peptide displayed on the surface of an M13 recombinant bacteriophage that interacts tightly with HBcAg was applied as a diagnostic reagent in IPCR. The phage displayed peptide and its encoding DNA can be used to replace monoclonal antibody (mAb) and chemically bound DNA, respectively. This method is able to detect as low as 10ng of HBcAg with 10(8)pfu/ml of the recombinant phage which is about 10,000 times more sensitive than the phage-ELISA. The PD-IPCR provides an alternative means for the detection of HBcAg in human serum samples. Copyright © 2012 Elsevier B.V. All rights reserved.

Characterization of the genome of the dairy Lactobacillus helveticus bacteriophage {Phi}AQ113.

PubMed

Zago, Miriam; Scaltriti, Erika; Rossetti, Lia; Guffanti, Alessandro; Armiento, Angelarita; Fornasari, Maria Emanuela; Grolli, Stefano; Carminati, Domenico; Brini, Elena; Pavan, Paolo; Felsani, Armando; D'Urzo, Annalisa; Moles, Anna; Claude, Jean-Baptiste; Grandori, Rita; Ramoni, Roberto; Giraffa, Giorgio

2013-08-01

The complete genomic sequence of the dairy Lactobacillus helveticus bacteriophage ΦAQ113 was determined. Phage ΦAQ113 is a Myoviridae bacteriophage with an isometric capsid and a contractile tail. The final assembled consensus sequence revealed a linear, circularly permuted, double-stranded DNA genome with a size of 36,566 bp and a G+C content of 37%. Fifty-six open reading frames (ORFs) were predicted, and a putative function was assigned to approximately 90% of them. The ΦAQ113 genome shows functionally related genes clustered together in a genome structure composed of modules for DNA replication/regulation, DNA packaging, head and tail morphogenesis, cell lysis, and lysogeny. The identification of genes involved in the establishment of lysogeny indicates that it may have originated as a temperate phage, even if it was isolated from natural cheese whey starters as a virulent phage, because it is able to propagate in a sensitive host strain. Additionally, we discovered that the ΦAQ113 phage genome is closely related to Lactobacillus gasseri phage KC5a and Lactobacillus johnsonii phage Lj771 genomes. The phylogenetic similarities between L. helveticus phage ΦAQ113 and two phages that belong to gut species confirm a possible common ancestral origin and support the increasing consideration of L. helveticus as a health-promoting organism.

Characterization of the Genome of the Dairy Lactobacillus helveticus Bacteriophage ΦAQ113

PubMed Central

Scaltriti, Erika; Rossetti, Lia; Guffanti, Alessandro; Armiento, Angelarita; Fornasari, Maria Emanuela; Grolli, Stefano; Carminati, Domenico; Brini, Elena; Pavan, Paolo; Felsani, Armando; D'Urzo, Annalisa; Moles, Anna; Claude, Jean-Baptiste; Grandori, Rita; Ramoni, Roberto; Giraffa, Giorgio

2013-01-01

The complete genomic sequence of the dairy Lactobacillus helveticus bacteriophage ΦAQ113 was determined. Phage ΦAQ113 is a Myoviridae bacteriophage with an isometric capsid and a contractile tail. The final assembled consensus sequence revealed a linear, circularly permuted, double-stranded DNA genome with a size of 36,566 bp and a G+C content of 37%. Fifty-six open reading frames (ORFs) were predicted, and a putative function was assigned to approximately 90% of them. The ΦAQ113 genome shows functionally related genes clustered together in a genome structure composed of modules for DNA replication/regulation, DNA packaging, head and tail morphogenesis, cell lysis, and lysogeny. The identification of genes involved in the establishment of lysogeny indicates that it may have originated as a temperate phage, even if it was isolated from natural cheese whey starters as a virulent phage, because it is able to propagate in a sensitive host strain. Additionally, we discovered that the ΦAQ113 phage genome is closely related to Lactobacillus gasseri phage KC5a and Lactobacillus johnsonii phage Lj771 genomes. The phylogenetic similarities between L. helveticus phage ΦAQ113 and two phages that belong to gut species confirm a possible common ancestral origin and support the increasing consideration of L. helveticus as a health-promoting organism. PMID:23728811

Genome packaging in EL and Lin68, two giant phiKZ-like bacteriophages of P. aeruginosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sokolova, O.S., E-mail: sokolova@mail.bio.msu.ru; A.V. Shoubnikov Institute of Crystallography RAS, Moscow; Shaburova, O.V.

A unique feature of the Pseudomonas aeruginosa giant phage phiKZ is its way of genome packaging onto a spool-like protein structure, the inner body. Until recently, no similar structures have been detected in other phages. We have studied DNA packaging in P. aeruginosa phages EL and Lin68 using cryo-electron microscopy and revealed the presence of inner bodies. The shape and positioning of the inner body and the density of the DNA packaging in EL are different from those found in phiKZ and Lin68. This internal organization explains how the shorter EL genome is packed into a large EL capsid, whichmore » has the same external dimensions as the capsids of phiKZ and Lin68. The similarity in the structural organization in EL and other phiKZ-like phages indicates that EL is phylogenetically related to other phiKZ-like phages, and, despite the lack of detectable DNA homology, EL, phiKZ, and Lin68 descend from a common ancestor. - Highlights: • We performed a comparative structural study of giant P. aeruginosa phages: EL, Lin68 and phiKZ. • We revealed that the inner body is a common feature in giant phages. • The phage genome size correlates with the overall dimensions of the inner body.« less

Role of Escherichia coli dnaG function in coliphage M13 DNA synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dasgupta, S.; Mitra, S.

Examination of the role of Escherichia coli dnaG function in different stages of M13 phage DNA synthesis by ultracentrifugal analysis of intracellular phage DNA in a thermosensitive dnaG mutant shows that: (a) the formation of parental double-strand replicative-form DNA (rfDNA) from the infecting virus is independent of dnaG function; (b) the synthesis of progeny rfDNA requires dnaG product; (c) after a pool of rfDNA is made up, dnaG function is not required for the progeny single-strand DNA (ssDNA) synthesis. The ssDNAs produced under nonpermissive condition are mostly circular and biologically functional.

Phage display for generating peptide reagents.

PubMed

Brigati, Jennifer R; Samoylova, Tatiana I; Jayanna, Prashanth K; Petrenko, Valery A

2008-02-01

This unit presents detailed protocols for selection and propagation of landscape phages, which are fusions of filamentous phage fd (or its close relatives M13 and f1) and foreign DNA that result in chimeric phage virions with foreign peptides (8 to 9 amino acids long) covering the entire surface of the phage particles. These landscape phages bind specifically to mammalian and bacterial cells, spores, or discrete molecular targets. (c) 2008 by John Wiley & Sons, Inc.

tif-Stimulated deoxyribonucleic acid repair in Escherichia coli K-12.

PubMed Central

Castellazzi, M; Jacques, M; George, J

1980-01-01

Bacterial survival is significantly increased after ultraviolet irradiation in tif sfi cells, provided that the thermosensitive tif mutation has been expressed at 41 degrees C before irradiation. This tif-mediated "reactivation of ultraviolet irradiated bacteria" needs de novo protein synthesis, as is the case for the tif-mediated reactivation of ultraviolet-irradiated phage lambda. However, in striking contrast to the phage reactivation process, this tif-mediated reactivation is no longer associated with mutagenesis. It also requires the presence of the uvrA+ excision function. These results strongly suggest the existence in Escherichia coli K-12 of a repair pathway acting on bacterial deoxyribonucleic acid which is inducible, error free, and uvr dependent. PMID:6451614

The Arabidopsis At1g30680 gene encodes a homologue to the phage T7 gp4 protein that has both DNA primase and DNA helicase activities.

PubMed

Diray-Arce, Joann; Liu, Bin; Cupp, John D; Hunt, Travis; Nielsen, Brent L

2013-03-04

The Arabidopsis thaliana genome encodes a homologue of the full-length bacteriophage T7 gp4 protein, which is also homologous to the eukaryotic Twinkle protein. While the phage protein has both DNA primase and DNA helicase activities, in animal cells Twinkle is localized to mitochondria and has only DNA helicase activity due to sequence changes in the DNA primase domain. However, Arabidopsis and other plant Twinkle homologues retain sequence homology for both functional domains of the phage protein. The Arabidopsis Twinkle homologue has been shown by others to be dual targeted to mitochondria and chloroplasts. To determine the functional activity of the Arabidopsis protein we obtained the gene for the full-length Arabidopsis protein and expressed it in bacteria. The purified protein was shown to have both DNA primase and DNA helicase activities. Western blot and qRT-PCR analysis indicated that the Arabidopsis gene is expressed most abundantly in young leaves and shoot apex tissue, as expected if this protein plays a role in organelle DNA replication. This expression is closely correlated with the expression of organelle-localized DNA polymerase in the same tissues. Homologues from other plant species show close similarity by phylogenetic analysis. The results presented here indicate that the Arabidopsis phage T7 gp4/Twinkle homologue has both DNA primase and DNA helicase activities and may provide these functions for organelle DNA replication.

A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria

PubMed Central

Quiles-Puchalt, Nuria; Tormo-Más, María Ángeles; Campoy, Susana; Toledo-Arana, Alejandro; Monedero, Vicente; Lasa, Íñigo; Novick, Richard P.; Christie, Gail E.; Penadés, José R.

2013-01-01

The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria. PMID:23771138

Isolation and Expression of the Lysis Genes of Actinomyces naeslundii Phage Av-1

PubMed Central

Delisle, Allan L.; Barcak, Gerard J.; Guo, Ming

2006-01-01

Like most gram-positive oral bacteria, Actinomyces naeslundii is resistant to salivary lysozyme and to most other lytic enzymes. We are interested in studying the lysins of phages of this important oral bacterium as potential diagnostic and therapeutic agents. To identify the Actinomyces phage genes encoding these species-specific enzymes in Escherichia coli, we constructed a new cloning vector, pAD330, that can be used to enrich for and isolate phage holin genes, which are located adjacent to the lysin genes in most phage genomes. Cloned holin insert sequences were used to design sequencing primers to identify nearby lysin genes by using whole phage DNA as the template. From partial digestions of A. naeslundii phage Av-1 genomic DNA we were able to clone, in independent experiments, inserts that complemented the defective λ holin in pAD330, as evidenced by extensive lysis after thermal induction. The DNA sequence of the inserts in these plasmids revealed that both contained the complete lysis region of Av-1, which is comprised of two holin-like genes, designated holA and holB, and an endolysin gene, designated lysA. We were able to subclone and express these genes and determine some of the functional properties of their gene products. PMID:16461656

TF1, the bacteriophage SPO1-encoded type II DNA-binding protein, is essential for viral multiplication.

PubMed

Sayre, M H; Geiduschek, E P

1988-09-01

The lytic Bacillus subtilis bacteriophage SPO1 encodes an abundant, 99-amino-acid type II DNA-binding protein, transcription factor 1 (TF1). TF1 is special in this family of procaryotic chromatin-forming proteins in its preference for hydroxymethyluracil-containing DNA, such as SPO1 DNA, and in binding with high affinity to specific sites in the SPO1 chromosome. We constructed recessive null alleles of the TF1 gene and introduced them into SPO1 chromosomes. Segregation analysis with partially diploid phage heterozygous for TF1 showed that phage bearing only these null alleles was inviable. Deletion of the nine C-proximal amino acids of TF1 prohibited phage multiplication in vivo and abolished its site-specific DNA-binding activity in vitro.

Megabase sequencing of human genome by ordered-shotgun-sequencing (OSS) strategy

NASA Astrophysics Data System (ADS)

Chen, Ellson Y.

1997-05-01

So far we have used OSS strategy to sequence over 2 megabases DNA in large-insert clones from regions of human X chromosomes with different characteristic levels of GC content. The method starts by randomly fragmenting a BAC, YAC or PAC to 8-12 kb pieces and subcloning those into lambda phage. Insert-ends of these clones are sequenced and overlapped to create a partial map. Complete sequencing is then done on a minimal tiling path of selected subclones, recursively focusing on those at the edges of contigs to facilitate mergers of clones across the entire target. To reduce manual labor, PCR processes have been adapted to prepare sequencing templates throughout the entire operation. The streamlined process can thus lend itself to further automation. The OSS approach is suitable for large- scale genomic sequencing, providing considerable flexibility in the choice of subclones or regions for more or less intensive sequencing. For example, subclones containing contaminating host cell DNA or cloning vector can be recognized and ignored with minimal sequencing effort; regions overlapping a neighboring clone already sequenced need not be redone; and segments containing tandem repeats or long repetitive sequences can be spotted early on and targeted for additional attention.

Activity of foreign proteins targeted within the bacteriophage T4 head and prohead: implications for packaged DNA structure.

PubMed

Mullaney, J M; Black, L W

1998-11-13

The phage-derived expression, packaging, and processing (PEPP) system was used to target foreign proteins into the bacteriophage capsid to probe the intracapsid environment and the structure of packaged DNA. Small proteins with minimal requirements for activity were selected, staphylococcal nuclease (SN) and green fluorescent protein (GFP). These proteins were targeted into the T4 head by means of IPIII (internal protein III) fusions or CTS (capsid targeting sequence) fusions. Additional evidence is provided that foreign proteins are targeted into T4 by the N-terminal ten amino acid residue consensus CTS of IPIII identified in previous work. Fusion proteins were produced within host bacteria by expression from plasmids or by produc tion from recombinant phage carrying the fusion genes. Packaged fusion proteins CTS IPIII SN, CTS IPIII TSN, CTS IPIII GFP, CTS IPIII TGFP, and CTS GFP, where [symbol: see text] indicates a linkage peptide sequence Leu(Ile)-N-Glu cleaved by the T4 head morphogenetic proteinase gp21 during head maturation, are observed to exhibit intracapsid activity. SN activity within the head is demonstrated by loss of phage viability and by digested genomic DNA patterns visualized by gel electrophoresis when viable phage are incubated in Ca2+. Green fluorescent phage result immediately after packaging GFP produced at 30 degreesC and below, and continue to give green fluorescence under 470 nm light after CsCl purification. Non-fluorescent GFP-fusions are produced in bacteria at 37 degreesC, and phage packaged with these proteins achieve a fluorescent state after incubation for several months at 4 degreesC. GFP-packaged phage and proheads analyzed by fluorescence spectroscopy show that the mature head and the DNA-empty prohead package identical numbers of GFP-fusion proteins. Encapsidated GFP and SN can be injected into bacteria and rapidly exhibit intracellular activity. In vivo SN digestion of encapsidated DNA gives an intriguing pattern of DNA fragments by gel analysis, predominantly a repeat pattern of 160 bp multiples, reminiscent of a nucleosome digestion ladder, This quasi-limit DNA digestion pattern, reached >100-fold more slowly than the loss of titer, is invariant over a range

Bacterial rep- mutations that block development of small DNA bacteriophages late in infection.

PubMed Central

Tessman, E S; Peterson, P K

1976-01-01

Several related mutants of Escherichia coli C have been isolated that block the growth of the small icosahedral DNA phages phiX174 and S13 late in infection. Phage G6 is also blocked, at a stage not yet known. Growth of the filamentous phage M13, though not blocked, is affected in these strains. These host mutations co-transduce with ilv at high frequency, as do rep- mutations. However, the new mutants, designated groL-, differ from previously studied rep- mutants in that they permit synthesis of progeny replicative-form DNA. The groL- mutants are blocked in synthesis of stable single-stranded DNA of phiX174 and related phages. They are gro+ for P2. Evidence that groL- mutations and rep- mutations are in the same gene is presented. Spontaneous mutants (ogr) of phiX174, S13, and the G phages can grow on groL- strains. The ogr mutations are located in the phage's major capsid gene, F, as determined by complementation tests. There are numerous sites for mutation to ogr. Some mutations in genes A and F interfere with the ogr property when combined with an ogr mutation on the same genome. The ogr mutations are cis acting in a groL- cell; i.e., an ogr mutant gives very poor rescue of a non-ogr mutant. The wild-type form of each G phage appears to be naturally in the ogr mutant state for one or more groL- strains. It is suggested that a complex between F and rep proteins is involved in phage maturation. The A protein appears to interact with this complex. PMID:789914

Bacteriophages of wastewater foaming-associated filamentous Gordonia reduce host levels in raw activated sludge

PubMed Central

Liu, Mei; Gill, Jason J.; Young, Ry; Summer, Elizabeth J.

2015-01-01

Filamentous bacteria are a normal and necessary component of the activated sludge wastewater treatment process, but the overgrowth of filamentous bacteria results in foaming and bulking associated disruptions. Bacteriophages, or phages, were investigated for their potential to reduce the titer of foaming bacteria in a mixed-microbial activated sludge matrix. Foaming-associated filamentous bacteria were isolated from activated sludge of a commercial wastewater treatment plan and identified as Gordonia species by 16S rDNA sequencing. Four representative phages were isolated that target G. malaquae and two un-named Gordonia species isolates. Electron microscopy revealed the phages to be siphophages with long tails. Three of the phages - GordTnk2, Gmala1, and GordDuk1 - had very similar ~76 kb genomes, with >93% DNA identity. These genomes shared limited synteny with Rhodococcus equi phage ReqiDocB7 and Gordonia phage GTE7. In contrast, the genome of phage Gsput1 was smaller (43 kb) and was not similar enough to any known phage to be placed within an established phage type. Application of these four phages at MOIs of 5–15 significantly reduced Gordonia host levels in a wastewater sludge model by approximately 10-fold as compared to non-phage treated reactors. Phage control was observed for nine days after treatment. PMID:26349678

Full shut-off of Escherichia coli RNA-polymerase by T7 phage requires a small phage-encoded DNA-binding protein

PubMed Central

Tabib-Salazar, Aline; Liu, Bing; Shadrin, Andrey; Burchell, Lynn; Wang, Zhexin; Wang, Zhihao; Goren, Moran G.; Yosef, Ido; Qimron, Udi; Severinov, Konstantin

2017-01-01

Abstract Infection of Escherichia coli by the T7 phage leads to rapid and selective inhibition of the bacterial RNA polymerase (RNAP) by the 7 kDa T7 protein Gp2. We describe the identification and functional and structural characterisation of a novel 7 kDa T7 protein, Gp5.7, which adopts a winged helix-turn-helix-like structure and specifically represses transcription initiation from host RNAP-dependent promoters on the phage genome via a mechanism that involves interaction with DNA and the bacterial RNAP. Whereas Gp2 is indispensable for T7 growth in E. coli, we show that Gp5.7 is required for optimal infection outcome. Our findings provide novel insights into how phages fine-tune the activity of the host transcription machinery to ensure both successful and efficient phage progeny development. PMID:28486695

Deoxyribonucleic Acid Replication and Expression of Early and Late Bacteriophage Functions in Bacillus subtilis

PubMed Central

Pène, Jacques J.; Marmur, Julius

1967-01-01

The role of deoxyribonucleic acid (DNA) replication in the control of the synthesis of deoxycytidylate (dCMP) deaminase and lysozyme in Bacillus subtilis infected with bacteriophage 2C has been studied. These phage-induced enzymes are synthesized at different times during the latent period. It was shown by actinomycin inhibition that the formation of the late enzyme (lysozyme) required messenger ribonucleic acid (mRNA) synthesized de novo after the initiation of translation of mRNA which specifies the early function (dCMP deaminase). The inhibition of phage DNA synthesis by mitomycin C prevented the synthesis of lysozyme only when added before the onset of phage DNA replication, but it did not affect the synthesis or action of dCMP deaminase when added at any time during the latent period. Treatment of infected cells with mitomycin C after phage DNA synthesis had reached 8 to 10% of its maximal rate resulted in the production of normal amounts of lysozyme. These observations suggest that mRNA specifying early enzymes can be transcribed from parental (and probably also from progeny) DNA, whereas late functional messengers can be transcribed only after the formation of progeny DNA. PMID:4990039

Genome sequence and analysis of a broad-host range lytic bacteriophage that infects the Bacillus cereus group

PubMed Central

2013-01-01

Background Comparatively little information is available on members of the Myoviridae infecting low G+C content, Gram-positive host bacteria of the family Firmicutes. While numerous Bacillus phages have been isolated up till now only very few Bacillus cereus phages have been characterized in detail. Results Here we present data on the large, virulent, broad-host-range B. cereus phage vB_BceM_Bc431v3 (Bc431v3). Bc431v3 features a 158,618 bp dsDNA genome, encompassing 239 putative open reading frames (ORFs) and, 20 tRNA genes encoding 17 different amino acids. Since pulsed-field gel electrophoresis indicated that the genome of this phage has a mass of 155-158 kb Bc431v3 DNA appears not to contain long terminal repeats that are found in the genome of Bacillus phage SPO1. Conclusions Bc431v3 displays significant sequence similarity, at the protein level, to B. cereus phage BCP78, Listeria phage A511 and Enterococcus phage ØEF24C and other morphologically related phages infecting Firmicutes such as Staphylococcus phage K and Lactobacillus phage LP65. Based on these data we suggest that Bc431v3 should be included as a member of the Spounavirinae; however, because of all the diverse taxonomical information has been addressed recently, it is difficult to determine the genus. The Bc431v3 phage contains some highly unusual genes such as gp143 encoding putative tRNAHis guanylyltransferase. In addition, it carries some genes that appear to be related to the host sporulation regulators. These are: gp098, which encodes a putative segregation protein related to FstK/SpoIIIE DNA transporters; gp105, a putative segregation protein; gp108, RNA polymerase sigma factor F/B; and, gp109 encoding RNA polymerase sigma factor G. PMID:23388049

Sak and Sak4 recombinases are required for bacteriophage replication in Staphylococcus aureus

PubMed Central

Neamah, Maan M.; Mir-Sanchis, Ignacio; López-Sanz, María; Acosta, Sonia; Baquedano, Ignacio; Haag, Andreas F.

2017-01-01

Abstract DNA-single strand annealing proteins (SSAPs) are recombinases frequently encoded in the genome of many bacteriophages. As SSAPs can promote homologous recombination among DNA substrates with an important degree of divergence, these enzymes are involved both in DNA repair and in the generation of phage mosaicisms. Here, analysing Sak and Sak4 as representatives of two different families of SSAPs present in phages infecting the clinically relevant bacterium Staphylococcus aureus, we demonstrate for the first time that these enzymes are absolutely required for phage reproduction. Deletion of the genes encoding these enzymes significantly reduced phage replication and the generation of infectious particles. Complementation studies revealed that these enzymes are required both in the donor (after prophage induction) and in the recipient strain (for infection). Moreover, our results indicated that to perform their function SSAPs require the activity of their cognate single strand binding (Ssb) proteins. Mutational studies demonstrated that the Ssb proteins are also required for phage replication, both in the donor and recipient strain. In summary, our results expand the functions attributed to the Sak and Sak4 proteins, and demonstrate that both SSAPs and Ssb proteins are essential for the life cycle of temperate staphylococcal phages. PMID:28475766

Phage-mediated horizontal gene transfer of both prophage and heterologous DNA by ϕBB-1, a bacteriophage of Borrelia burgdorferi.

PubMed

Eggers, Christian H; Gray, Carlie M; Preisig, Alexander M; Glenn, Danielle M; Pereira, Jessica; Ayers, Ryan W; Alshahrani, Mohammad; Acabbo, Christopher; Becker, Maria R; Bruenn, Kimberly N; Cheung, Timothy; Jendras, Taylor M; Shepley, Aron B; Moeller, John T

2016-12-01

Horizontal gene transfer (HGT) in Borrelia burgdorferi, the Lyme disease agent, is likely mediated by bacteriophage. Studies of the B. burgdorferi phage, ϕBB-1 and its role in HGT have been hindered by the lack of an assay for readily characterizing phage-mediated DNA movement (transduction). Here we describe an in vitro assay in which a clone of B. burgdorferi strain CA-11.2A encoding kanamycin resistance on a ϕBB-1 prophage is co-cultured with different clones encoding gentamicin resistance on a shuttle vector; transduction is monitored by enumerating colonies selected in the presence of both kanamycin and gentamicin. When both clones used in the assay were derived from CA-11.2A, the frequency of transduction was 1.23 × 10 -6 transductants per cell, and could be increased 5-fold by exposing the phage-producing strain to 5% ethanol. Transduction was also demonstrated between the CA-11.2A clone and clones of both high-passage B. burgdorferi strain B31 and low-passage, virulent B. burgdorferi strain 297, although with lower transduction frequencies. The transductant in the 297 background produced phage capable of transducing another B. burgdorferi clone: this is the first experimental demonstration of transduction from a clone of a virulent strain. In addition to prophage DNA, small Escherichia coli-derived shuttle vectors were also transduced between co-cultured B. burgdorferi strains, suggesting both a broad role for the phage in the HGT of heterologous DNA and a potential use of the phage as a molecular tool. These results enhance our understanding of phage-mediated transduction as a mechanism of HGT in the Lyme disease spirochetes. Furthermore, the reagents and techniques developed herein will facilitate future studies of phage-mediated HGT, especially within the tick vector and vertebrate host. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The actin-like MreB cytoskeleton organizes viral DNA replication in bacteria.

PubMed

Muñoz-Espín, Daniel; Daniel, Richard; Kawai, Yoshikazu; Carballido-López, Rut; Castilla-Llorente, Virginia; Errington, Jeff; Meijer, Wilfried J J; Salas, Margarita

2009-08-11

Little is known about the organization or proteins involved in membrane-associated replication of prokaryotic genomes. Here we show that the actin-like MreB cytoskeleton of the distantly related bacteria Escherichia coli and Bacillus subtilis is required for efficient viral DNA replication. Detailed analyses of B. subtilis phage ϕ29 showed that the MreB cytoskeleton plays a crucial role in organizing phage DNA replication at the membrane. Thus, phage double-stranded DNA and components of the ϕ29 replication machinery localize in peripheral helix-like structures in a cytoskeleton-dependent way. Importantly, we show that MreB interacts directly with the ϕ29 membrane-protein p16.7, responsible for attaching viral DNA at the cell membrane. Altogether, the results reveal another function for the MreB cytoskeleton and describe a mechanism by which viral DNA replication is organized at the bacterial membrane.

Large-scale maps of variable infection efficiencies in aquatic Bacteroidetes phage-host model systems: Variable phage-host infection interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holmfeldt, Karin; Solonenko, Natalie; Howard-Varona, Cristina

Microbes drive ecosystem functioning and their viruses modulate these impacts through mortality, gene transfer and metabolic reprogramming. Despite the importance of virus-host interactions and likely variable infection efficiencies of individual phages across hosts, such variability is seldom quantified. In this paper, we quantify infection efficiencies of 38 phages against 19 host strains in aquatic Cellulophaga (Bacteroidetes) phage-host model systems. Binary data revealed that some phages infected only one strain while others infected 17, whereas quantitative data revealed that efficiency of infection could vary 10 orders of magnitude, even among phages within one population. This provides a baseline for understanding andmore » modeling intrapopulation host range variation. Genera specific host ranges were also informative. For example, the Cellulophaga Microviridae, showed a markedly broader intra-species host range than previously observed in Escherichia coli systems. Further, one phage genus, Cba41, was examined to investigate nonheritable changes in plating efficiency and burst size that depended on which host strain it most recently infected. While consistent with host modification of phage DNA, no differences in nucleotide sequence or DNA modifications were detected, leaving the observation repeatable, but the mechanism unresolved. Overall, this study highlights the importance of quantitatively considering replication variations in studies of phage-host interactions.« less

Large-scale maps of variable infection efficiencies in aquatic Bacteroidetes phage-host model systems: Variable phage-host infection interactions

DOE PAGES

Holmfeldt, Karin; Solonenko, Natalie; Howard-Varona, Cristina; ...

2016-06-28

Microbes drive ecosystem functioning and their viruses modulate these impacts through mortality, gene transfer and metabolic reprogramming. Despite the importance of virus-host interactions and likely variable infection efficiencies of individual phages across hosts, such variability is seldom quantified. In this paper, we quantify infection efficiencies of 38 phages against 19 host strains in aquatic Cellulophaga (Bacteroidetes) phage-host model systems. Binary data revealed that some phages infected only one strain while others infected 17, whereas quantitative data revealed that efficiency of infection could vary 10 orders of magnitude, even among phages within one population. This provides a baseline for understanding andmore » modeling intrapopulation host range variation. Genera specific host ranges were also informative. For example, the Cellulophaga Microviridae, showed a markedly broader intra-species host range than previously observed in Escherichia coli systems. Further, one phage genus, Cba41, was examined to investigate nonheritable changes in plating efficiency and burst size that depended on which host strain it most recently infected. While consistent with host modification of phage DNA, no differences in nucleotide sequence or DNA modifications were detected, leaving the observation repeatable, but the mechanism unresolved. Overall, this study highlights the importance of quantitatively considering replication variations in studies of phage-host interactions.« less

Advances in the T7 phage display system (Review).

PubMed

Deng, Xiangying; Wang, Li; You, Xiaolong; Dai, Pei; Zeng, Yanhua

2018-01-01

The present review describes the advantages and updated applications of the T7 phage display system in bioscience and medical science. Current phage display systems are based on various bacteriophage vectors, including M13, T7, T4 and f1. Of these, the M13 phage display is the most frequently used, however, the present review highlights the advantages of the T7 system. As a phage display platform, M13 contains single‑stranded DNA, while the T7 phage consists of double‑stranded DNA, which exhibits increased stability and is less prone to mutation during replication. Additional characteristics of the T7 phage include the following: The T7 phage does not depend on a protein secretion pathway in the lytic cycle; expressed peptides and proteins are usually located on the C‑terminal region of capsid protein gp10B, which avoids problems associated with steric hindrance; and T7 phage particles exhibit high stability under various extreme conditions, including high temperature and low pH, which facilitates effective high‑throughput affinity elutriation. Recent applications of the T7 phage display system have been instrumental in uncovering mechanisms of molecular interaction, particularly in the fields of antigen discovery, vaccine development, protein interaction, and cancer diagnosis and treatment.

Phage as a template to grow bone mineral nanocrystals.

PubMed

Cao, Binrui; Xu, Hong; Mao, Chuanbin

2014-01-01

Phage display is a biotechnique that fuses functional peptides on the outer surface of filamentous phage by inserting DNA encoding the peptides into the genes of its coat proteins. The resultant peptide-displayed phage particles have been widely used as biotemplates for the synthesis of functional hybrid nanomaterials. Here, we describe the bioengineering of M13 filamentous phage to surface-display bone mineral (hydroxyapatite (HAP))-nucleating peptides derived from dentin matrix protein-1 and using the engineered phage as a biotemplate to grow HAP nanocrystals.

Complete Genome Sequence of Pseudomonas aeruginosa Phage AAT-1

PubMed Central

Andrade-Domínguez, Andrés

2016-01-01

Aspects of the interaction between phages and animals are of interest and importance for medical applications. Here, we report the genome sequence of the lytic Pseudomonas phage AAT-1, isolated from mammalian serum. AAT-1 is a double-stranded DNA phage, with a genome of 57,599 bp, containing 76 predicted open reading frames. PMID:27563032

DNA packaging and the pathway of bacteriophage T4 head assembly.

PubMed Central

Hsiao, C L; Black, L W

1977-01-01

A cold-sensitive mutation in the structural gene for a minor phage T4 capsid protein (p20) leads to formation of heads containing p20 and cleaved head proteins and empty of DNA. Such heads can be filled with DNA and converted to active phages in vivo uponshift to high temperature. It appears that p20 has two distinct roles in head assembly: first, in construction of the prehead shell (blocked by ts and am mutation) and, second,in DNA packaging (blocked by cs mutation). The latter function is closely associated with gene 17 product, previously known to be required for DNA packagaing. Temperature shift studies of cs-ts double mutants and other observations allow determination of phage function required for DNA packaging. Contrary to previous proposals, we find that T4 DNA packaging is not directly coupled to and can follow DNA synthesis, protein cleavage, prehead core removal, and gene 21-mediated cleavage-induced increase in head volume. Our evidence suggests that an altered head assembly pathway exists and that DNA packaging is probably initiated by DNA-capsid (p20) interaction. Images PMID:269421

A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes.

PubMed Central

Tabor, S; Richardson, C C

1985-01-01

The RNA polymerase gene of bacteriophage T7 has been cloned into the plasmid pBR322 under the inducible control of the lambda PL promoter. After induction, T7 RNA polymerase constitutes 20% of the soluble protein of Escherichia coli, a 200-fold increase over levels found in T7-infected cells. The overproduced enzyme has been purified to homogeneity. During extraction the enzyme is sensitive to a specific proteolysis, a reaction that can be prevented by a modification of lysis conditions. The specificity of T7 RNA polymerase for its own promoters, combined with the ability to inhibit selectively the host RNA polymerase with rifampicin, permits the exclusive expression of genes under the control of a T7 RNA polymerase promoter. We describe such a coupled system and its use to express high levels of phage T7 gene 5 protein, a subunit of T7 DNA polymerase. Images PMID:3156376

Detection and Characterization of Streptococcus thermophilus Bacteriophages by Use of the Antireceptor Gene Sequence

PubMed Central

Binetti, Ana G.; Del Río, Beatriz; Martín, M. Cruz; Álvarez, Miguel A.

2005-01-01

In the dairy industry, the characterization of Streptococcus thermophilus phage types is very important for the selection and use of efficient starter cultures. The aim of this study was to develop a characterization system useful in phage control programs in dairy plants. A comparative study of phages of different origins was initially performed based on their morphology, DNA restriction profiles, DNA homology, structural proteins, packaging mechanisms, and lifestyles and on the presence of a highly conserved DNA fragment of the replication module. However, these traditional criteria were of limited industrial value, mainly because there appeared to be no correlation between these variables and host ranges. We therefore developed a PCR method to amplify VR2, a variable region of the antireceptor gene, which allowed rapid detection of S. thermophilus phages and classification of these phages. This method has a significant advantage over other grouping criteria since our results suggest that there is a correlation between typing profiles and host ranges. This association could be valuable for the dairy industry by allowing a rational starter rotation system to be established and by helping in the selection of more suitable starter culture resistance mechanisms. The method described here is also a useful tool for phage detection, since specific PCR amplification was possible when phage-contaminated milk was used as a template (detection limit, 105 PFU ml−1). PMID:16204526

Single-stranded DNA binding protein Gp5 of Bacillus subtilis phage Φ29 is required for viral DNA replication in growth-temperature dependent fashion.

PubMed

Tone, Takahiro; Takeuchi, Ari; Makino, Osamu

2012-01-01

In the absence of viral single-stranded DNA binding protein gp5, Bacillus subtilis phage φ29 failed to grow and to replicate its genome at 45 °C, while it grew and replicated normally at 30 °C and 42 °C. This indicates that gp5 is dispensable for φ29 DNA replication at 42 °C and lower temperatures.

Origins of the Xylella fastidiosa prophage-like regions and their impact in genome differentiation.

PubMed

de Mello Varani, Alessandro; Souza, Rangel Celso; Nakaya, Helder I; de Lima, Wanessa Cristina; Paula de Almeida, Luiz Gonzaga; Kitajima, Elliot Watanabe; Chen, Jianchi; Civerolo, Edwin; Vasconcelos, Ana Tereza Ribeiro; Van Sluys, Marie-Anne

2008-01-01

Xylella fastidiosa is a Gram negative plant pathogen causing many economically important diseases, and analyses of completely sequenced X. fastidiosa genome strains allowed the identification of many prophage-like elements and possibly phage remnants, accounting for up to 15% of the genome composition. To better evaluate the recent evolution of the X. fastidiosa chromosome backbone among distinct pathovars, the number and location of prophage-like regions on two finished genomes (9a5c and Temecula1), and in two candidate molecules (Ann1 and Dixon) were assessed. Based on comparative best bidirectional hit analyses, the majority (51%) of the predicted genes in the X. fastidiosa prophage-like regions are related to structural phage genes belonging to the Siphoviridae family. Electron micrograph reveals the existence of putative viral particles with similar morphology to lambda phages in the bacterial cell in planta. Moreover, analysis of microarray data indicates that 9a5c strain cultivated under stress conditions presents enhanced expression of phage anti-repressor genes, suggesting switches from lysogenic to lytic cycle of phages under stress-induced situations. Furthermore, virulence-associated proteins and toxins are found within these prophage-like elements, thus suggesting an important role in host adaptation. Finally, clustering analyses of phage integrase genes based on multiple alignment patterns reveal they group in five lineages, all possessing a tyrosine recombinase catalytic domain, and phylogenetically close to other integrases found in phages that are genetic mosaics and able to perform generalized and specialized transduction. Integration sites and tRNA association is also evidenced. In summary, we present comparative and experimental evidence supporting the association and contribution of phage activity on the differentiation of Xylella genomes.

Origins of the Xylella fastidiosa Prophage-Like Regions and Their Impact in Genome Differentiation

PubMed Central

de Mello Varani, Alessandro; Souza, Rangel Celso; Nakaya, Helder I.; de Lima, Wanessa Cristina; Paula de Almeida, Luiz Gonzaga; Kitajima, Elliot Watanabe; Chen, Jianchi; Civerolo, Edwin; Vasconcelos, Ana Tereza Ribeiro; Van Sluys, Marie-Anne

2008-01-01

Xylella fastidiosa is a Gram negative plant pathogen causing many economically important diseases, and analyses of completely sequenced X. fastidiosa genome strains allowed the identification of many prophage-like elements and possibly phage remnants, accounting for up to 15% of the genome composition. To better evaluate the recent evolution of the X. fastidiosa chromosome backbone among distinct pathovars, the number and location of prophage-like regions on two finished genomes (9a5c and Temecula1), and in two candidate molecules (Ann1 and Dixon) were assessed. Based on comparative best bidirectional hit analyses, the majority (51%) of the predicted genes in the X. fastidiosa prophage-like regions are related to structural phage genes belonging to the Siphoviridae family. Electron micrograph reveals the existence of putative viral particles with similar morphology to lambda phages in the bacterial cell in planta. Moreover, analysis of microarray data indicates that 9a5c strain cultivated under stress conditions presents enhanced expression of phage anti-repressor genes, suggesting switches from lysogenic to lytic cycle of phages under stress-induced situations. Furthermore, virulence-associated proteins and toxins are found within these prophage-like elements, thus suggesting an important role in host adaptation. Finally, clustering analyses of phage integrase genes based on multiple alignment patterns reveal they group in five lineages, all possessing a tyrosine recombinase catalytic domain, and phylogenetically close to other integrases found in phages that are genetic mosaics and able to perform generalized and specialized transduction. Integration sites and tRNA association is also evidenced. In summary, we present comparative and experimental evidence supporting the association and contribution of phage activity on the differentiation of Xylella genomes. PMID:19116666

Electron microscopic studies of bacteriophage M13 DNA replication. [Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Allison, D.P.; Ganesan, A.T.; Olson, A.C.

Intracellular forms of M13 phage DNA isolated after infection of Escherichia coli with wild-type phage have been studied by electron microscopy and ultracentrifugation. The data indicate the involvement of rolling-circle intermediates in single-stranded DNA synthesis. In addition to single-stranded, circular DNA, we observed covalently closed and nicked replicative-form (RF) DNAs, dimer RF DNAs, concatenated RF DNAs, RF DNAs with single-stranded tails (sigma, rolling circles), and, occasionally, RF DNAs with theta structures. The tails in sigma molecules are always single stranded and are never longer than the DNA from mature phage; the proportion of sigma to other RF molecules does notmore » change significantly with time after infection. The origin of single-stranded DNA synthesis has been mapped by electron microscopy at a unique location on RF DNA by use of partial denaturation mapping and restriction endonuclease digestion. This location is between gene IV and gene II, and synthesis proceeds in a counterclockwise direction on the conventional genetic map.« less

Agarose Gel Electrophoresis Reveals Structural Fluidity of a Phage T3 DNA Packaging Intermediate

PubMed Central

Serwer, Philip; Wright, Elena T.

2012-01-01

We find a new aspect of DNA packaging-associated structural fluidity for phage T3 capsids. The procedure is (1) glutaraldehyde cross-linking of in vivo DNA packaging intermediates for stabilization of structure and then (2) determining of effective radius by two-dimensional agarose gel electrophoresis (2d-AGE). The intermediates are capsids with incompletely packaged DNA (ipDNA) and without an external DNA segment; these intermediates are called ipDNA-capsids. We initially increase production of ipDNA-capsids by raising NaCl concentration during in vivo DNA packaging. By 2d-AGE, we find a new state of contracted shell for some particles of one previously identified ipDNA-capsid. The contracted shell-state is found when ipDNA length/mature DNA length (F) is above 0.17, but not at lower F. Some contracted-shell ipDNA-capsids have the phage tail; others do not. The contracted-shell ipDNA-capsids are explained by premature DNA maturation cleavage that makes accessible a contracted-shell intermediate of a cycle of the T3 DNA packaging motor. The analysis of ipDNA-capsids, rather than intermediates with uncleaved DNA, provides a simplifying strategy for a complete biochemical analysis of in vivo DNA packaging. PMID:22222979

Genomes and Characterization of Phages Bcep22 and BcepIL02, Founders of a Novel Phage Type in Burkholderia cenocepacia▿†

PubMed Central

Gill, Jason J.; Summer, Elizabeth J.; Russell, William K.; Cologna, Stephanie M.; Carlile, Thomas M.; Fuller, Alicia C.; Kitsopoulos, Kate; Mebane, Leslie M.; Parkinson, Brandi N.; Sullivan, David; Carmody, Lisa A.; Gonzalez, Carlos F.; LiPuma, John J.; Young, Ry

2011-01-01

Within the Burkholderia cepacia complex, B. cenocepacia is the most common species associated with aggressive infections in the lungs of cystic fibrosis patients, causing disease that is often refractive to treatment by antibiotics. Phage therapy may be a potential alternative form of treatment for these infections. Here we describe the genome of the previously described therapeutic B. cenocepacia podophage BcepIL02 and its close relative, Bcep22. Phage Bcep22 was found to contain a circularly permuted genome of 63,882 bp containing 77 genes; BcepIL02 was found to be 62,714 bp and contains 76 predicted genes. Major virion-associated proteins were identified by proteomic analysis. We propose that these phages comprise the founding members of a novel podophage lineage, the Bcep22-like phages. Among the interesting features of these phages are a series of tandemly repeated putative tail fiber genes that are similar to each other and also to one or more such genes in the other phages. Both phages also contain an extremely large (ca. 4,600-amino-acid), virion-associated, multidomain protein that accounts for over 20% of the phages' coding capacity, is widely distributed among other bacterial and phage genomes, and may be involved in facilitating DNA entry in both phage and other mobile DNA elements. The phages, which were previously presumed to be virulent, show evidence of a temperate lifestyle but are apparently unable to form stable lysogens in their hosts. This ambiguity complicates determination of a phage lifestyle, a key consideration in the selection of therapeutic phages. PMID:21804006

Genome packaging in EL and Lin68, two giant phiKZ-like bacteriophages of P. aeruginosa.

PubMed

Sokolova, O S; Shaburova, O V; Pechnikova, E V; Shaytan, A K; Krylov, S V; Kiselev, N A; Krylov, V N

2014-11-01

A unique feature of the Pseudomonas aeruginosa giant phage phiKZ is its way of genome packaging onto a spool-like protein structure, the inner body. Until recently, no similar structures have been detected in other phages. We have studied DNA packaging in P. aeruginosa phages EL and Lin68 using cryo-electron microscopy and revealed the presence of inner bodies. The shape and positioning of the inner body and the density of the DNA packaging in EL are different from those found in phiKZ and Lin68. This internal organization explains how the shorter EL genome is packed into a large EL capsid, which has the same external dimensions as the capsids of phiKZ and Lin68. The similarity in the structural organization in EL and other phiKZ-like phages indicates that EL is phylogenetically related to other phiKZ-like phages, and, despite the lack of detectable DNA homology, EL, phiKZ, and Lin68 descend from a common ancestor. Copyright © 2014 Elsevier Inc. All rights reserved.

PuLSE: Quality control and quantification of peptide sequences explored by phage display libraries.

PubMed

Shave, Steven; Mann, Stefan; Koszela, Joanna; Kerr, Alastair; Auer, Manfred

2018-01-01

The design of highly diverse phage display libraries is based on assumption that DNA bases are incorporated at similar rates within the randomized sequence. As library complexity increases and expected copy numbers of unique sequences decrease, the exploration of library space becomes sparser and the presence of truly random sequences becomes critical. We present the program PuLSE (Phage Library Sequence Evaluation) as a tool for assessing randomness and therefore diversity of phage display libraries. PuLSE runs on a collection of sequence reads in the fastq file format and generates tables profiling the library in terms of unique DNA sequence counts and positions, translated peptide sequences, and normalized 'expected' occurrences from base to residue codon frequencies. The output allows at-a-glance quantitative quality control of a phage library in terms of sequence coverage both at the DNA base and translated protein residue level, which has been missing from toolsets and literature. The open source program PuLSE is available in two formats, a C++ source code package for compilation and integration into existing bioinformatics pipelines and precompiled binaries for ease of use.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bradbury, Andrew M.

The invention relates to a novel phagemid display system for packaging phagemid DNA into phagemid particles which completely avoids the use of helper phage. The system of the invention incorporates the use of bacterial packaging cell lines which have been transformed with helper plasmids containing all required phage proteins but not the packaging signals. The absence of packaging signals in these helper plasmids prevents their DNA from being packaged in the bacterial cell, which provides a number of significant advantages over the use of both standard and modified helper phage. Packaged phagemids expressing a protein or peptide of interest, inmore » fusion with a phage coat protein such as g3p, are generated simply by transfecting phagemid into the packaging cell line.« less

The identification of cellular targets of 17β estradiol using a lytic (T7) cDNA phage display approach.

PubMed

Van Dorst, Bieke; Mehta, Jaytry; Rouah-Martin, Elsa; De Coen, Wim; Blust, Ronny; Robbens, Johan

2011-02-01

To unravel the mechanism of action of chemical compounds, it is crucial to know their cellular targets. A novel in vitro tool that can be used as a fast, simple and cost effective alternative is cDNA phage display. This tool is used in our study to select cellular targets of 17β estradiol (E2). It was possible to select two potential cellular targets of E2 out of the T7 Select™ Human Breast cDNA phage library. The selected cellular targets, autophagy/beclin-1 regulator 1 (beclin 1) and ATP synthase F(0) subunit 6 (ATP6) have so far been unknown as binding proteins of E2. To confirm the E2 binding properties of these selected proteins, surface plasmon resonance (SPR) was used. With SPR the K(d) values were determined to be 0.178±0.031 and 0.401±0.142 nM for the ATP6 phage and beclin 1 phage, respectively. These K(d) values in the low nM range verify that the selected cellular proteins are indeed binding proteins for E2. The selection and identification of these two potential cellular targets of E2, can enhance our current understanding of its mechanism of action. This illustrates the potential of lytic (T7) cDNA phage display in toxicology, to provide important information about cellular targets of chemical compounds. Copyright © 2010 Elsevier Ltd. All rights reserved.

Phage particles harboring antibiotic resistance genes in fresh-cut vegetables and agricultural soil.

PubMed

Larrañaga, Olatz; Brown-Jaque, Maryury; Quirós, Pablo; Gómez-Gómez, Clara; Blanch, Anicet R; Rodríguez-Rubio, Lorena; Muniesa, Maite

2018-06-01

Bacteriophages are ubiquitously distributed prokaryotic viruses that are more abundant than bacteria. As a consequence of their life cycle, phages can kidnap part of their host's genetic material, including antibiotic resistance genes (ARGs), which released phage particles transfer in a process called transduction. The spread of ARGs among pathogenic bacteria currently constitutes a serious global health problem. In this study, fresh vegetables (lettuce, spinach and cucumber), and cropland soil were screened by qPCR for ten ARGs (bla TEM , bla CTX-M-1 group, bla CTX-M-9 group, bla OXA-48 , bla VIM , mecA, sul1, qnrA, qnrS and armA) in their viral DNA fraction. The presence of ARGs in the phage DNA was analyzed before and after propagation experiments in an Escherichia coli host strain to evaluate the ability of the phage particles to infect a host. ARGs were found in the phage DNA fraction of all matrices, although with heterogeneous values. ARG prevalence was significantly higher in lettuce and soil, and the most common overall were β-lactamases. After propagation experiments, an increase in ARG densities in phage particles was observed in samples of all four matrices, confirming that part of the isolated phage particles were infectious. This study reveals the abundance of free, replicative ARG-containing phage particles in vegetable matrices and cropland soil. The particles are proposed as vehicles for resistance transfer in these environments, where they can persist for a long time, with the possibility of generating new resistant bacterial strains. Ingestion of these mobile genetic elements may also favor the emergence of new resistances, a risk not previously considered. Copyright © 2018 Elsevier Ltd. All rights reserved.

Anti-liver-kidney microsome antibody type 1 recognizes human cytochrome P450 db1.

PubMed

Gueguen, M; Yamamoto, A M; Bernard, O; Alvarez, F

1989-03-15

Anti-liver-kidney microsome antibody type 1 (LKM1), present in the sera of a group of children with autoimmune hepatitis, was recently shown to recognize a 50 kDa protein identified as rat liver cytochromes P450 db1 and db2. High homology between these two members of the rat P450 IID subfamily and human P450 db1 suggested that anti-LKM1 antibody is directed against this human protein. To test this hypothesis, a human liver cDNA expression library in phage lambda GT-11 was screened using rat P450 db1 cDNA as a probe. Two human cDNA clones were found to be identical to human P450 db1 by restriction mapping. Immunoblot analysis using as antigen, the purified fusion protein from one of the human cDNA clones showed that only anti-LKM1 with anti-50 kDa reactivity recognized the fusion protein. This fusion protein was further used to develop an ELISA test that was shown to be specific for sera of children with this disease. These results: 1) identify the human liver antigen recognized by anti-LKM1 auto-antibodies as cytochrome P450 db1, 2) allow to speculate that mutation on the human P450 db1 gene could alter its expression in the hepatocyte and make it auto-antigenic, 3) provide a simple and specific diagnostic test for this disease.

An integrated optics microfluidic device for detecting single DNA molecules.

PubMed

Krogmeier, Jeffrey R; Schaefer, Ian; Seward, George; Yantz, Gregory R; Larson, Jonathan W

2007-12-01

A fluorescence-based integrated optics microfluidic device is presented, capable of detecting single DNA molecules in a high throughput and reproducible manner. The device integrates microfluidics for DNA stretching with two optical elements for single molecule detection (SMD): a plano-aspheric refractive lens for fluorescence excitation (illuminator) and a solid parabolic reflective mirror for fluorescence collection (collector). Although miniaturized in size, both optical components were produced and assembled onto the microfluidic device by readily manufacturable fabrication techniques. The optical resolution of the device is determined by the small and relatively low numerical aperture (NA) illuminator lens (0.10 effective NA, 4.0 mm diameter) that delivers excitation light to a diffraction limited 2.0 microm diameter spot at full width half maximum within the microfluidic channel. The collector (0.82 annular NA, 15 mm diameter) reflects the fluorescence over a large collection angle, representing 71% of a hemisphere, toward a single photon counting module in an infinity-corrected scheme. As a proof-of-principle experiment for this simple integrated device, individual intercalated lambda-phage DNA molecules (48.5 kb) were stretched in a mixed elongational-shear microflow, detected, and sized with a fluorescence signal to noise ratio of 9.9 +/-1.0. We have demonstrated that SMD does not require traditional high numerical aperture objective lenses and sub-micron positioning systems conventionally used in many applications. Rather, standard manufacturing processes can be combined in a novel way that promises greater accessibility and affordability for microfluidic-based single molecule applications.

Constitutive Expression of a Transcription Termination Factor by a Repressed Prophage: Promoters for Transcribing the Phage HK022 nun Gene

PubMed Central

King, Rodney A.; Madsen, Peter L.; Weisberg, Robert A.

2000-01-01

Lysogens of phage HK022 are resistant to infection by phage λ. Lambda resistance is caused by the action of the HK022 Nun protein, which prematurely terminates early λ transcripts. We report here that transcription of the nun gene initiates at a constitutive prophage promoter, PNun, located just upstream of the protein coding sequence. The 5′ end of the transcript was determined by primer extension analysis of RNA isolated from HK022 lysogens or RNA made in vitro by transcribing a template containing the promoter with purified Escherichia coli RNA polymerase. Inactivation of PNun by mutation greatly reduced Nun activity and Nun antigen in an HK022 lysogen. However, a low level of residual activity was detected, suggesting that a secondary promoter also contributes to nun expression. We found one possible secondary promoter, PNun′, just upstream of PNun. Neither promoter is likely to increase the expression of other phage genes in a lysogen because their transcripts should be terminated downstream of nun. We estimate that HK022 lysogens in stationary phase contain several hundred molecules of Nun per cell and that cells in exponential phase probably contain fewer. PMID:10629193

Communication: Origin of the contributions to DNA structure in phages

PubMed Central

Myers, Christopher G.; Pettitt, B. Montgomery

2013-01-01

Cryo electron microscopy (cryo-EM) data of the interior of phages show ordering of the interior DNA that has been interpreted as a nearly perfectly ordered polymer. We show surface-induced correlations, excluded volume, and electrostatic forces are sufficient to predict most of the major features of the current structural data for DNA packaged within viral capsids without additional ordering due to elastic bending forces for the polymer. Current models assume highly-ordered, even spooled, hexagonally packed conformations based on interpretation of cryo-EM density maps. We show herein that the surface induced packing of short (6mer), unconnected DNA polymer segments is the only necessary ingredient in creating ringed densities consistent with experimental density maps. This implies the ensemble of possible conformations of polymeric DNA within the capsid that are consistent with cryo-EM data may be much larger than implied by traditional interpretations where such rings can only result from highly-ordered spool-like conformations. This opens the possibility of a more disordered, entropically-driven view of phage packaging thermodynamics. We also show the electrostatics of the DNA contributes a large portion of the internal hydrostatic and osmotic pressures of a phage virion, suggesting that nonlinear elastic anomalies might reduce the overall elastic bending enthalpy of more disordered conformations to have allowable free energies. PMID:23444988

Communication: Origin of the contributions to DNA structure in phages.

PubMed

Myers, Christopher G; Pettitt, B Montgomery

2013-02-21

Cryo electron microscopy (cryo-EM) data of the interior of phages show ordering of the interior DNA that has been interpreted as a nearly perfectly ordered polymer. We show surface-induced correlations, excluded volume, and electrostatic forces are sufficient to predict most of the major features of the current structural data for DNA packaged within viral capsids without additional ordering due to elastic bending forces for the polymer. Current models assume highly-ordered, even spooled, hexagonally packed conformations based on interpretation of cryo-EM density maps. We show herein that the surface induced packing of short (6mer), unconnected DNA polymer segments is the only necessary ingredient in creating ringed densities consistent with experimental density maps. This implies the ensemble of possible conformations of polymeric DNA within the capsid that are consistent with cryo-EM data may be much larger than implied by traditional interpretations where such rings can only result from highly-ordered spool-like conformations. This opens the possibility of a more disordered, entropically-driven view of phage packaging thermodynamics. We also show the electrostatics of the DNA contributes a large portion of the internal hydrostatic and osmotic pressures of a phage virion, suggesting that nonlinear elastic anomalies might reduce the overall elastic bending enthalpy of more disordered conformations to have allowable free energies.

Isolation, Characterization, and Bioinformatic Analyses of Lytic Salmonella Enteritidis Phages and Tests of Their Antibacterial Activity in Food.

PubMed

Han, Han; Wei, Xiaoting; Wei, Yi; Zhang, Xiufeng; Li, Xuemin; Jiang, Jinzhong; Wang, Ran

2017-02-01

Salmonella Enteritidis remains a major threat for food safety. To take efforts to develop phage-based biocontrol for S. Enteritidis contamination in food, in this study, the phages against S. Enteritidis were isolated from sewage samples, characterized by host range assays, DNA restriction enzyme pattern analyses, and transmission electron microscope observations, and tested for antibacterial activity in food; some potent phages were further characterized by bioinformatic analyses. Results showed that based on the plaque quality and host range, seven lytic phages targeting S. Enteritidis were selected, considered as seven distinct phages through DNA physical maps, and classified as Myoviridae or Siphoviridae family by morphologic observations; the combined use of such seven strain phages as a "food additive" could succeed in controlling the artificial S. Enteritidis contamination in the different physical forms of food at a range of temperatures; by bioinformatic analyses, both selected phage BPS 11 Q 3 and BPS 15 Q 2 seemed to be newfound obligate lytic phage strains with no indications for any potentially harmful genes in their genomes. In conclusion, our results showed a potential of isolated phages as food additives for controlling S. Enteritidis contamination in some salmonellosis outbreak-associated food vehicles, and there could be minimized potential risk associated with using BPS 11 Q 3 and BPS 15 Q 2 in food.

Sak and Sak4 recombinases are required for bacteriophage replication in Staphylococcus aureus.

PubMed

Neamah, Maan M; Mir-Sanchis, Ignacio; López-Sanz, María; Acosta, Sonia; Baquedano, Ignacio; Haag, Andreas F; Marina, Alberto; Ayora, Silvia; Penadés, José R

2017-06-20

DNA-single strand annealing proteins (SSAPs) are recombinases frequently encoded in the genome of many bacteriophages. As SSAPs can promote homologous recombination among DNA substrates with an important degree of divergence, these enzymes are involved both in DNA repair and in the generation of phage mosaicisms. Here, analysing Sak and Sak4 as representatives of two different families of SSAPs present in phages infecting the clinically relevant bacterium Staphylococcus aureus, we demonstrate for the first time that these enzymes are absolutely required for phage reproduction. Deletion of the genes encoding these enzymes significantly reduced phage replication and the generation of infectious particles. Complementation studies revealed that these enzymes are required both in the donor (after prophage induction) and in the recipient strain (for infection). Moreover, our results indicated that to perform their function SSAPs require the activity of their cognate single strand binding (Ssb) proteins. Mutational studies demonstrated that the Ssb proteins are also required for phage replication, both in the donor and recipient strain. In summary, our results expand the functions attributed to the Sak and Sak4 proteins, and demonstrate that both SSAPs and Ssb proteins are essential for the life cycle of temperate staphylococcal phages. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Full shut-off of Escherichia coli RNA-polymerase by T7 phage requires a small phage-encoded DNA-binding protein.

PubMed

Tabib-Salazar, Aline; Liu, Bing; Shadrin, Andrey; Burchell, Lynn; Wang, Zhexin; Wang, Zhihao; Goren, Moran G; Yosef, Ido; Qimron, Udi; Severinov, Konstantin; Matthews, Steve J; Wigneshweraraj, Sivaramesh

2017-07-27

Infection of Escherichia coli by the T7 phage leads to rapid and selective inhibition of the bacterial RNA polymerase (RNAP) by the 7 kDa T7 protein Gp2. We describe the identification and functional and structural characterisation of a novel 7 kDa T7 protein, Gp5.7, which adopts a winged helix-turn-helix-like structure and specifically represses transcription initiation from host RNAP-dependent promoters on the phage genome via a mechanism that involves interaction with DNA and the bacterial RNAP. Whereas Gp2 is indispensable for T7 growth in E. coli, we show that Gp5.7 is required for optimal infection outcome. Our findings provide novel insights into how phages fine-tune the activity of the host transcription machinery to ensure both successful and efficient phage progeny development. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Bacteriophage T4 capsid packaging and unpackaging of DNA and proteins.

PubMed

Mullaney, Julienne M; Black, Lindsay W

2014-01-01

Bacteriophage T4 has proven itself readily amenable to phage-based DNA and protein packaging, expression, and display systems due to its physical resiliency and genomic flexibility. As a large dsDNA phage with dispensable internal proteins and dispensable outer capsid proteins it can be adapted to package both DNA and proteins of interest within the capsid and to display peptides and proteins externally on the capsid. A single 170 kb linear DNA, or single or multiple copies of shorter linear DNAs, of any sequence can be packaged by the large terminase subunit in vitro into protein-containing proheads and give full or partially full capsids. The prohead receptacles for DNA packaging can also display peptides or full-length proteins from capsid display proteins HOC and SOC. Our laboratory has also developed a protein expression, packaging, and processing (PEPP) system which we have found to have advantages over mammalian and bacterial cell systems, including high yield, increased stability, and simplified downstream processing. Proteins that we have produced by the phage PEPP platform include human HIV-1 protease, micrococcal endonuclease from Staphylococcus aureus, restriction endonuclease EcoRI, luciferase, human granulocyte colony stimulating factor (GCSF), green fluorescent protein (GFP), and the 99 amino acid C-terminus of amyloid precursor protein (APP). Difficult to produce proteins that are toxic in mammalian protein expression systems are easily produced, packaged, and processed with the PEPP platform. APP is one example of such a highly refractory protein that has been produced successfully. The methods below describe the procedures for in vitro packaging of proheads with DNA and for producing recombinant T4 phage that carry a gene of interest in the phage genome and produce and internally package the corresponding protein of interest.

Bacteriophage prevalence in the genus Azospirillum and analysis of the first genome sequence of an Azospirillum brasilense integrative phage.

PubMed

Boyer, Mickaël; Haurat, Jacqueline; Samain, Sylvie; Segurens, Béatrice; Gavory, Frédérick; González, Víctor; Mavingui, Patrick; Rohr, René; Bally, René; Wisniewski-Dyé, Florence

2008-02-01

The prevalence of bacteriophages was investigated in 24 strains of four species of plant growth-promoting rhizobacteria belonging to the genus Azospirillum. Upon induction by mitomycin C, the release of phage particles was observed in 11 strains from three species. Transmission electron microscopy revealed two distinct sizes of particles, depending on the identity of the Azospirillum species, typical of the Siphoviridae family. Pulsed-field gel electrophoresis and hybridization experiments carried out on phage-encapsidated DNAs revealed that all phages isolated from A. lipoferum and A. doebereinerae strains had a size of about 10 kb whereas all phages isolated from A. brasilense strains displayed genome sizes ranging from 62 to 65 kb. Strong DNA hybridizing signals were shown for most phages hosted by the same species whereas no homology was found between phages harbored by different species. Moreover, the complete sequence of the A. brasilense Cd bacteriophage (phiAb-Cd) genome was determined as a double-stranded DNA circular molecule of 62,337 pb that encodes 95 predicted proteins. Only 14 of the predicted proteins could be assigned functions, some of which were involved in DNA processing, phage morphogenesis, and bacterial lysis. In addition, the phiAb-Cd complete genome was mapped as a prophage on a 570-kb replicon of strain A. brasilense Cd, and a region of 27.3 kb of phiAb-Cd was found to be duplicated on the 130-kb pRhico plasmid previously sequenced from A. brasilense Sp7, the parental strain of A. brasilense Cd.

An RNA Phage Lab: MS2 in Walter Fiers' laboratory of molecular biology in Ghent, from genetic code to gene and genome, 1963-1976.

PubMed

Pierrel, Jérôme

2012-01-01

The importance of viruses as model organisms is well-established in molecular biology and Max Delbrück's phage group set standards in the DNA phage field. In this paper, I argue that RNA phages, discovered in the 1960s, were also instrumental in the making of molecular biology. As part of experimental systems, RNA phages stood for messenger RNA (mRNA), genes and genome. RNA was thought to mediate information transfers between DNA and proteins. Furthermore, RNA was more manageable at the bench than DNA due to the availability of specific RNases, enzymes used as chemical tools to analyse RNA. Finally, RNA phages provided scientists with a pure source of mRNA to investigate the genetic code, genes and even a genome sequence. This paper focuses on Walter Fiers' laboratory at Ghent University (Belgium) and their work on the RNA phage MS2. When setting up his Laboratory of Molecular Biology, Fiers planned a comprehensive study of the virus with a strong emphasis on the issue of structure. In his lab, RNA sequencing, now a little-known technique, evolved gradually from a means to solve the genetic code, to a tool for completing the first genome sequence. Thus, I follow the research pathway of Fiers and his 'RNA phage lab' with their evolving experimental system from 1960 to the late 1970s. This study illuminates two decisive shifts in post-war biology: the emergence of molecular biology as a discipline in the 1960s in Europe and of genomics in the 1990s.

Diagnostic value of protein chips constructed by lung-cancer-associated markers selected by the T7 phage display library.

PubMed

Li, Hong-Mei; Guo, Kang; Yu, Zhuang; Feng, Rui; Xu, Ping

2015-07-01

Traditional diagnostic technology with tumor biomarkers is inefficient, expensive and requires a large number of serum samples. The purpose of this study was to construct human lung cancer protein chips with new lung cancer biomarkers screened by the T7-phage display library, and improve the early diagnosis rate of lung cancer. A T7-phage cDNA display library was constructed of fresh samples from 30 lung cancer patients. With biopanning and high-throughput screening, we gained the immunogenic phage clones from the cDNA library. The insert of selected phage was blasted at GeneBank for alignment to find the exact or the most similar known genes. Protein chips were then constructed and used to assay their expression level in lung cancer serum from 217 cases of lung cancer groups:80 cases of benign lung disease and 220 healthy controls. After four rounds of Biopanning and two rounds of enzyme-linked immunosorbent assay, 12 phage monoclonal samples were selected from 2880 phage monoclonal samples. After blasting at GeneBank, six similar genes were used to construct diagnostic protein chips. The protein chips were then used to assay expression level in lung cancer serum. The expression level of six genes in lung cancer groups was significantly higher than those in the other two groups (P < 0.05). In this study, we successfully constructed diagnostic protein chips with biomarkers selected from the lung cancer T7-phage cDNA library, which can be used for the early screening of lung cancer patients.

Kilo-sequencing: an ordered strategy for rapid DNA sequence data acquisition.

PubMed Central

Barnes, W M; Bevan, M

1983-01-01

A strategy for rapid DNA sequence acquisition in an ordered, nonrandom manner, while retaining all of the conveniences of the dideoxy method with M13 transducing phage DNA template, is described. Target DNA 3 to 14 kb in size can be stably carried by our M13 vectors. Suitable targets are stretches of DNA which lack an enzyme recognition site which is unique on our cloning vectors and adjacent to the sequencing primer; current sites that are so useful when lacking are Pst, Xba, HindIII, BglII, EcoRI. By an in vitro procedure, we cut RF DNA once randomly and once specifically, to create thousands of deletions which start at the unique restriction site adjacent to the dideoxy sequencing primer and extend various distances across the target DNA. Phage carrying a desired size of deletions, whose DNA as template will give rise to DNA sequence data in a desired location along the target DNA, may be purified by electrophoresis alive on agarose gels. Phage running in the same location on the agarose gel thus conveniently give rise to nucleotide sequence data from the same kilobase of target DNA. Images PMID:6298723

Overexpression of the DNA mismatch repair factor, PMS2, confers hypermutability and DNA damage tolerance.

PubMed

Gibson, Shannon L; Narayanan, Latha; Hegan, Denise Campisi; Buermeyer, Andrew B; Liskay, R Michael; Glazer, Peter M

2006-12-08

Inherited defects in genes associated with DNA mismatch repair (MMR) have been linked to familial colorectal cancer. Cells deficient in MMR are genetically unstable and demonstrate a tolerance phenotype in response to certain classes of DNA damage. Some sporadic human cancers also show abnormalities in MMR gene function, typically due to diminished expression of one of the MutL homologs, MLH1. Here, we report that overexpression of the MutL homolog, human PMS2, can also cause a disruption of the MMR pathway in mammalian cells, resulting in hypermutability and DNA damage tolerance. A mouse fibroblast cell line carrying a recoverable lambda phage shuttle vector for mutation detection was transfected with either a vector designed to express hPMS2 or with an empty vector control. Cells overexpressing hPMS2 were found to have elevated spontaneous mutation frequencies at the cII reporter gene locus. They also showed an increase in the level of mutations induced by the alkylating agent, methynitrosourea (MNU). Clonogenic survival assays demonstrated increased survival of the PMS2-overexpressing cells following exposure to MNU, consistent with the induction of a damage tolerance phenotype. Similar results were seen in cells expressing a mutant PMS2 gene, containing a premature stop codon at position 134 and representing a variant found in an individual with familial colon cancer. These results show that dysregulation of PMS2 gene expression can disrupt MMR function in mammalian cells and establish an additional carcinogenic mechanism by which cells can develop genetic instability and acquire resistance to cytotoxic cancer therapies.

Complete Genome Sequence of Pseudomonas aeruginosa Phage AAT-1.

PubMed

Andrade-Domínguez, Andrés; Kolter, Roberto

2016-08-25

Aspects of the interaction between phages and animals are of interest and importance for medical applications. Here, we report the genome sequence of the lytic Pseudomonas phage AAT-1, isolated from mammalian serum. AAT-1 is a double-stranded DNA phage, with a genome of 57,599 bp, containing 76 predicted open reading frames. Copyright © 2016 Andrade-Domínguez and Kolter.

Evolved Populations of Shigella flexneri Phage Sf6 Acquire Large Deletions, Altered Genomic Architecture, and Faster Life Cycles.

PubMed

Dover, John A; Burmeister, Alita R; Molineux, Ian J; Parent, Kristin N

2016-09-19

Genomic architecture is the framework within which genes and regulatory elements evolve and where specific constructs may constrain or potentiate particular adaptations. One such construct is evident in phages that use a headful packaging strategy that results in progeny phage heads packaged with DNA until full rather than encapsidating a simple unit-length genome. Here, we investigate the evolution of the headful packaging phage Sf6 in response to barriers that impede efficient phage adsorption to the host cell. Ten replicate populations evolved faster Sf6 life cycles by parallel mutations found in a phage lysis gene and/or by large, 1.2- to 4.0-kb deletions that remove a mobile genetic IS911 element present in the ancestral phage genome. The fastest life cycles were found in phages that acquired both mutations. No mutations were found in genes encoding phage structural proteins, which were a priori expected from the experimental design that imposed a challenge for phage adsorption by using a Shigella flexneri host lacking receptors preferred by Sf6. We used DNA sequencing, molecular approaches, and physiological experiments on 82 clonal isolates taken from all 10 populations to reveal the genetic basis of the faster Sf6 life cycle. The majority of our isolates acquired deletions in the phage genome. Our results suggest that deletions are adaptive and can influence the duration of the phage life cycle while acting in conjunction with other lysis time-determining point mutations. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

The Caulobacter crescentus phage phiCbK: genomics of a canonical phage

PubMed Central

2012-01-01

Background The bacterium Caulobacter crescentus is a popular model for the study of cell cycle regulation and senescence. The large prolate siphophage phiCbK has been an important tool in C. crescentus biology, and has been studied in its own right as a model for viral morphogenesis. Although a system of some interest, to date little genomic information is available on phiCbK or its relatives. Results Five novel phiCbK-like C. crescentus bacteriophages, CcrMagneto, CcrSwift, CcrKarma, CcrRogue and CcrColossus, were isolated from the environment. The genomes of phage phiCbK and these five environmental phage isolates were obtained by 454 pyrosequencing. The phiCbK-like phage genomes range in size from 205 kb encoding 318 proteins (phiCbK) to 280 kb encoding 448 proteins (CcrColossus), and were found to contain nonpermuted terminal redundancies of 10 to 17 kb. A novel method of terminal ligation was developed to map genomic termini, which confirmed termini predicted by coverage analysis. This suggests that sequence coverage discontinuities may be useable as predictors of genomic termini in phage genomes. Genomic modules encoding virion morphogenesis, lysis and DNA replication proteins were identified. The phiCbK-like phages were also found to encode a number of intriguing proteins; all contain a clearly T7-like DNA polymerase, and five of the six encode a possible homolog of the C. crescentus cell cycle regulator GcrA, which may allow the phage to alter the host cell’s replicative state. The structural proteome of phage phiCbK was determined, identifying the portal, major and minor capsid proteins, the tail tape measure and possible tail fiber proteins. All six phage genomes are clearly related; phiCbK, CcrMagneto, CcrSwift, CcrKarma and CcrRogue form a group related at the DNA level, while CcrColossus is more diverged but retains significant similarity at the protein level. Conclusions Due to their lack of any apparent relationship to other described phages, this group is proposed as the founding cohort of a new phage type, the phiCbK-like phages. This work will serve as a foundation for future studies on morphogenesis, infection and phage-host interactions in C. crescentus. PMID:23050599

An Empirically Calibrated Model of Cell Fate Decision Following Viral Infection

NASA Astrophysics Data System (ADS)

Coleman, Seth; Igoshin, Oleg; Golding, Ido

The life cycle of the virus (phage) lambda is an established paradigm for the way genetic networks drive cell fate decisions. But despite decades of interrogation, we are still unable to theoretically predict whether the infection of a given cell will result in cell death or viral dormancy. The poor predictive power of current models reflects the absence of quantitative experimental data describing the regulatory interactions between different lambda genes. To address this gap, we are constructing a theoretical model that captures the known interactions in the lambda network. Model assumptions and parameters are calibrated using new single-cell data from our lab, describing the activity of lambda genes at single-molecule resolution. We began with a mean-field model, aimed at exploring the population averaged gene-expression trajectories under different initial conditions. Next, we will develop a stochastic formulation, to capture the differences between individual cells within the population. The eventual goal is to identify how the post-infection decision is driven by the interplay between network topology, initial conditions, and stochastic effects. The insights gained here will inform our understanding of cell fate choices in more complex cellular systems.

Complete genome sequence of a phage hyperparasite of Candidatus Xenohaliotis californiensis (Rickettsiales) - a pathogen of Haliotis spp (Gasteropoda).

PubMed

Cruz-Flores, Roberto; Cáceres-Martínez, Jorge; Del Río-Portilla, Miguel Ángel; Licea-Navarro, Alexei F; Gonzales-Sánchez, Ricardo; Guerrero, Abraham

2018-04-01

Bacteriophages are recognized as major mortality agents of microbes, among them intracellular marine rickettsiales-like bacteria. Recently, a phage hyperparasite of Candidatus Xenohaliotis californiensis (CXc) has been described. This bacterium is considered the causal agent of Withering Syndrome (WS) which is a chronic and potentially lethal disease of abalone species from California, USA and the peninsula of Baja California, Mexico. This hyperparasite which infects CXc could be used as a biocontrol agent for WS. Therefore, it is necessary to obtain genomic information to characterize this phage. In this study, the first complete genome sequence of a novel phage, Xenohaliotis phage (pCXc) was determined. The complete genome of pCXc from red abalone (Haliotis rufescens) is 35,728 bp, while the complete genome of pCXc from yellow abalone (Haliotis corrugata) is 35,736 bp. Both phage genomes consist of double-stranded DNA with a G + C content of 38.9%. In both genomes 33 open reading frames (ORFs) were predicted. Only 10 ORFs encode proteins that have identifiable functional homologues. These 10 ORFs were classified by function, including structural, DNA replication, DNA packaging, nucleotide transport and metabolism, life cycle regulation, recombination and repair, and additional functions. A PCR method for the specific detection of pCXc was developed. This information will help to understand a new group of phages that infect intracellular marine rickettsiales-like bacteria in mollusks.

Locating and Activating Molecular ‘Time Bombs’: Induction of Mycolata Prophages

PubMed Central

Dyson, Zoe A.; Brown, Teagan L.; Farrar, Ben; Doyle, Stephen R.; Tucci, Joseph; Seviour, Robert J.; Petrovski, Steve

2016-01-01

Little is known about the prevalence, functionality and ecological roles of temperate phages for members of the mycolic acid producing bacteria, the Mycolata. While many lytic phages infective for these organisms have been isolated, and assessed for their suitability for use as biological control agents of activated sludge foaming, no studies have investigated how temperate phages might be induced for this purpose. Bioinformatic analysis using the PHAge Search Tool (PHAST) on Mycolata whole genome sequence data in GenBank for members of the genera Gordonia, Mycobacterium, Nocardia, Rhodococcus, and Tsukamurella revealed 83% contained putative prophage DNA sequences. Subsequent prophage inductions using mitomycin C were conducted on 17 Mycolata strains. This led to the isolation and genome characterization of three novel Caudovirales temperate phages, namely GAL1, GMA1, and TPA4, induced from Gordonia alkanivorans, Gordonia malaquae, and Tsukamurella paurometabola, respectively. All possessed highly distinctive dsDNA genome sequences. PMID:27487243

Role of osmotic and hydrostatic pressures in bacteriophage genome ejection

NASA Astrophysics Data System (ADS)

Lemay, Serge G.; Panja, Debabrata; Molineux, Ian J.

2013-02-01

A critical step in the bacteriophage life cycle is genome ejection into host bacteria. The ejection process for double-stranded DNA phages has been studied thoroughly in vitro, where after triggering with the cellular receptor the genome ejects into a buffer. The experimental data have been interpreted in terms of the decrease in free energy of the densely packed DNA associated with genome ejection. Here we detail a simple model of genome ejection in terms of the hydrostatic and osmotic pressures inside the phage, a bacterium, and a buffer solution or culture medium. We argue that the hydrodynamic flow associated with the water movement from the buffer solution into the phage capsid and further drainage into the bacterial cytoplasm, driven by the osmotic gradient between the bacterial cytoplasm and culture medium, provides an alternative mechanism for phage genome ejection in vivo; the mechanism is perfectly consistent with phage genome ejection in vitro.

Genomes of the T4-related bacteriophages as windows on microbial genome evolution.

PubMed

Petrov, Vasiliy M; Ratnayaka, Swarnamala; Nolan, James M; Miller, Eric S; Karam, Jim D

2010-10-28

The T4-related bacteriophages are a group of bacterial viruses that share morphological similarities and genetic homologies with the well-studied Escherichia coli phage T4, but that diverge from T4 and each other by a number of genetically determined characteristics including the bacterial hosts they infect, the sizes of their linear double-stranded (ds) DNA genomes and the predicted compositions of their proteomes. The genomes of about 40 of these phages have been sequenced and annotated over the last several years and are compared here in the context of the factors that have determined their diversity and the diversity of other microbial genomes in evolution. The genomes of the T4 relatives analyzed so far range in size between ~160,000 and ~250,000 base pairs (bp) and are mosaics of one another, consisting of clusters of homology between them that are interspersed with segments that vary considerably in genetic composition between the different phage lineages. Based on the known biological and biochemical properties of phage T4 and the proteins encoded by the T4 genome, the T4 relatives reviewed here are predicted to share a genetic core, or "Core Genome" that determines the structural design of their dsDNA chromosomes, their distinctive morphology and the process of their assembly into infectious agents (phage morphogenesis). The Core Genome appears to be the most ancient genetic component of this phage group and constitutes a mere 12-15% of the total protein encoding potential of the typical T4-related phage genome. The high degree of genetic heterogeneity that exists outside of this shared core suggests that horizontal DNA transfer involving many genetic sources has played a major role in diversification of the T4-related phages and their spread to a wide spectrum of bacterial species domains in evolution. We discuss some of the factors and pathways that might have shaped the evolution of these phages and point out several parallels between their diversity and the diversity generally observed within all groups of interrelated dsDNA microbial genomes in nature.

Genomes of the T4-related bacteriophages as windows on microbial genome evolution

PubMed Central

2010-01-01

The T4-related bacteriophages are a group of bacterial viruses that share morphological similarities and genetic homologies with the well-studied Escherichia coli phage T4, but that diverge from T4 and each other by a number of genetically determined characteristics including the bacterial hosts they infect, the sizes of their linear double-stranded (ds) DNA genomes and the predicted compositions of their proteomes. The genomes of about 40 of these phages have been sequenced and annotated over the last several years and are compared here in the context of the factors that have determined their diversity and the diversity of other microbial genomes in evolution. The genomes of the T4 relatives analyzed so far range in size between ~160,000 and ~250,000 base pairs (bp) and are mosaics of one another, consisting of clusters of homology between them that are interspersed with segments that vary considerably in genetic composition between the different phage lineages. Based on the known biological and biochemical properties of phage T4 and the proteins encoded by the T4 genome, the T4 relatives reviewed here are predicted to share a genetic core, or "Core Genome" that determines the structural design of their dsDNA chromosomes, their distinctive morphology and the process of their assembly into infectious agents (phage morphogenesis). The Core Genome appears to be the most ancient genetic component of this phage group and constitutes a mere 12-15% of the total protein encoding potential of the typical T4-related phage genome. The high degree of genetic heterogeneity that exists outside of this shared core suggests that horizontal DNA transfer involving many genetic sources has played a major role in diversification of the T4-related phages and their spread to a wide spectrum of bacterial species domains in evolution. We discuss some of the factors and pathways that might have shaped the evolution of these phages and point out several parallels between their diversity and the diversity generally observed within all groups of interrelated dsDNA microbial genomes in nature. PMID:21029436

Ionic switch controls the DNA state in phage λ

PubMed Central

Li, Dong; Liu, Ting; Zuo, Xiaobing; Li, Tao; Qiu, Xiangyun; Evilevitch, Alex

2015-01-01

We have recently found that DNA packaged in phage λ undergoes a disordering transition triggered by temperature, which results in increased genome mobility. This solid-to-fluid like DNA transition markedly increases the number of infectious λ particles facilitating infection. However, the structural transition strongly depends on temperature and ionic conditions in the surrounding medium. Using titration microcalorimetry combined with solution X-ray scattering, we mapped both energetic and structural changes associated with transition of the encapsidated λ-DNA. Packaged DNA needs to reach a critical stress level in order for transition to occur. We varied the stress on DNA in the capsid by changing the temperature, packaged DNA length and ionic conditions. We found striking evidence that the intracapsid DNA transition is ‘switched on’ at the ionic conditions mimicking those in vivo and also at the physiologic temperature of infection at 37°C. This ion regulated on-off switch of packaged DNA mobility in turn affects viral replication. These results suggest a remarkable adaptation of phage λ to the environment of its host bacteria in the human gut. The metastable DNA state in the capsid provides a new paradigm for the physical evolution of viruses. PMID:26092697

Ionic switch controls the DNA state in phage λ

DOE PAGES

Li, Dong; Liu, Ting; Zuo, Xiaobing; ...

2015-06-19

We have recently found that DNA packaged in phage λ undergoes a disordering transition triggered by temperature, which results in increased genome mobility. This solid-to-fluid like DNA transition markedly increases the number of infectious λ particles facilitating infection. However, the structural transition strongly depends on temperature and ionic conditions in the surrounding medium. Using titration microcalorimetry combined with solution X-ray scattering, we mapped both energetic and structural changes associated with transition of the encapsidated λ-DNA. Packaged DNA needs to reach a critical stress level in order for transition to occur. We varied the stress on DNA in the capsid bymore » changing the temperature, packaged DNA length and ionic conditions. We found striking evidence that the intracapsid DNA transition is ‘switched on’ at the ionic conditions mimicking those in vivo and also at the physiologic temperature of infection at 37°C. This ion regulated on-off switch of packaged DNA mobility in turn affects viral replication. The results suggest a remarkable adaptation of phage λ to the environment of its host bacteria in the human gut. The metastable DNA state in the capsid provides a new paradigm for the physical evolution of viruses.« less

Ionic switch controls the DNA state in phage λ

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Dong; Liu, Ting; Zuo, Xiaobing

We have recently found that DNA packaged in phage λ undergoes a disordering transition triggered by temperature, which results in increased genome mobility. This solid-to-fluid like DNA transition markedly increases the number of infectious λ particles facilitating infection. However, the structural transition strongly depends on temperature and ionic conditions in the surrounding medium. Using titration microcalorimetry combined with solution X-ray scattering, we mapped both energetic and structural changes associated with transition of the encapsidated λ-DNA. Packaged DNA needs to reach a critical stress level in order for transition to occur. We varied the stress on DNA in the capsid bymore » changing the temperature, packaged DNA length and ionic conditions. We found striking evidence that the intracapsid DNA transition is ‘switched on’ at the ionic conditions mimicking those in vivo and also at the physiologic temperature of infection at 37°C. This ion regulated on-off switch of packaged DNA mobility in turn affects viral replication. The results suggest a remarkable adaptation of phage λ to the environment of its host bacteria in the human gut. The metastable DNA state in the capsid provides a new paradigm for the physical evolution of viruses.« less

Inhibitory effect of tocotrienol on eukaryotic DNA polymerase {lambda} and angiogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mizushina, Yoshiyuki; Nakagawa, Kiyotaka; Shibata, Akira

2006-01-20

Tocotrienols, vitamin E compounds that have an unsaturated side chain with three double bonds, selectively inhibited the activity of mammalian DNA polymerase {lambda} (pol {lambda}) in vitro. These compounds did not influence the activities of replicative pols such as {alpha}, {delta}, and {epsilon}, or even the activity of pol {beta} which is thought to have a very similar three-dimensional structure to the pol {beta}-like region of pol {lambda}. Since {delta}-tocotrienol had the strongest inhibitory effect among the four ({alpha}- to {delta}-) tocotrienols, the isomer's structure might be an important factor in the inhibition of pol {lambda}. The inhibitory effect ofmore » {delta}-tocotrienol on both intact pol {lambda} (residues 1-575) and a truncated pol {lambda} lacking the N-terminal BRCA1 C-terminus (BRCT) domain (residues 133-575, del-1 pol {lambda}) was dose-dependent, with 50% inhibition observed at a concentration of 18.4 and 90.1 {mu}M, respectively. However, del-2 pol {lambda} (residues 245-575) containing the C-terminal pol {beta}-like region was unaffected. Tocotrienols also inhibited the proliferation of and formation of tubes by bovine aortic endothelial cells, with {delta}-tocotrienol having the greatest effect. These results indicated that tocotrienols targeted both pol {lambda} and angiogenesis as anti-cancer agents. The relationship between the inhibition of pol {lambda} and anti-angiogenesis by {delta}-tocotrienol was discussed.« less

Novel Optical Metamaterials and Approaches for Fabrication

DTIC Science & Technology

2012-08-01

phage display , we have also identified peptides that bind with nanoparticles and glass substrates. This is a critical step in engineering M13 ...with 2-mercaptoethanol ............................................... 12 Figure 13: DNA sequence of the three rounds of phage display selection with...corresponding amino acids ................................................................................ 13 Figure 14: M13 Phage bound to silicon

Structure-function analysis of the DNA translocating portal of the bacteriophage T4 packaging machine.

PubMed

Padilla-Sanchez, Victor; Gao, Song; Kim, Hyung Rae; Kihara, Daisuke; Sun, Lei; Rossmann, Michael G; Rao, Venigalla B

2014-03-06

Tailed bacteriophages and herpesviruses consist of a structurally well conserved dodecameric portal at a special 5-fold vertex of the capsid. The portal plays critical roles in head assembly, genome packaging, neck/tail attachment, and genome ejection. Although the structures of portals from phages φ29, SPP1, and P22 have been determined, their mechanistic roles have not been well understood. Structural analysis of phage T4 portal (gp20) has been hampered because of its unusual interaction with the Escherichia coli inner membrane. Here, we predict atomic models for the T4 portal monomer and dodecamer, and we fit the dodecamer into the cryo-electron microscopy density of the phage portal vertex. The core structure, like that from other phages, is cone shaped with the wider end containing the "wing" and "crown" domains inside the phage head. A long "stem" encloses a central channel, and a narrow "stalk" protrudes outside the capsid. A biochemical approach was developed to analyze portal function by incorporating plasmid-expressed portal protein into phage heads and determining the effect of mutations on head assembly, DNA translocation, and virion production. We found that the protruding loops of the stalk domain are involved in assembling the DNA packaging motor. A loop that connects the stalk to the channel might be required for communication between the motor and the portal. The "tunnel" loops that project into the channel are essential for sealing the packaged head. These studies established that the portal is required throughout the DNA packaging process, with different domains participating at different stages of genome packaging. © 2013.

To Be or Not To Be T4: Evidence of a Complex Evolutionary Pathway of Head Structure and Assembly in Giant Salmonella Virus SPN3US

PubMed Central

Ali, Bazla; Desmond, Maxim I.; Mallory, Sara A.; Benítez, Andrea D.; Buckley, Larry J.; Weintraub, Susan T.; Osier, Michael V.; Black, Lindsay W.; Thomas, Julie A.

2017-01-01

Giant Salmonella phage SPN3US has a 240-kb dsDNA genome and a large complex virion composed of many proteins for which the functions of most are undefined. We recently determined that SPN3US shares a core set of genes with related giant phages and sequenced and characterized 18 amber mutants to facilitate its use as a genetic model system. Notably, SPN3US and related giant phages contain a bolus of ejection proteins within their heads, including a multi-subunit virion RNA polymerase (vRNAP), that enter the host cell with the DNA during infection. In this study, we characterized the SPN3US virion using mass spectrometry to gain insight into its head composition and the features that its head shares with those of related giant phages and with T4 phage. SPN3US has only homologs to the T4 proteins critical for prohead shell formation, the portal and major capsid proteins, as well as to the major enzymes essential for head maturation, the prohead protease and large terminase subunit. Eight of ~50 SPN3US head proteins were found to undergo proteolytic processing at a cleavage motif by the prohead protease gp245. Gp245 undergoes auto-cleavage of its C-terminus, suggesting this is a conserved activation and/or maturation feature of related phage proteases. Analyses of essential head gene mutants showed that the five subunits of the vRNAP must be assembled for any subunit to be incorporated into the prohead, although the assembled vRNAP must then undergo subsequent major conformational rearrangements in the DNA packed capsid to allow ejection through the ~30 Å diameter tail tube for transcription from the injected DNA. In addition, ejection protein candidate gp243 was found to play a critical role in head assembly. Our analyses of the vRNAP and gp243 mutants highlighted an unexpected dichotomy in giant phage head maturation: while all analyzed giant phages have a homologous protease that processes major capsid and portal proteins, processing of ejection proteins is not always a stable/defining feature. Our identification in SPN3US, and related phages, of a diverged paralog to the prohead protease further hints toward a complicated evolutionary pathway for giant phage head structure and assembly. PMID:29187846

Rapid, Multiplexed Microfluidic Phage Display

DTIC Science & Technology

2012-01-01

affinity phage- displayed peptides for multiple targets in just a single round and without the need for bacterial infection. The chip is shown to be able...by bacterial titer and amplification, and at least two additional rounds of selection. After the final round of biopan- ning, eluted phage are grown on...agar plates, and individual plaques are selected for DNA characterization to determine the amino acid sequence of the phage-displayed peptides. While

DNA Packaging Specificity of Bacteriophage N15 with an Excursion into the Genetics of a Cohesive End Mismatch

PubMed Central

Feiss, Michael; Young Min, Jea; Sultana, Sawsan; Patel, Priyal; Sippy, Jean

2015-01-01

During DNA replication by the λ-like bacteriophages, immature concatemeric DNA is produced by rolling circle replication. The concatemers are processed into mature chromosomes with cohesive ends, and packaged into prohead shells, during virion assembly. Cohesive ends are generated by the viral enzyme terminase, which introduces staggered nicks at cos, an approx. 200 bp-long sequence containing subsites cosQ, cosN and cosB. Interactions of cos subsites of immature concatemeric DNA with terminase orchestrate DNA processing and packaging. To initiate DNA packaging, terminase interacts with cosB and nicks cosN. The cohesive ends of N15 DNA differ from those of λ at 2/12 positions. Genetic experiments show that phages with chromosomes containing mismatched cohesive ends are functional. In at least some infections, the cohesive end mismatch persists through cyclization and replication, so that progeny phages of both allelic types are produced in the infected cell. N15 possesses an asymmetric packaging specificity: N15 DNA is not packaged by phages λ or 21, but surprisingly, N15-specific terminase packages λ DNA. Implications for genetic interactions among λ-like bacteriophages are discussed. PMID:26633301

Characterisation of a novel enterobacteria phage, CAjan, isolated from rat faeces.

PubMed

Carstens, Alexander B; Kot, Witold; Lametsch, Rene; Neve, Horst; Hansen, Lars H

2016-08-01

In this study, we describe the isolation and characterisation of the novel enterobacteria phage CAjan. This phage belongs to the order Caudovirales and the family Siphoviridae. The phage possesses a linear, double-stranded DNA genome consisting of 59,670 bp with a G+C content of 44.7 % and 91 predicted open reading frames (ORFs). Putative functions were assigned to 39 of the ORFs (37.4 %). The phage structural genes were furthermore functionally characterised by LC MS/MS. CAjan, together with Escherichia phage Seurat and Escherichia phage slur01, represent a novel and genetically distinct clade of Siphoviridae phages that could be considered to constitute a new phage genus. Despite limited sequence similarity, the phages in this group share a number of other common features, including genome structure and the presence of queuosine biosynthesis genes.

The ltp gene of temperate Streptococcus thermophilus phage TP-J34 confers superinfection exclusion to Streptococcus thermophilus and Lactococcus lactis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun Xingmin; Goehler, Andre; Heller, Knut J.

2006-06-20

The ltp gene, located within the lysogeny module of temperate Streptococcus thermophilus phage TP-J34, has been shown to be expressed in lysogenic strain S. thermophilus J34. It codes for a lipoprotein, as demonstrated by inhibition of cleavage of the signal sequence by globomycin. Exposure of Ltp on the surface of Lactococcus lactis protoplasts bearing a plasmid-encoded copy of ltp has been demonstrated by immunogold labeling and electron microscopy. Expression of ltp in prophage- and plasmid-cured S. thermophilus J34-6f interfered with TP-J34 infection. While plating efficiency was reduced by a factor of about 40 and lysis of strain J34-6f in liquidmore » medium was delayed considerably, phage adsorption was not affected at all. Intracellular accumulation of phage DNA was shown to be inhibited by Ltp. This indicates interference of Ltp with infection at the stage of triggering DNA release and injection into the cell, indicating a role of Ltp in superinfection exclusion. Expression of ltp in L. lactis Bu2-60 showed that the same superinfection exclusion mechanism was strongly effective against phage P008, a member of the lactococcal 936 phage species: no plaque-formation was detectable with even 10{sup 9} phage per ml applied, and lysis in liquid medium did not occur. In Lactococcus also, Ltp apparently inhibited phage DNA release and/or injection. Ltp appears to be a member of a family of small, secreted proteins with a 42 amino acids repeat structure encoded by genes of Gram-positive bacteria. Some of these homologous genes are part of the genomes of prophages.« less

Genome of a SAR116 bacteriophage shows the prevalence of this phage type in the oceans.

PubMed

Kang, Ilnam; Oh, Hyun-Myung; Kang, Dongmin; Cho, Jang-Cheon

2013-07-23

The abundance, genetic diversity, and crucial ecological and evolutionary roles of marine phages have prompted a large number of metagenomic studies. However, obtaining a thorough understanding of marine phages has been hampered by the low number of phage isolates infecting major bacterial groups other than cyanophages and pelagiphages. Therefore, there is an urgent requirement for the isolation of phages that infect abundant marine bacterial groups. In this study, we isolated and characterized HMO-2011, a phage infecting a bacterium of the SAR116 clade, one of the most abundant marine bacterial lineages. HMO-2011, which infects "Candidatus Puniceispirillum marinum" strain IMCC1322, has an ~55-kb dsDNA genome that harbors many genes with novel features rarely found in cultured organisms, including genes encoding a DNA polymerase with a partial DnaJ central domain and an atypical methanesulfonate monooxygenase. Furthermore, homologs of nearly all HMO-2011 genes were predominantly found in marine metagenomes rather than cultured organisms, suggesting the novelty of HMO-2011 and the prevalence of this phage type in the oceans. A significant number of the viral metagenome sequences obtained from the ocean surface were best assigned to the HMO-2011 genome. The number of reads assigned to HMO-2011 accounted for 10.3%-25.3% of the total reads assigned to viruses in seven viromes from the Pacific and Indian Oceans, making the HMO-2011 genome the most or second-most frequently assigned viral genome. Given its ability to infect the abundant SAR116 clade and its widespread distribution, Puniceispirillum phage HMO-2011 could be an important resource for marine virus research.

Interaction between transposable phages: cip locus of prophage D3112, responsible for inhibition of integration and transposition of the related phage B39 of Pseudomonas aeruginosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gerasimov, V.A.; Yanenko, A.S.; Akhverdyan, V.Z.

1986-04-01

Bacteriophage D3112 forms two types of PA01 (D3112) lysogens: those that partially, or completely, limit the growth of the related heteroimmune phage B39. DNA/DNA hybridization has shown that the lysogens of the first type always contain one copy of prophage D3112 (monolysogens), and the lysogens of the second type contain two or more copies of prophage D3112. Limitation of the growth of phage B39 on PA01 (D3112) lysogens is associated with the functioning of the locus of prophage D3112, designated as cip (control of interaction of phages). Using deletion derivatives of plasmid RP4::D3112, the cip locus was mapped at anmore » interval of 1.3-2.45 kb of the D3112 genome. The expression of the cip locus occurs only if the D3112 genome is at the prophage state. The function of the Cip prophage of D3112 exerts an influence on early stages of development of phage B39, decreasing the efficiency of the integration and transposition processes of phage B39.« less

A novel chimeric prophage vB_LdeS-phiJB from commercial Lactobacillus delbrueckii subsp. bulgaricus.

PubMed

Guo, Tingting; Zhang, Chenchen; Xin, Yongping; Xin, Min; Kong, Jian

2016-05-01

Prophage vB_LdeS-phiJB (phiJB) was induced by mitomycin C and UV radiation from the Lactobacillus delbrueckii subsp. bulgaricus SDMCC050201 isolated from a Chinese yoghurt sample. It has an isometric head and a non-contractile tail with 36,969 bp linear double-stranded DNA genome, which is classified into the group a of Lb. delbrueckii phages. The genome of phiJB is highly modular with functionally related genes clustered together. Unexpectedly, there is no similarity of its DNA replication module to any phages that have been reported, while it consists of open-reading frames homologous to the proteins of Lactobacillus strains. Comparative genomic analysis indicated that its late gene clusters, integration/lysogeny modules and DNA replication module derived from different evolutionary ancestors and integrated into a chimera. Our results revealed a novel chimeric phage of commercial Lb. delbrueckii and will broaden the knowledge of phage diversity in the dairy industry.

Type III CRISPR-Cas systems can provide redundancy to counteract viral escape from type I systems.

PubMed

Silas, Sukrit; Lucas-Elio, Patricia; Jackson, Simon A; Aroca-Crevillén, Alejandra; Hansen, Loren L; Fineran, Peter C; Fire, Andrew Z; Sánchez-Amat, Antonio

2017-08-17

CRISPR-Cas-mediated defense utilizes information stored as spacers in CRISPR arrays to defend against genetic invaders. We define the mode of target interference and role in antiviral defense for two CRISPR-Cas systems in Marinomonas mediterranea . One system (type I-F) targets DNA. A second system (type III-B) is broadly capable of acquiring spacers in either orientation from RNA and DNA, and exhibits transcription-dependent DNA interference. Examining resistance to phages isolated from Mediterranean seagrass meadows, we found that the type III-B machinery co-opts type I-F CRISPR-RNAs. Sequencing and infectivity assessments of related bacterial and phage strains suggests an 'arms race' in which phage escape from the type I-F system can be overcome through use of type I-F spacers by a horizontally-acquired type III-B system. We propose that the phage-host arms race can drive selection for horizontal uptake and maintenance of promiscuous type III interference modules that supplement existing host type I CRISPR-Cas systems.

Thioredoxin is required for filamentous phage assembly.

PubMed Central

Russel, M; Model, P

1985-01-01

Sequence comparisons show that the fip gene product of Escherichia coli, which is required for filamentous phage assembly, is thioredoxin. Thioredoxin serves as a cofactor for reductive processes in many cell types and is a constituent of phage T7 DNA polymerase. The fip-1 mutation makes filamentous phage and T7 growth temperature sensitive in cells that carry it. The lesion lies within a highly conserved thioredoxin active site. Thioredoxin reductase (NADPH), as well as thioredoxin, is required for efficient filamentous phage production. Mutant phages defective in phage gene I are particularly sensitive to perturbations in the fip-thioredoxin system. A speculative model is presented in which thioredoxin reductase, thioredoxin, and the gene I protein interact to drive an engine for filamentous phage assembly. Images PMID:3881756

Metagenomic detection of phage-encoded platelet-binding factors in the human oral cavity

PubMed Central

Willner, Dana; Furlan, Mike; Schmieder, Robert; Grasis, Juris A.; Pride, David T.; Relman, David A.; Angly, Florent E.; McDole, Tracey; Mariella, Ray P.; Rohwer, Forest; Haynes, Matthew

2011-01-01

The human oropharynx is a reservoir for many potential pathogens, including streptococcal species that cause endocarditis. Although oropharyngeal microbes have been well described, viral communities are essentially uncharacterized. We conducted a metagenomic study to determine the composition of oropharyngeal DNA viral communities (both phage and eukaryotic viruses) in healthy individuals and to evaluate oropharyngeal swabs as a rapid method for viral detection. Viral DNA was extracted from 19 pooled oropharyngeal swabs and sequenced. Viral communities consisted almost exclusively of phage, and complete genomes of several phage were recovered, including Escherichia coli phage T3, Propionibacterium acnes phage PA6, and Streptococcus mitis phage SM1. Phage relative abundances changed dramatically depending on whether samples were chloroform treated or filtered to remove microbial contamination. pblA and pblB genes of phage SM1 were detected in the metagenomes. pblA and pblB mediate the attachment of S. mitis to platelets and play a significant role in S. mitis virulence in the endocardium, but have never previously been detected in the oral cavity. These genes were also identified in salivary metagenomes from three individuals at three time points and in individual saliva samples by PCR. Additionally, we demonstrate that phage SM1 can be induced by commonly ingested substances. Our results indicate that the oral cavity is a reservoir for pblA and pblB genes and for phage SM1 itself. Further studies will determine the association between pblA and pblB genes in the oral cavity and the risk of endocarditis. PMID:20547834

Efficacy of potential phage cocktails against Vibrio harveyi and closely related Vibrio species isolated from shrimp aquaculture environment in the south east coast of India.

PubMed

Stalin, Nattan; Srinivasan, Pappu

2017-08-01

A diverse set of novel phages infecting the marine pathogenic Vibrio harveyi was isolated from shrimp aquaculture environments in the south east coast of India. Based on initial screening, three phages with a broad host range revealed that the growth inhibition of phage is relatively specific to V. harveyi. They were also able to infect V. alginolyticus and V. parahemolyticus that belonged to the Harveyi clade species from shrimp pond and sea coast environment samples. However, the impact of these phages on their host bacterium are well understood; a one-step growth curve experiment and transmission electron microscope (TEM) revealed three phages grouped under the Myoviridae (VHM1 and VHM2); Siphoviridae (VHS1) family. These phages were further molecular characterized with respect to phage genomic DNA isolates. The randomly amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) digestion with HindIII, and major structural proteins were distinguished by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) clearly indicated that all the phage isolates were different, even when they came from the same source, giving an insight into the diversity of phages. Evaluation of microcosm studies of Penaeus monodon larvae infected with V. harveyi (105 CFU mL-1) showed that larvae survival after 96 h in the presence of phage treatment at 109 PFU mL-1 was enhanced when compared with the control. The resolution in over survival highly recommended that this study provides the phage-based therapy which could be an innovative and eco-friendly solution against Vibrio disease in shrimp aquaculture and in the natural environment. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of Five Novel Brevibacillus Bacteriophages and Genomic Comparison of Brevibacillus Phages

PubMed Central

Berg, Jordan A.; Merrill, Bryan D.; Crockett, Justin T.; Esplin, Kyle P.; Evans, Marlee R.; Heaton, Karli E.; Hilton, Jared A.; Hyde, Jonathan R.; McBride, Morgan S.; Schouten, Jordan T.; Simister, Austin R.; Thurgood, Trever L.; Ward, Andrew T.; Breakwell, Donald P.; Hope, Sandra; Grose, Julianne H.

2016-01-01

Brevibacillus laterosporus is a spore-forming bacterium that causes a secondary infection in beehives following European Foulbrood disease. To better understand the contributions of Brevibacillus bacteriophages to the evolution of their hosts, five novel phages (Jenst, Osiris, Powder, SecTim467, and Sundance) were isolated and characterized. When compared with the five Brevibacillus phages currently in NCBI, these phages were assigned to clusters based on whole genome and proteome synteny. Powder and Osiris, both myoviruses, were assigned to the previously described Jimmer-like cluster. SecTim467 and Jenst, both siphoviruses, formed a novel phage cluster. Sundance, a siphovirus, was assigned as a singleton phage along with the previously isolated singleton, Emery. In addition to characterizing the basic relationships between these phages, several genomic features were observed. A motif repeated throughout phages Jenst and SecTim467 was frequently upstream of genes predicted to function in DNA replication, nucleotide metabolism, and transcription, suggesting transcriptional co-regulation. In addition, paralogous gene pairs that encode a putative transcriptional regulator were identified in four Brevibacillus phages. These paralogs likely evolved to bind different DNA sequences due to variation at amino acid residues predicted to bind specific nucleotides. Finally, a putative transposable element was identified in SecTim467 and Sundance that carries genes homologous to those found in Brevibacillus chromosomes. Remnants of this transposable element were also identified in phage Jenst. These discoveries provide a greater understanding of the diversity of phages, their behavior, and their evolutionary relationships to one another and to their host. In addition, they provide a foundation with which further Brevibacillus phages can be compared. PMID:27304881

Characterization of Five Podoviridae Phages Infecting Citrobacter freundii

PubMed Central

Hamdi, Sana; Rousseau, Geneviève M.; Labrie, Simon J.; Kourda, Rim S.; Tremblay, Denise M.; Moineau, Sylvain; Slama, Karim B.

2016-01-01

Citrobacter freundii causes opportunistic infections in humans and animals, which are becoming difficult to treat due to increased antibiotic resistance. The aim of this study was to explore phages as potential antimicrobial agents against this opportunistic pathogen. We isolated and characterized five new virulent phages, SH1, SH2, SH3, SH4, and SH5 from sewage samples in Tunisia. Morphological and genomic analyses revealed that the five C. freundii phages belong to the Caudovirales order, Podoviridae family, and Autographivirinae subfamily. Their linear double-stranded DNA genomes range from 39,158 to 39,832 bp and are terminally redundant with direct repeats between 183 and 242 bp. The five genomes share the same organization as coliphage T7. Based on genomic comparisons and on the phylogeny of the DNA polymerases, we assigned the five phages to the T7virus genus but separated them into two different groups. Phages SH1 and SH2 are very similar to previously characterized phages phiYeO3-12 and phiSG-JL2, infecting, respectively, Yersinia enterocolitica and Salmonella enterica, as well as sharing more than 80% identity with most genes of coliphage T7. Phages SH3, SH4, and SH5 are very similar to phages K1F and Dev2, infecting, respectively, Escherichia coli and Cronobacter turicensis. Several structural proteins of phages SH1, SH3, and SH4 were detected by mass spectrometry. The five phages were also stable from pH 5 to 10. No genes coding for known virulence factors or integrases were found, suggesting that the five isolated phages could be good candidates for therapeutic applications to prevent or treat C. freundii infections. In addition, this study increases our knowledge about the evolutionary relationships within the T7virus genus. PMID:27446058

Melting of genomic DNA: Predictive modeling by nonlinear lattice dynamics

NASA Astrophysics Data System (ADS)

Theodorakopoulos, Nikos

2010-08-01

The melting behavior of long, heterogeneous DNA chains is examined within the framework of the nonlinear lattice dynamics based Peyrard-Bishop-Dauxois (PBD) model. Data for the pBR322 plasmid and the complete T7 phage have been used to obtain model fits and determine parameter dependence on salt content. Melting curves predicted for the complete fd phage and the Y1 and Y2 fragments of the ϕX174 phage without any adjustable parameters are in good agreement with experiment. The calculated probabilities for single base-pair opening are consistent with values obtained from imino proton exchange experiments.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Serwer, Philip, E-mail: serwer@uthscsa.edu; Wright, Elena T.; Liu, Zheng

DNA packaging of phages phi29, T3 and T7 sometimes produces incompletely packaged DNA with quantized lengths, based on gel electrophoretic band formation. We discover here a packaging ATPase-free, in vitro model for packaged DNA length quantization. We use directed evolution to isolate a five-site T3 point mutant that hyper-produces tail-free capsids with mature DNA (heads). Three tail gene mutations, but no head gene mutations, are present. A variable-length DNA segment leaks from some mutant heads, based on DNase I-protection assay and electron microscopy. The protected DNA segment has quantized lengths, based on restriction endonuclease analysis: six sharp bands of DNAmore » missing 3.7–12.3% of the last end packaged. Native gel electrophoresis confirms quantized DNA expulsion and, after removal of external DNA, provides evidence that capsid radius is the quantization-ruler. Capsid-based DNA length quantization possibly evolved via selection for stalling that provides time for feedback control during DNA packaging and injection. - Graphical abstract: Highlights: • We implement directed evolution- and DNA-sequencing-based phage assembly genetics. • We purify stable, mutant phage heads with a partially leaked mature DNA molecule. • Native gels and DNase-protection show leaked DNA segments to have quantized lengths. • Native gels after DNase I-removal of leaked DNA reveal the capsids to vary in radius. • Thus, we hypothesize leaked DNA quantization via variably quantized capsid radius.« less

Molecular Determinants of Estrogen Receptor Alpha Stability

DTIC Science & Technology

2008-07-01

presence of E2. This question can be addressed by a T7 phage display screen using a breast cancer cell library and DNA-bound ERα in the presence of...conformation of ERα induced by 27HC versus E2. To accomplish this, we performed combinatorial phage display using a modified M13 phage display screen

Homologous recombination between overlapping thymidine kinase gene fragments stably inserted into a mouse cell genome.

PubMed

Lin, F L; Sternberg, N

1984-05-01

We have constructed a substrate to study homologous recombination between adjacent segments of chromosomal DNA. This substrate, designated lambda tk2 , consists of one completely defective and one partially defective herpes simplex virus thymidine kinase (tk) gene cloned in bacteriophage lambda DNA. The two genes have homologous 984-base-pair sequences and are separated by 3 kilobases of largely vector DNA. When lambda tk2 DNA was transferred into mouse LMtk- cells by the calcium phosphate method, rare TK+ transformants were obtained that contained many (greater than 40) copies of the unrecombined DNA. Tk- revertants, which had lost most of the copies of unrecombined DNA, were isolated from these TK+-transformed lines. Two of these Tk- lines were further studied by analysis of their reversion back to the Tk+ phenotype. They generated ca. 200 Tk+ revertants per 10(8) cells after growth in nonselecting medium for 5 days. All of these Tk+ revertants have an intact tk gene reconstructed by homologous recombination; they also retain various amounts of unrecombined lambda tk2 DNA. Southern blot analysis suggested that at least some of the recombination events involve unequal sister chromatid exchanges. We also tested three agents, mitomycin C, 12-O-tetradecanoyl-phorbol-13-acetate, and mezerein, that are thought to stimulate recombination to determine whether they affect the reversion from Tk- to Tk+. Only mitomycin C increased the number of Tk+ revertants.

Structural relationship of curcumin derivatives binding to the BRCT domain of human DNA polymerase lambda.

PubMed

Takeuchi, Toshifumi; Ishidoh, Tomomi; Iijima, Hiroshi; Kuriyama, Isoko; Shimazaki, Noriko; Koiwai, Osamu; Kuramochi, Kouji; Kobayashi, Susumu; Sugawara, Fumio; Sakaguchi, Kengo; Yoshida, Hiromi; Mizushina, Yoshiyuki

2006-03-01

We previously reported that phenolic compounds, petasiphenol and curcumin (diferuloylmethane), were a selective inhibitor of DNA polymerase lambda (pol lambda) in vitro. The purpose of this study was to investigate the molecular structural relationship of curcumin and 13 chemically synthesized derivatives of curcumin. The inhibitory effect on pol lambda (full-length, i.e. intact pol lambda including the BRCA1 C- terminal [BRCT] domain) by some derivatives was stronger than that by curcumin, and monoacetylcurcumin (compound 13) was the strongest pol lambda inhibitor of all the compounds tested, achieving 50% inhibition at a concentration of 3.9 microm. The compound did not influence the activities of replicative pols such as alpha, delta, and epsilon. It had no effect on pol beta activity either, although the three-dimensional structure of pol beta is thought to be highly similar to that of pol lambda. Compound 13 did not inhibit the activity of the C-terminal catalytic domain of pol lambda including the pol beta-like core, in which the BRCT motif was deleted from its N-terminal region. MALDI-TOF MS analysis demonstrated that compound 13 bound selectively to the N-terminal domain of pol lambda, but did not bind to the C-terminal region. Based on these results, the pol lambda-inhibitory mechanism of compound 13 is discussed.

Methylation of DNA

PubMed Central

Gold, Marvin; Gefter, Malcolm; Hausmann, Rudolph; Hurwitz, Jerard

1966-01-01

The methylated bases of DNA are formed by the transfer of the methyl group from S-adenosylmethionine to a polynucleotide acceptor. This transfer is catalyzed by highly specific enzymes which recognize a limited number of available sites in the DNA. The mechanism for the recognition is presently unknown. In some instances, there is evidence that other cellular components, such as lipopolysaccharides, can influence the methylation reaction. Certain bacteriophages induce new methylases upon infection of their hosts. Phage T3 is unique in establishing an environment in which methylation of neither the phage nor the host nucleic acid can occur. By superinfecting T3-infected cells with other phages, the latter can be obtained with methyl-deficient DNA. Although a great deal is known about the enzymology of the methylation reaction, and there appears to be a strong correlation between the in vitro and in vivo reactions, studies in which DNA is either supermethylated or totally unmethylated have not yielded any insight as to what the possible function of the methylated bases may be. PMID:5338563

Cloning vector

DOEpatents

Guilfoyle, Richard A.; Smith, Lloyd M.

1994-01-01

A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site.

Cloning vector

DOEpatents

Guilfoyle, R.A.; Smith, L.M.

1994-12-27

A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site. 2 figures.

Bacteriophage Prevalence in the Genus Azospirillum and Analysis of the First Genome Sequence of an Azospirillum brasilense Integrative Phage▿

PubMed Central

Boyer, Mickaël; Haurat, Jacqueline; Samain, Sylvie; Segurens, Béatrice; Gavory, Frédérick; González, Víctor; Mavingui, Patrick; Rohr, René; Bally, René; Wisniewski-Dyé, Florence

2008-01-01

The prevalence of bacteriophages was investigated in 24 strains of four species of plant growth-promoting rhizobacteria belonging to the genus Azospirillum. Upon induction by mitomycin C, the release of phage particles was observed in 11 strains from three species. Transmission electron microscopy revealed two distinct sizes of particles, depending on the identity of the Azospirillum species, typical of the Siphoviridae family. Pulsed-field gel electrophoresis and hybridization experiments carried out on phage-encapsidated DNAs revealed that all phages isolated from A. lipoferum and A. doebereinerae strains had a size of about 10 kb whereas all phages isolated from A. brasilense strains displayed genome sizes ranging from 62 to 65 kb. Strong DNA hybridizing signals were shown for most phages hosted by the same species whereas no homology was found between phages harbored by different species. Moreover, the complete sequence of the A. brasilense Cd bacteriophage (ΦAb-Cd) genome was determined as a double-stranded DNA circular molecule of 62,337 pb that encodes 95 predicted proteins. Only 14 of the predicted proteins could be assigned functions, some of which were involved in DNA processing, phage morphogenesis, and bacterial lysis. In addition, the ΦAb-Cd complete genome was mapped as a prophage on a 570-kb replicon of strain A. brasilense Cd, and a region of 27.3 kb of ΦAb-Cd was found to be duplicated on the 130-kb pRhico plasmid previously sequenced from A. brasilense Sp7, the parental strain of A. brasilense Cd. PMID:18065619

Complete Genome Sequences of 38 Gordonia sp. Bacteriophages

PubMed Central

Montgomery, Matthew T.; Bonilla, J. Alfred; Dejong, Randall; Garlena, Rebecca A.; Guerrero Bustamante, Carlos; Klyczek, Karen K.; Russell, Daniel A.; Wertz, John T.; Jacobs-Sera, Deborah; Hatfull, Graham F.

2017-01-01

ABSTRACT We report here the genome sequences of 38 newly isolated bacteriophages using Gordonia terrae 3612 (ATCC 25594) and Gordonia neofelifaecis NRRL59395 as bacterial hosts. All of the phages are double-stranded DNA (dsDNA) tail phages with siphoviral morphologies, with genome sizes ranging from 17,118 bp to 93,843 bp and spanning considerable nucleotide sequence diversity. PMID:28057748

Somatic diversification of chicken immunoglobulin light chains by point mutations.

PubMed

Parvari, R; Ziv, E; Lantner, F; Heller, D; Schechter, I

1990-04-01

The light-chain locus of chicken has 1 functional V lambda 1 gene, 1 J gene, and 25 pseudo-V lambda-genes (where V = variable and J = joining). A major problem is which somatic mechanisms expand this extremely limited germ-line information to generate many different antibodies. Weill's group [Reynaud, C. A., Anquez, V., Grimal, H. & Weill, J. C. (1987) Cell 48, 379-388] has shown that the pseudo-V lambda-genes diversify the rearranged V lambda 1 by gene conversion. Here we demonstrate that chicken light chains are further diversified by somatic point mutations and by V lambda 1-J flexible joining. Somatic point mutations were identified in the J and 3' noncoding DNA of rearranged light-chain genes of chicken. These regions were analyzed because point mutations in V lambda 1 are obscured by gene conversion; the J and 3' noncoding DNA are presented in one copy per haploid genome and are not subject to gene conversion. In rodents point mutations occur as frequently in the V-J coding regions as in the adjacent flanking DNA. Therefore, we conclude that somatic point mutations diversify the V lambda 1 of chicken. The frequency (0-1%) and distribution of the mutations (decreasing in number with increased distance from the V lambda 1 segment) in chicken were as observed in rodents. Sequence variability at the V lambda 1-J junctions could be attributed to imprecise joining of the V lambda 1 and J genes. The modification by gene conversion of rearranged V lambda 1 genes in the bursa was similar in chicken aged 3 months (9.5%) or 3 weeks (9.1%)--i.e., gene conversion that generates the preimmune repertoire in the bursa seems to level off around 3 weeks of age. This preimmune repertoire can be further diversified by somatic point mutations that presumably lead to the formation of antibodies with increased affinity. A segment with structural features of a matrix association region [(A + T)-rich and four topoisomerase II binding sites] was identified in the middle of the J-C lambda intron (where C = constant).

CRISPR-Cas-Mediated Phage Resistance Enhances Horizontal Gene Transfer by Transduction.

PubMed

Watson, Bridget N J; Staals, Raymond H J; Fineran, Peter C

2018-02-13

A powerful contributor to prokaryotic evolution is horizontal gene transfer (HGT) through transformation, conjugation, and transduction, which can be advantageous, neutral, or detrimental to fitness. Bacteria and archaea control HGT and phage infection through CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) adaptive immunity. Although the benefits of resisting phage infection are evident, this can come at a cost of inhibiting the acquisition of other beneficial genes through HGT. Despite the ability of CRISPR-Cas to limit HGT through conjugation and transformation, its role in transduction is largely overlooked. Transduction is the phage-mediated transfer of bacterial DNA between cells and arguably has the greatest impact on HGT. We demonstrate that in Pectobacterium atrosepticum , CRISPR-Cas can inhibit the transduction of plasmids and chromosomal loci. In addition, we detected phage-mediated transfer of a large plant pathogenicity genomic island and show that CRISPR-Cas can inhibit its transduction. Despite these inhibitory effects of CRISPR-Cas on transduction, its more common role in phage resistance promotes rather than diminishes HGT via transduction by protecting bacteria from phage infection. This protective effect can also increase transduction of phage-sensitive members of mixed populations. CRISPR-Cas systems themselves display evidence of HGT, but little is known about their lateral dissemination between bacteria and whether transduction can contribute. We show that, through transduction, bacteria can acquire an entire chromosomal CRISPR-Cas system, including cas genes and phage-targeting spacers. We propose that the positive effect of CRISPR-Cas phage immunity on enhancing transduction surpasses the rarer cases where gene flow by transduction is restricted. IMPORTANCE The generation of genetic diversity through acquisition of DNA is a powerful contributor to microbial evolution and occurs through transformation, conjugation, and transduction. Of these, transduction, the phage-mediated transfer of bacterial DNA, is arguably the major route for genetic exchange. CRISPR-Cas adaptive immune systems control gene transfer by conjugation and transformation, but transduction has been mostly overlooked. Our results indicate that CRISPR-Cas can impede, but typically enhances the transduction of plasmids, chromosomal genes, and pathogenicity islands. By limiting wild-type phage replication, CRISPR-Cas immunity increases transduction in both phage-resistant and -sensitive members of mixed populations. Furthermore, we demonstrate mobilization of a chromosomal CRISPR-Cas system containing phage-targeting spacers by generalized transduction, which might partly account for the uneven distribution of these systems in nature. Overall, the ability of CRISPR-Cas to promote transduction reveals an unexpected impact of adaptive immunity on horizontal gene transfer, with broader implications for microbial evolution. Copyright © 2018 Watson et al.

Characterization of Sinorhizobium sp. LM21 Prophages and Virus-Encoded DNA Methyltransferases in the Light of Comparative Genomic Analyses of the Sinorhizobial Virome

PubMed Central

Decewicz, Przemyslaw; Radlinska, Monika; Dziewit, Lukasz

2017-01-01

The genus Sinorhizobium/Ensifer mostly groups nitrogen-fixing bacteria that create root or stem nodules on leguminous plants and transform atmospheric nitrogen into ammonia, which improves the productivity of the plants. Although these biotechnologically-important bacteria are commonly found in various soil environments, little is known about their phages. In this study, the genome of Sinorhizobium sp. LM21 isolated from a heavy-metal-contaminated copper mine in Poland was investigated for the presence of prophages and DNA methyltransferase-encoding genes. In addition to the previously identified temperate phage, ΦLM21, and the phage-plasmid, pLM21S1, the analysis revealed the presence of three prophage regions. Moreover, four novel phage-encoded DNA methyltransferase (MTase) genes were identified and the enzymes were characterized. It was shown that two of the identified viral MTases methylated the same target sequence (GANTC) as cell cycle-regulated methyltransferase (CcrM) of the bacterial host strain, LM21. This discovery was recognized as an example of the evolutionary convergence between enzymes of sinorhizobial viruses and their host, which may play an important role in virus cycle. In the last part of the study, thorough comparative analyses of 31 sinorhizobial (pro)phages (including active sinorhizobial phages and novel putative prophages retrieved and manually re-annotated from Sinorhizobium spp. genomes) were performed. The networking analysis revealed the presence of highly conserved proteins (e.g., holins and endolysins) and a high diversity of viral integrases. The analysis also revealed a large number of viral DNA MTases, whose genes were frequently located within the predicted replication modules of analyzed prophages, which may suggest their important regulatory role. Summarizing, complex analysis of the phage protein similarity network enabled a new insight into overall sinorhizobial virome diversity. PMID:28672885

Nucleotide excision repair and recombination are engaged in repair of trans-4-hydroxy-2-nonenal adducts to DNA bases in Escherichia coli.

PubMed

Janowska, Beata; Komisarski, Marek; Prorok, Paulina; Sokołowska, Beata; Kuśmierek, Jarosław; Janion, Celina; Tudek, Barbara

2009-09-23

One of the major products of lipid peroxidation is trans-4-hydroxy-2-nonenal (HNE). HNE forms highly mutagenic and genotoxic adducts to all DNA bases. Using M13 phage lacZ system, we studied the mutagenesis and repair of HNE treated phage DNA in E. coli wild-type or uvrA, recA, and mutL mutants. These studies revealed that: (i) nucleotide excision and recombination, but not mismatch repair, are engaged in repair of HNE adducts when present in phage DNA replicating in E. coli strains; (ii) in the single uvrA mutant, phage survival was drastically decreased while mutation frequency increased, and recombination events constituted 48% of all mutations; (iii) in the single recA mutant, the survival and mutation frequency of HNE-modified M13 phage was slightly elevated in comparison to that in the wild-type bacteria. The majority of mutations in recA(-) strain were G:C --> T:A transversions, occurring within the sequence which in recA(+) strains underwent RecA-mediated recombination, and the entire sequence was deleted; (iv) in the double uvrA recA mutant, phage survival was the same as in the wild-type although the mutation frequency was higher than in the wild-type and recA single mutant, but lower than in the single uvrA mutant. The majority of mutations found in the latter strain were base substitutions, with G:C --> A:T transitions prevailing. These transitions could have resulted from high reactivity of HNE with G and C, and induction of SOS-independent mutations.

Nucleotide excision repair and recombination are engaged in repair of trans-4-hydroxy-2-nonenal adducts to DNA bases in Escherichia coli

PubMed Central

Janowska, Beata; Komisarski, Marek; Prorok, Paulina; Sokołowska, Beata; Kuśmierek, Jarosław; Janion, Celina; Tudek, Barbara

2009-01-01

One of the major products of lipid peroxidation is trans-4-hydroxy-2-nonenal (HNE). HNE forms highly mutagenic and genotoxic adducts to all DNA bases. Using M13 phage lacZ system, we studied the mutagenesis and repair of HNE treated phage DNA in E. coli wild-type or uvrA, recA, and mutL mutants. These studies revealed that: (i) nucleotide excision and recombination, but not mismatch repair, are engaged in repair of HNE adducts when present in phage DNA replicating in E. coli strains; (ii) in the single uvrA mutant, phage survival was drastically decreased while mutation frequency increased, and recombination events constituted 48 % of all mutations; (iii) in the single recA mutant, the survival and mutation frequency of HNE-modified M13 phage was slightly elevated in comparison to that in the wild-type bacteria. The majority of mutations in recA- strain were G:C → T:A transversions, occurring within the sequence which in recA+ strains underwent RecA-mediated recombination, and the entire sequence was deleted; (iv) in the double uvrA recA mutant, phage survival was the same as in the wild-type although the mutation frequency was higher than in the wild-type and recA single mutant, but lower than in the single uvrA mutant. The majority of mutations found in the latter strain were base substitutions, with G:C → A:T transitions prevailing. These transitions could have resulted from high reactivity of HNE with G and C, and induction of SOS-independent mutations. PMID:19834545

Structure-Function Analysis of the DNA Translocating Portal of the Bacteriophage T4 Packaging Machine

PubMed Central

Padilla-Sanchez, Victor; Gao, Song; Kim, Hyung Rae; Kihara, Daisuke; Sun, Lei; Rossmann, Michael G.; Rao, Venigalla B.

2013-01-01

Tailed bacteriophages and herpesviruses consist of a structurally well conserved dodecameric portal at a special five-fold vertex of the capsid. The portal plays critical roles in head assembly, genome packaging, neck/tail attachment, and genome ejection. Although the structures of portals from phages φ29, SPP1 and P22 have been determined, their mechanistic roles have not been well understood. Structural analysis of phage T4 portal (gp20) has been hampered because of its unusual interaction with the E. coli inner membrane. Here, we predict atomic models for the T4 portal monomer and dodecamer, and fit the dodecamer into the cryoEM density of the phage portal vertex. The core structure, like that from other phages, is cone-shaped with the wider end containing the “wing” and “crown” domains inside the phage head. A long “stem” encloses a central channel, and a narrow “stalk” protrudes outside the capsid. A biochemical approach was developed to analyze portal function by incorporating plasmid-expressed portal protein into phage heads and determining the effect of mutations on head assembly, DNA translocation, and virion production. We found that the protruding loops of the stalk domain are involved in assembling the DNA packaging motor. A loop that connects the stalk to the channel might be required for communication between the motor and portal. The “tunnel” loops that project into the channel are essential for sealing the packaged head. These studies established that the portal is required throughout the DNA packaging process, with different domains participating at different stages of genome packaging. PMID:24126213

Characterization of bacteriophages Cp1 and Cp2, the strain-typing agents for Xanthomonas axonopodis pv. citri.

PubMed

Ahmad, Abdelmonim Ali; Ogawa, Megumi; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

2014-01-01

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

Characterization of the relationship between integrase, excisionase and antirepressor activities associated with a superinfecting Shiga toxin encoding bacteriophage

PubMed Central

Fogg, P. C. M.; Rigden, D. J.; Saunders, J. R.; McCarthy, A. J.; Allison, H. E.

2011-01-01

Shigatoxigenic Escherichia coli emerged as new food borne pathogens in the early 1980s, primarily driven by the dispersal of Shiga toxin-encoding lambdoid bacteriophages. At least some of these Stx phages display superinfection phenotypes, which differ significantly from lambda phage itself, driving through in situ recombination further phage evolution, increasing host range and potentially increasing the host's pathogenic profile. Here, increasing levels of Stx phage Φ24B integrase expression in multiple lysogen cultures are demonstrated along with apparently negligible repression of integrase expression by the cognate CI repressor. The Φ24B int transcription start site and promoter region were identified and found to differ from in silico predictions. The unidirectional activity of this integrase was determined in an in situ, inducible tri-partite reaction. This indicated that Φ24B must encode a novel directionality factor that is controlling excision events during prophage induction. This excisionase was subsequently identified and characterized through complementation experiments. In addition, the previous proposal that a putative antirepressor was responsible for the lack of immunity to superinfection through inactivation of CI has been revisited and a new hypothesis involving the role of this protein in promoting efficient induction of the Φ24B prophage is proposed. PMID:21062824

Molecular Characterization of a Novel Temperate Sinorhizobium Bacteriophage, ФLM21, Encoding DNA Methyltransferase with CcrM-Like Specificity

PubMed Central

Dziewit, Lukasz; Oscik, Karolina; Bartosik, Dariusz

2014-01-01

ABSTRACT ΦLM21 is a temperate phage isolated from Sinorhizobium sp. strain LM21 (Alphaproteobacteria). Genomic analysis and electron microscopy suggested that ΦLM21 is a member of the family Siphoviridae. The phage has an isometric head and a long noncontractile tail. The genome of ΦLM21 has 50,827 bp of linear double-stranded DNA encoding 72 putative proteins, including proteins responsible for the assembly of the phage particles, DNA packaging, transcription, replication, and lysis. Virion proteins were characterized using mass spectrometry, leading to the identification of the major capsid and tail components, tape measure, and a putative portal protein. We have confirmed the activity of two gene products, a lytic enzyme (a putative chitinase) and a DNA methyltransferase, sharing sequence specificity with the cell cycle-regulating methyltransferase (CcrM) of the bacterial host. Interestingly, the genome of Sinorhizobium phage ΦLM21 shows very limited similarity to other known phage genome sequences and is thus considered unique. IMPORTANCE Prophages are known to play an important role in the genomic diversification of bacteria via horizontal gene transfer. The influence of prophages on pathogenic bacteria is very well documented. However, our knowledge of the overall impact of prophages on the survival of their lysogenic, nonpathogenic bacterial hosts is still limited. In particular, information on prophages of the agronomically important Sinorhizobium species is scarce. In this study, we describe the isolation and molecular characterization of a novel temperate bacteriophage, ΦLM21, of Sinorhizobium sp. LM21. Since we have not found any similar sequences, we propose that this bacteriophage is a novel species. We conducted a functional analysis of selected proteins. We have demonstrated that the phage DNA methyltransferase has the same sequence specificity as the cell cycle-regulating methyltransferase CcrM of its host. We point out that this phenomenon of mimicking the host regulatory mechanisms by viruses is quite common in bacteriophages. PMID:25187538

[Construction of the new Escherichia coli K-12 wild-type strain with improved growth characteristics for application in metabolic engineering].

PubMed

Biriukova, I V; Krylov, A A; Kiseleva, E M; Minaeva, N I; Mashko, S V

2010-03-01

MG1655 of Escherichia coli K-12 is frequently used in metabolic engineering as the wild-type strain. However, its two mutations, ilvG and rph-1 provide a negative effect on culture growth. The "polar effect" of rph-1 decreases the level of pyrE expression, causing partial auxotrophy for pyrimidines. Mutation ilvG leading to the appearance of Val(S) phenotype causes retardation of cell growth rate on media containing amino acids. In this work, the substitution of two loci in the genome of MG1655 with the recovery of the wild-type phenotype was accomplished. Gene rph(wt) from the chromosome of E. coli TG1 was marked via Red-dependent integration of DNA fragment carrying lambda attL-Cm(R)-lambda attR and transduced with phage P1 into MG1655; later, the Cm(R) marker was removed with the use of lambda Xis/Int recombinase. Parallel to this procedure, a spontaneous Val(R) mutant of E. coli MG1655 yielding colonies of maximal size on M9 medium with glucose in the presence of Val (50 microg/ml) was isolated. It was shown that a nucleotide deletion in the isolated Val(R) strain had been generated in the region of the identified E. coli K-12 ilvG mutation, which led to the recovery of the reading frame and active protein synthesis. This mutation named ilvG-15, which is the only reason for the Val(R) phenotype in the obtained strain, was transferred to MG1655-rph(wt) using cotransduction, by analogy to the transfer of rph(wt). Evaluation of rates of aerobically growing cells (microm, hour(-1)) on M9 medium with glucose produced the following values: 0.56, 0.69, and 0.73 for strains MG1655, MG1655-rph(wt), and MG1655-(rph(wt), ilvG-15), respectively.

Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions

PubMed Central

Guo, Fei; Liu, Zheng; Fang, Ping-An; Zhang, Qinfen; Wright, Elena T.; Wu, Weimin; Zhang, Ci; Vago, Frank; Ren, Yue; Jakana, Joanita; Chiu, Wah; Serwer, Philip; Jiang, Wen

2014-01-01

Many dsDNA viruses first assemble a DNA-free procapsid, using a scaffolding protein-dependent process. The procapsid, then, undergoes dramatic conformational maturation while packaging DNA. For bacteriophage T7 we report the following four single-particle cryo-EM 3D reconstructions and the derived atomic models: procapsid (4.6-Å resolution), an early-stage DNA packaging intermediate (3.5 Å), a later-stage packaging intermediate (6.6 Å), and the final infectious phage (3.6 Å). In the procapsid, the N terminus of the major capsid protein, gp10, has a six-turn helix at the inner surface of the shell, where each skewed hexamer of gp10 interacts with two scaffolding proteins. With the exit of scaffolding proteins during maturation the gp10 N-terminal helix unfolds and swings through the capsid shell to the outer surface. The refolded N-terminal region has a hairpin that forms a novel noncovalent, joint-like, intercapsomeric interaction with a pocket formed during shell expansion. These large conformational changes also result in a new noncovalent, intracapsomeric topological linking. Both interactions further stabilize the capsids by interlocking all pentameric and hexameric capsomeres in both DNA packaging intermediate and phage. Although the final phage shell has nearly identical structure to the shell of the DNA-free intermediate, surprisingly we found that the icosahedral faces of the phage are slightly (∼4 Å) contracted relative to the faces of the intermediate, despite the internal pressure from the densely packaged DNA genome. These structures provide a basis for understanding the capsid maturation process during DNA packaging that is essential for large numbers of dsDNA viruses. PMID:25313071

Structure of nascent replicative form DNA of coliphage M13

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dasgupta, S.; Mitra, S.

Nascent replicative form type II (RFII) DNA of coliphage M13 synthesized in an Escherichia coli mutant deficient in the 5' ..-->.. 3' exonuclease associated with DNA polymerase I contains ribonucleotides that are retained in the covalently closed RFI DNA sealed in vitro by the joint action of T5 phage DNA polymerase and T4 phage DNA ligase. These RFI molecules are labile to alkali and RNase H, unlike the RFI produced either in vivo or from RFII with E. coli DNA polymerase I and E. coli DNA ligase. The ribonucleotides are located at one site and predominantly in one strand ofmore » the nascent RF DNA. Furthermore, these molecules contain multiple small gaps, randomly located, and one large gap in the intracistronic region.« less

New Small Polypeptides Associated with DNA-Dependent RNA Polymerase of Escherichia coli after Infection with Bacteriophage T4

PubMed Central

Stevens, Audrey

1972-01-01

Four new small polypeptides are associated with DNA-dependent RNA polymerase from E. coli after infection with T4 phage. The new polypeptides are easily detected in RNA polymerase from E. coli cells labeled with amino acids after phage infection. Their molecular weights range from 10,000 to 22,000, as detected by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. All four polypeptides are found after infection with either wild-type T4 phage or T4 early amber mutants in genes 44, 42, 47, and 46. None of the polypeptides is labeled significantly before 5 min after infection at 30°. When two maturation-defective amber mutants in gene 55 of T4 phage are used for infection, a polypeptide with a molecular weight of 22,000 is absent. When a maturation-defective amber mutant in gene 33 of T4 phage is used, another small protein is absent. PMID:4551978

Temperate bacteriophage {phi}O18P from an Aeromonas media isolate: Characterization and complete genome sequence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beilstein, Frauke; Dreiseikelmann, Brigitte

2008-03-30

A group of 74 Aeromonas isolates from surface water of three ponds in Bielefeld, Germany was screened for prophage induction after UV irradiation. The phage {phi}O18P was induced from the Aeromonas media isolate O18. {phi}O18P belongs to the Myoviridae phage family. The complete nucleotide sequence of the double stranded DNA genome of bacteriophage {phi}O18P consists of 33,985 bp. The genome has 5' protruding cohesive ends of 16 bases. On the {phi}O18P genome 46 open reading frames (orfs) were identified which are organized in the modules integration and regulation, replication, head, packaging, tail and lysis. Additionally the phage DNA includes amore » methylase gene. Comparison of the genome architecture with those of other bacteriophages revealed significant similarities to the P2 phage family and especially to the prophages of Aeromonas salmonicida and the Vibrio cholerae phage K139.« less

The complete genome sequence and proteomics of Yersinia pestis phage Yep-phi.

PubMed

Zhao, Xiangna; Wu, Weili; Qi, Zhizhen; Cui, Yujun; Yan, Yanfeng; Guo, Zhaobiao; Wang, Zuyun; Wang, Hu; Deng, Haijun; Xue, Yan; Chen, Weijun; Wang, Xiaoyi; Yang, Ruifu

2011-01-01

Yep-phi, a lytic phage of Yersinia pestis, was isolated in China and is routinely used as a diagnostic phage for the identification of the plague pathogen. Yep-phi has an isometric hexagonal head containing dsDNA and a short non-contractile conical tail. In this study, we sequenced the Yep-phi genome (GenBank accession no. HQ333270) and performed proteomics analysis. The genome consists of 38 ,616 bp of DNA, including direct terminal repeats of 222 bp, and is predicted to contain 45 ORFs. Most structural proteins were identified by proteomics analysis. Compared with the three available genome sequences of lytic phages for Y. pestis, the phages could be divided into two subgroups. Yep-phi displays marked homology to the bacteriophages Berlin (GenBank accession no. AM183667) and Yepe2 (GenBank accession no. EU734170), and these comprise one subgroup. The other subgroup is represented by bacteriophage ΦA1122 (GenBank accession no. AY247822). Potential recombination was detected among the Yep-phi subgroup.

Interaction Analysis through Proteomic Phage Display

PubMed Central

2014-01-01

Phage display is a powerful technique for profiling specificities of peptide binding domains. The method is suited for the identification of high-affinity ligands with inhibitor potential when using highly diverse combinatorial peptide phage libraries. Such experiments further provide consensus motifs for genome-wide scanning of ligands of potential biological relevance. A complementary but considerably less explored approach is to display expression products of genomic DNA, cDNA, open reading frames (ORFs), or oligonucleotide libraries designed to encode defined regions of a target proteome on phage particles. One of the main applications of such proteomic libraries has been the elucidation of antibody epitopes. This review is focused on the use of proteomic phage display to uncover protein-protein interactions of potential relevance for cellular function. The method is particularly suited for the discovery of interactions between peptide binding domains and their targets. We discuss the largely unexplored potential of this method in the discovery of domain-motif interactions of potential biological relevance. PMID:25295249

Phage T4 endonuclease SegD that is similar to group I intron endonucleases does not initiate homing of its own gene.

PubMed

Sokolov, Andrey S; Latypov, Oleg R; Kolosov, Peter M; Shlyapnikov, Michael G; Bezlepkina, Tamara A; Kholod, Natalia S; Kadyrov, Farid A; Granovsky, Igor E

2018-02-01

Homing endonucleases are a group of site-specific endonucleases that initiate homing, a nonreciprocal transfer of its own gene into a new allele lacking this gene. This work describes a novel phage T4 endonuclease, SegD, which is homologous to the GIY-YIG family of homing endonucleases. Like other T4 homing endonucleases SegD recognizes an extended, 16bp long, site, cleaves it asymmetrically to form 3'-protruding ends and digests both unmodified DNA and modified T-even phage DNA with similar efficiencies. Surprisingly, we revealed that SegD cleavage site was identical in the genomes of segD - and segD + phages. We found that segD gene was expressed during the T4 developmental cycle. Nevertheless, endonuclease SegD was not able to initiate homing of its own gene as well as genetic recombination between phages in its site inserted into the rII locus. Copyright © 2018 Elsevier Inc. All rights reserved.

[Bacteriophage λ: electrostatic properties of the genome and its elements].

PubMed

Krutinina, G G; Krutinin, E A; Kamzolova, S G; Osypov, A A

2015-01-01

Bacteriophage λ is a classical model object in molecular biology, but little is still known on the physical properties of its DNA and regulatory elements. A study was made of the electrostatic properties of phage λ DNA and regulatory elements. A global electrostatic potential distribution along the phage genome was found to be nonuniform with main regulatory elements being located in a limited region with a high potential. The RNA polymerase binding frequency on the linearized phage chromosome directly correlates with its local potential. Strong promoters of the phage and its host Escherichia coli have distinct electrostatic upstream elements, which differ in nucleotide sequence. Attachment and recombination sites of phage λ and its host have a higher potential, which possibly facilitates their recognition by integrase. Phage λ and host Rho-independent terminators have a symmetrical M-shaped potential profile, which only slightly depends on the annotated terminator palindrome length, and occur in a region with a substantially higher potential, which may cause polymerase retention, facilitating the formation of a terminator hairpin in RNA. It was concluded that virtually all elements of phage λ genome have potential distribution specifics, which are related to their structural properties and may play a role in their biological function. The global potential distribution along the phage genome reflects the architecture of the regulation of its transcription and integration in the host genome.

Complete nucleotide sequence of the gene for human heparin cofactor II and mapping to chromosomal band 22q11

DOE Office of Scientific and Technical Information (OSTI.GOV)

Herzog, R.; Lutz, S.; Blin, N.

1991-02-05

Heparin cofactor II (HCII) is a 66-kDa plasma glycoprotein that inhibits thrombin rapidly in the presence of dermatan sulfate or heparin. Clones comprising the entire HCII gene were isolated from a human leukocyte genomic library in EMBL-3 {lambda} phage. The sequence of the gene was determined on both strands of DNA (15,849 bp) and included 1,749 bp of 5{prime}-flanking sequence, five exons, four introns, and 476 bp of DNA 3{prime} to the polyadenylation site. Ten complete and one partial Alu repeats were identified in the introns and 5{prime}-flanking region. The HCII gene was regionally mapped on chromosome 22 using rodent-humanmore » somatic cell hybrids, carrying only parts of human chromosome 22, and the chronic myelogenous leukemia cell line K562. With the cDNA probe HCII7.2, containing the entire coding region of the gene, the HCII gene was shown to be amplified 10-20-fold in K562 cells by Southern analysis and in situ hybridization. From these data, the authors concluded that the HCII gene is localized on the chromosomal band 22q11 proximal to the breakpoint cluster region (BCR). Analysis by pulsed-field gel electrophoresis indicated that the amplified HCII gene in K562 cells maps at least 2 Mbp proximal to BCR-1. Furthermore, the HCII7.2 cDNA probe detected two frequent restriction fragment length polymorphisms with the restriction enzymes BamHI and Hind III.« less

DNA-psoralen interaction: a single molecule experiment.

PubMed

Rocha, M S; Viana, N B; Mesquita, O N

2004-11-15

By attaching one end of a single lambda-DNA molecule to a microscope coverslip and the other end to a polystyrene microsphere trapped by an optical tweezers, we can study the entropic elasticity of the lambda-DNA by measuring force versus extension as we stretch the molecule. This powerful method permits single molecule studies. We are particularly interested in the effects of the photosensitive drug psoralen on the elasticity of the DNA molecule. We have illuminated the sample with different light sources, studying how the different wavelengths affect the psoralen-DNA linkage. To do this, we measure the persistence length of individual DNA-psoralen complexes.

Phages and the Evolution of Bacterial Pathogens: from Genomic Rearrangements to Lysogenic Conversion

PubMed Central

Brüssow, Harald; Canchaya, Carlos; Hardt, Wolf-Dietrich

2004-01-01

Comparative genomics demonstrated that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving. This process is most evident for bacterial pathogens where the majority contain prophages or phage remnants integrated into the bacterial DNA. Many prophages from bacterial pathogens encode virulence factors. Two situations can be distinguished: Vibrio cholerae, Shiga toxin-producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum depend on a specific prophage-encoded toxin for causing a specific disease, whereas Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar Typhimurium harbor a multitude of prophages and each phage-encoded virulence or fitness factor makes an incremental contribution to the fitness of the lysogen. These prophages behave like “swarms” of related prophages. Prophage diversification seems to be fueled by the frequent transfer of phage material by recombination with superinfecting phages, resident prophages, or occasional acquisition of other mobile DNA elements or bacterial chromosomal genes. Prophages also contribute to the diversification of the bacterial genome architecture. In many cases, they actually represent a large fraction of the strain-specific DNA sequences. In addition, they can serve as anchoring points for genome inversions. The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework. PMID:15353570

The role of prophage for genome diversification within a clonal lineage of Lactobacillus johnsonii: characterization of the defective prophage LJ771.

PubMed

Denou, Emmanuel; Pridmore, Raymond David; Ventura, Marco; Pittet, Anne-Cécile; Zwahlen, Marie-Camille; Berger, Bernard; Barretto, Caroline; Panoff, Jean-Michel; Brüssow, Harald

2008-09-01

Two independent isolates of the gut commensal Lactobacillus johnsonii were sequenced. These isolates belonged to the same clonal lineage and differed mainly by a 40.8-kb prophage, LJ771, belonging to the Sfi11 phage lineage. LJ771 shares close DNA sequence identity with Lactobacillus gasseri prophages. LJ771 coexists as an integrated prophage and excised circular phage DNA, but phage DNA packaged into extracellular phage particles was not detected. Between the phage lysin gene and attR a likely mazE ("antitoxin")/pemK ("toxin") gene cassette was detected in LJ771 but not in the L. gasseri prophages. Expressed pemK could be cloned in Escherichia coli only together with the mazE gene. LJ771 was shown to be highly stable and could be cured only by coexpression of mazE from a plasmid. The prophage was integrated into the methionine sulfoxide reductase gene (msrA) and complemented the 5' end of this gene, creating a protein with a slightly altered N-terminal sequence. The two L. johnsonii strains had identical in vitro growth and in vivo gut persistence phenotypes. Also, in an isogenic background, the presence of the prophage resulted in no growth disadvantage.

Excision of thymine dimers in vitro by extracts of bacteriophage-infected Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Friedberg, E.C.; Minton, K.; Pawl, G.

1974-05-01

Extracts of DNA polymerase I defective Escherichia coli infected with phage T4 contain an exonuclease activity that removes thymine dimers from uv- irradiated DNA previously nicked with T4 uv endonuclease. This activity is not expressed if cells are infected in the presence of chloramphenicol. The enzyme has a requirement for divalent cation and is not affected by caffeine, but excision is inhibited in the presence of proflavine. The enzyme is present in all phage T4 mutants thus far examined, including 25 uv-sensitive mutants isolated during the course of the experiments, all of which are defective in the v gene. Amore » similar activity can be detected in cells infected with phages T2, T3, and T6, but not in cells infected with phage T7. (auth)« less

Genomic analysis of Staphylococcus phage Stau2 isolated from medical specimen.

PubMed

Hsieh, Sue-Er; Tseng, Yi-Hsiung; Lo, Hsueh-Hsia; Chen, Shui-Tu; Wu, Cheng-Nan

2016-02-01

Stau2 is a lytic myophage of Staphylococcus aureus isolated from medical specimen. Exhibiting a broad host range against S. aureus clinical isolates, Stau2 is potentially useful for topical phage therapy or as an additive in food preservation. In this study, Stau2 was firstly revealed to possess a circularly permuted linear genome of 133,798 bp, with low G + C content, containing 146 open reading frames, but encoding no tRNA. The genome is organized into several modules containing genes for packaging, structural proteins, replication/transcription and host-cell-lysis, with the structural proteins and DNA polymerase modules being organized similarly to that in Twort-like phages of Staphylococcus. With the encoded DNA replication genes, Stau2 can possibly use its own system for replication. In addition, analysis in silico found several introns in seven genes, including those involved in DNA metabolism, packaging, and structure, while one of them (helicase gene) is experimentally confirmed to undergo splicing. Furthermore, phylogenetic analysis suggested Stau2 to be most closely related to Staphylococcus phages SA11 and Remus, members of Twort-like phages. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis showed 14 structural proteins of Stau2 and N-terminal sequencing identified three of them. Importantly, this phage does not encode any proteins which are known or suspected to be involved in toxicity, pathogenicity, or antibiotic resistance. Therefore, further investigations of feasible therapeutic application of Stau2 are needed.

Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage.

PubMed

Brok-Volchanskaya, Vera S; Kadyrov, Farid A; Sivogrivov, Dmitry E; Kolosov, Peter M; Sokolov, Andrey S; Shlyapnikov, Michael G; Kryukov, Valentine M; Granovsky, Igor E

2008-04-01

Homing endonucleases initiate nonreciprocal transfer of DNA segments containing their own genes and the flanking sequences by cleaving the recipient DNA. Bacteriophage T4 segB gene, which is located in a cluster of tRNA genes, encodes a protein of unknown function, homologous to homing endonucleases of the GIY-YIG family. We demonstrate that SegB protein is a site-specific endonuclease, which produces mostly 3' 2-nt protruding ends at its DNA cleavage site. Analysis of SegB cleavage sites suggests that SegB recognizes a 27-bp sequence. It contains 11-bp conserved sequence, which corresponds to a conserved motif of tRNA TpsiC stem-loop, whereas the remainder of the recognition site is rather degenerate. T4-related phages T2L, RB1 and RB3 contain tRNA gene regions that are homologous to that of phage T4 but lack segB gene and several tRNA genes. In co-infections of phages T4 and T2L, segB gene is inherited with nearly 100% of efficiency. The preferred inheritance depends absolutely on the segB gene integrity and is accompanied by the loss of the T2L tRNA gene region markers. We suggest that SegB is a homing endonuclease that functions to ensure spreading of its own gene and the surrounding tRNA genes among T4-related phages.

Deep-sea vent phage DNA polymerase specifically initiates DNA synthesis in the absence of primers.

PubMed

Zhu, Bin; Wang, Longfei; Mitsunobu, Hitoshi; Lu, Xueling; Hernandez, Alfredo J; Yoshida-Takashima, Yukari; Nunoura, Takuro; Tabor, Stanley; Richardson, Charles C

2017-03-21

A DNA polymerase is encoded by the deep-sea vent phage NrS-1. NrS-1 has a unique genome organization containing genes that are predicted to encode a helicase and a single-stranded DNA (ssDNA)-binding protein. The gene for an unknown protein shares weak homology with the bifunctional primase-polymerases (prim-pols) from archaeal plasmids but is missing the zinc-binding domain typically found in primases. We show that this gene product has efficient DNA polymerase activity and is processive in DNA synthesis in the presence of the NrS-1 helicase and ssDNA-binding protein. Remarkably, this NrS-1 DNA polymerase initiates DNA synthesis from a specific template DNA sequence in the absence of any primer. The de novo DNA polymerase activity resides in the N-terminal domain of the protein, whereas the C-terminal domain enhances DNA binding.

Forces and pressures in DNA packaging and release from viral capsids.

PubMed

Tzlil, Shelly; Kindt, James T; Gelbart, William M; Ben-Shaul, Avinoam

2003-03-01

In a previous communication (Kindt et al., 2001) we reported preliminary results of Brownian dynamics simulation and analytical theory which address the packaging and ejection forces involving DNA in bacteriophage capsids. In the present work we provide a systematic formulation of the underlying theory, featuring the energetic and structural aspects of the strongly confined DNA. The free energy of the DNA chain is expressed as a sum of contributions from its encapsidated and released portions, each expressed as a sum of bending and interstrand energies but subjected to different boundary conditions. The equilibrium structure and energy of the capsid-confined and free chain portions are determined, for each ejected length, by variational minimization of the free energy with respect to their shape profiles and interaxial spacings. Numerical results are derived for a model system mimicking the lambda-phage. We find that the fully encapsidated genome is highly compressed and strongly bent, forming a spool-like condensate, storing enormous elastic energy. The elastic stress is rapidly released during the first stage of DNA injection, indicating the large force (tens of pico Newtons) needed to complete the (inverse) loading process. The second injection stage sets in when approximately 1/3 of the genome has been released, and the interaxial distance has nearly reached its equilibrium value (corresponding to that of a relaxed torus in solution); concomitantly the encapsidated genome begins a gradual morphological transformation from a spool to a torus. We also calculate the loading force, the average pressure on the capsid's walls, and the anisotropic pressure profile within the capsid. The results are interpreted in terms of the (competing) bending and interaction components of the packing energy, and are shown to be in good agreement with available experimental data.

Molecular epidemiology of Escherichia coli O157:H7 strains by bacteriophage lambda restriction fragment length polymorphism analysis: application to a multistate foodborne outbreak and a day-care center cluster.

PubMed

Samadpour, M; Grimm, L M; Desai, B; Alfi, D; Ongerth, J E; Tarr, P I

1993-12-01

Genomic DNAs prepared from 168 isolates of Escherichia coli O157:H7 were analyzed for restriction fragment length polymorphisms on Southern blots probed with bacteriophage lambda DNA. The isolates analyzed included strains from a recent large multistate outbreak of E. coli O157:H7 infection associated with consumption of poorly cooked beef in restaurants, a day-care center cluster, and temporally and geographically unrelated isolates. E. coli O157:H7 isolates recovered from the incriminated meat and from 61 (96.8%) of 63 patients from Washington and Nevada possessed identical lambda restriction fragment length patterns. The lambda restriction fragment length polymorphisms observed in 11 (91.7%) of 12 day-care center patients were identical, but they differed from that of the strain associated with the multistate outbreak. E. coli O157:H7 from 42 patients temporally or geographically unrelated to either cluster of infection possessed unique and different lambda restriction fragment length patterns, except for paired isolates from three separate clusters of infection. These data demonstrate that the hybridization of DNA digests of E. coli O157:H7 with radiolabelled bacteriophage lambda DNA can be a useful, stable, and discriminatory epidemiologic tool for analyzing the linkage between strains of E. coli O157:H7.

Isolation and Characterization of Lytic Phage vB_EcoM_JS09 against Clinically Isolated Antibiotic-Resistant Avian Pathogenic Escherichia coli and Enterotoxigenic Escherichia coli.

PubMed

Zhou, Yan; Bao, Hongduo; Zhang, Hui; Wang, Ran

2015-01-01

To characterize the lytic coliphage vB_EcoM_JS09 (phage JS09) isolated from sewage samples of a swine farm in Jiangsu Province, China, which infects antibiotic-resistant avian pathogenic Escherichia coli (APEC) and enterotoxigenic E. coli (ETEC). Transmission electron microscopy revealed that phage JS09 has an isometric icosahedral head (76 nm in diameter) and a long contractile tail (140 nm in length) and features a T-even morphology. Its latent period was 30 min and the average burst size was 79 phage particles per infected cell. It attached to the host cells within 9 min. JS09 could infect 16 clinically isolated APEC and ETEC strains and the laboratory-engineered E. coli K and B strains. Ten of the clinical isolates of E. coli were resistant to antibiotics. At a multiplicity of infection of 10, 3, 1, or 0.3, the phage caused rapid cell lysis within 2 h, resulting in 5- to 10-fold reductions in cell concentration. Sequencing of the JS09 genome revealed a 169.148-kb linear but circularly permuted and terminally redundant dsDNA with 37.98% G+C content. Two hundred seventy-three open reading frames were predicted to be coding sequences, 135 of which were functionally defined and organized in a modular format which includes modules for DNA replication, DNA packaging, structural proteins, and host cell lysis proteins. Phage JS09 is assigned to the Caudovirales order (Myoviridae phage family), and it is considered a T4-like phage based on its morphological, genomic, and growth characteristics. JS09 gp37, a receptor-binding protein (RBP) important for host cell infection, shares little homology with other RBP in the NCBI database, which suggests that the variable regions in gp37 determine the unique host range of phage JS09. Protein sequence comparisons cluster the putative 'RBP' of JS09 much more closely with those of Yersinia phage phiD1, phage TuIa, and phage TuIb. A novel lytic coliphage named JS09 was isolated from sewage samples of a swine farm in Jiangsu Province, China. It could infect antibiotic-resistant APEC and ETEC. The morphological, genomic, and growth characteristics of JS09 were studied, and this will be helpful for phage therapy in controlling diseases caused by APEC and ETEC. © 2015 S. Karger AG, Basel.

Homologous recombination between overlapping thymidine kinase gene fragments stably inserted into a mouse cell genome.

PubMed Central

Lin, F L; Sternberg, N

1984-01-01

We have constructed a substrate to study homologous recombination between adjacent segments of chromosomal DNA. This substrate, designated lambda tk2 , consists of one completely defective and one partially defective herpes simplex virus thymidine kinase (tk) gene cloned in bacteriophage lambda DNA. The two genes have homologous 984-base-pair sequences and are separated by 3 kilobases of largely vector DNA. When lambda tk2 DNA was transferred into mouse LMtk- cells by the calcium phosphate method, rare TK+ transformants were obtained that contained many (greater than 40) copies of the unrecombined DNA. Tk- revertants, which had lost most of the copies of unrecombined DNA, were isolated from these TK+-transformed lines. Two of these Tk- lines were further studied by analysis of their reversion back to the Tk+ phenotype. They generated ca. 200 Tk+ revertants per 10(8) cells after growth in nonselecting medium for 5 days. All of these Tk+ revertants have an intact tk gene reconstructed by homologous recombination; they also retain various amounts of unrecombined lambda tk2 DNA. Southern blot analysis suggested that at least some of the recombination events involve unequal sister chromatid exchanges. We also tested three agents, mitomycin C, 12-O-tetradecanoyl-phorbol-13-acetate, and mezerein, that are thought to stimulate recombination to determine whether they affect the reversion from Tk- to Tk+. Only mitomycin C increased the number of Tk+ revertants. Images PMID:6328272

Breaking Transmission with Vaccines: The Case of Tuberculosis.

PubMed

Gonzalo-Asensio, Jesus; Aguilo, Nacho; Marinova, Dessislava; Martin, Carlos

2017-07-01

Members of the Mycobacterium tuberculosis complex (MTBC) have evolved causing tuberculosis (TB) in different mammalian hosts. MTBC ecotypes have adapted to diverse animal species, with M. bovis being the most common cause of TB in livestock. Cattle-to-human transmission of M. bovis through ingestion of raw milk was common before introduction of the pasteurization process. TB in humans is mainly caused by M. tuberculosis . This bacterium is considered a genetically clonal pathogen that has coevolved with humans due to its ability to manipulate and subvert the immune response. TB is a major public health problem due to airborne person-to-person transmission of M. tuberculosis . The essential yet unanswered question on the natural history of TB is when M. tuberculosis decides to establish latent infection in the host (resambling the lysogenic cycle of lambda phage) or to cause pulmonary disease (comparable to the lytic cycle of lambda phage). In this latter case, M. tuberculosis kills the host with the aim of achieving transmission to new hosts. Combating the TB epidemic requires stopping transmission. M. bovis BCG, the present vaccine against TB, is derived from M. bovis and only protects against disseminated forms of TB. Thus, a priority in TB research is development of new effective vaccines to prevent pulmonary disease. Attenuated vaccines based on M. tuberculosis as MTBVAC are potential candidates that could contribute to break the TB transmission cycle.

Type III CRISPR-Cas systems can provide redundancy to counteract viral escape from type I systems

PubMed Central

Silas, Sukrit; Lucas-Elio, Patricia; Jackson, Simon A; Aroca-Crevillén, Alejandra; Hansen, Loren L; Fineran, Peter C

2017-01-01

CRISPR-Cas-mediated defense utilizes information stored as spacers in CRISPR arrays to defend against genetic invaders. We define the mode of target interference and role in antiviral defense for two CRISPR-Cas systems in Marinomonas mediterranea. One system (type I-F) targets DNA. A second system (type III-B) is broadly capable of acquiring spacers in either orientation from RNA and DNA, and exhibits transcription-dependent DNA interference. Examining resistance to phages isolated from Mediterranean seagrass meadows, we found that the type III-B machinery co-opts type I-F CRISPR-RNAs. Sequencing and infectivity assessments of related bacterial and phage strains suggests an ‘arms race’ in which phage escape from the type I-F system can be overcome through use of type I-F spacers by a horizontally-acquired type III-B system. We propose that the phage-host arms race can drive selection for horizontal uptake and maintenance of promiscuous type III interference modules that supplement existing host type I CRISPR-Cas systems. PMID:28826484

Bacteriophages of Yersinia pestis.

PubMed

Zhao, Xiangna; Skurnik, Mikael

2016-01-01

Bacteriophage play many varied roles in microbial ecology and evolution. This chapter collates a vast body of knowledge and expertise on Yersinia pestis phages, including the history of their isolation and classical methods for their isolation and identification. The genomic diversity of Y. pestis phage and bacteriophage islands in the Y. pestis genome are also discussed because all phage research represents a branch of genetics. In addition, our knowledge of the receptors that are recognized by Y. pestis phage, advances in phage therapy for Y. pestis infections, the application of phage in the detection of Y. pestis, and clustered regularly interspaced short palindromic repeats (CRISPRs) sequences of Y. pestis from prophage DNA are all reviewed here.

Complete Genome Sequences of 44 Arthrobacter Phages.

PubMed

Klyczek, Karen K; Jacobs-Sera, Deborah; Adair, Tamarah L; Adams, Sandra D; Ball, Sarah L; Benjamin, Robert C; Bonilla, J Alfred; Breitenberger, Caroline A; Daniels, Charles J; Gaffney, Bobby L; Harrison, Melinda; Hughes, Lee E; King, Rodney A; Krukonis, Gregory P; Lopez, A Javier; Monsen-Collar, Kirsten; Pizzorno, Marie C; Rinehart, Claire A; Staples, Amanda K; Stowe, Emily L; Garlena, Rebecca A; Russell, Daniel A; Cresawn, Steven G; Pope, Welkin H; Hatfull, Graham F

2018-02-01

We report here the complete genome sequences of 44 phages infecting Arthrobacter sp. strain ATCC 21022. These phages have double-stranded DNA genomes with sizes ranging from 15,680 to 70,707 bp and G+C contents from 45.1% to 68.5%. All three tail types (belonging to the families Siphoviridae , Myoviridae , and Podoviridae ) are represented. Copyright © 2018 Klyczek et al.

Complete Genome Sequences of 44 Arthrobacter Phages

PubMed Central

Klyczek, Karen K.; Adair, Tamarah L.; Adams, Sandra D.; Ball, Sarah L.; Benjamin, Robert C.; Bonilla, J. Alfred; Breitenberger, Caroline A.; Daniels, Charles J.; Gaffney, Bobby L.; Harrison, Melinda; Hughes, Lee E.; King, Rodney A.; Krukonis, Gregory P.; Lopez, A. Javier; Monsen-Collar, Kirsten; Pizzorno, Marie C.; Staples, Amanda K.; Stowe, Emily L.; Garlena, Rebecca A.; Russell, Daniel A.

2018-01-01

ABSTRACT We report here the complete genome sequences of 44 phages infecting Arthrobacter sp. strain ATCC 21022. These phages have double-stranded DNA genomes with sizes ranging from 15,680 to 70,707 bp and G+C contents from 45.1% to 68.5%. All three tail types (belonging to the families Siphoviridae, Myoviridae, and Podoviridae) are represented. PMID:29437090

Screening by imaging: scaling up single-DNA-molecule analysis with a novel parabolic VA-TIRF reflector and noise-reduction techniques.

PubMed

van 't Hoff, Marcel; Reuter, Marcel; Dryden, David T F; Oheim, Martin

2009-09-21

Bacteriophage lambda-DNA molecules are frequently used as a scaffold to characterize the action of single proteins unwinding, translocating, digesting or repairing DNA. However, scaling up such single-DNA-molecule experiments under identical conditions to attain statistically relevant sample sizes remains challenging. Additionally the movies obtained are frequently noisy and difficult to analyse with any precision. We address these two problems here using, firstly, a novel variable-angle total internal reflection fluorescence (VA-TIRF) reflector composed of a minimal set of optical reflective elements, and secondly, using single value decomposition (SVD) to improve the signal-to-noise ratio prior to analysing time-lapse image stacks. As an example, we visualize under identical optical conditions hundreds of surface-tethered single lambda-DNA molecules, stained with the intercalating dye YOYO-1 iodide, and stretched out in a microcapillary flow. Another novelty of our approach is that we arrange on a mechanically driven stage several capillaries containing saline, calibration buffer and lambda-DNA, respectively, thus extending the approach to high-content, high-throughput screening of single molecules. Our length measurements of individual DNA molecules from noise-reduced kymograph images using SVD display a 6-fold enhanced precision compared to raw-data analysis, reaching approximately 1 kbp resolution. Combining these two methods, our approach provides a straightforward yet powerful way of collecting statistically relevant amounts of data in a semi-automated manner. We believe that our conceptually simple technique should be of interest for a broader range of single-molecule studies, well beyond the specific example of lambda-DNA shown here.

Direct Cloning of Yeast Genes from an Ordered Set of Lambda Clones in Saccharomyces Cerevisiae by Recombination in Vivo

PubMed Central

Erickson, J. R.; Johnston, M.

1993-01-01

We describe a technique that facilitates the isolation of yeast genes that are difficult to clone. This technique utilizes a plasmid vector that rescues lambda clones as yeast centromere plasmids. The source of these lambda clones is a set of clones whose location in the yeast genome has been determined by L. Riles et al. in 1993. The Esherichia coli-yeast shuttle plasmid carries URA3, ARS4 and CEN6, and contains DNA fragments from the lambda vector that flank the cloned yeast insert. When yeast is cotransformed with linearized plasmid and lambda clone DNA, Ura(+) transformants are obtained by a recombination event between the lambda clone and the plasmid vector that generates an autonomously replicating plasmid containing the cloned yeast DNA sequences. Genes whose genetic map positions are known can easily be identified and recovered in this plasmid by testing only those lambda clones that map to the relevant region of the yeast genome for their ability to complement the mutant phenotype. This technique facilitates the isolation of yeast genes that resist cloning either because (1) they are underrepresented in yeast genomic libraries amplified in E. coli, (2) they provide phenotypes that are too marginal to allow selection of the gene by genetic complementation or (3) they provide phenotypes that are laborious to score. We demonstrate the utility of this technique by isolating three genes, GAL83, SSN2 and MAK7, each of which presents one of these problems for cloning. PMID:8514124

Genomic analysis of WCP30 Phage of Weissella cibaria for Dairy Fermented Foods.

PubMed

Lee, Young-Duck; Park, Jong-Hyun

2017-01-01

In this study, we report the morphogenetic analysis and genome sequence of a new WCP30 phage of Weissella cibaria , isolated from a fermented food. Based on its morphology, as observed by transmission electron microscopy, WCP30 phage belongs to the family Siphoviridae . Genomic analysis of WCP30 phage showed that it had a 33,697-bp double-stranded DNA genome with 41.2% G+C content. Bioinformatics analysis of the genome revealed 35 open reading frames. A BLASTN search showed that WCP30 phage had low sequence similarity compared to other phages infecting lactic acid bacteria. This is the first report of the morphological features and complete genome sequence of WCP30 phage, which may be useful for controlling the fermentation of dairy foods.

Phage display as a technology delivering on the promise of peptide drug discovery.

PubMed

Hamzeh-Mivehroud, Maryam; Alizadeh, Ali Akbar; Morris, Michael B; Church, W Bret; Dastmalchi, Siavoush

2013-12-01

Phage display represents an important approach in the development pipeline for producing peptides and peptidomimetics therapeutics. Using randomly generated DNA sequences and molecular biology techniques, large diverse peptide libraries can be displayed on the phage surface. The phage library can be incubated with a target of interest and the phage which bind can be isolated and sequenced to reveal the displayed peptides' primary structure. In this review, we focus on the 'mechanics' of the phage display process, whilst highlighting many diverse and subtle ways it has been used to further the drug-development process, including the potential for the phage particle itself to be used as a drug carrier targeted to a particular pathogen or cell type in the body. Copyright © 2013 Elsevier Ltd. All rights reserved.

[Peptide phage display in biotechnology and biomedicine].

PubMed

Kuzmicheva, G A; Belyavskaya, V A

2016-07-01

To date peptide phage display is one of the most common combinatorial methods used for identifying specific peptide ligands. Phage display peptide libraries containing billions different clones successfully used for selection of ligands with high affinity and selectivity toward wide range of targets including individual proteins, bacteria, viruses, spores, different kind of cancer cells and variety of nonorganic targets (metals, alloys, semiconductors etc.) Success of using filamentous phage in phage display technologies relays on the robustness of phage particles and a possibility to genetically modify its DNA to construct new phage variants with novel properties. In this review we are discussing characteristics of the most known non-commercial peptide phage display libraries of different formats (landscape libraries in particular) and their successful applications in several fields of biotechnology and biomedicine: discovery of peptides with diagnostic values against different pathogens, discovery and using of peptides recognizing cancer cells, trends in using of phage display technologies in human interactome studies, application of phage display technologies in construction of novel nano materials.

Characterization of a ViI-like Phage Specific to Escherichia coli O157:H7

PubMed Central

2011-01-01

Phage vB_EcoM_CBA120 (CBA120), isolated against Escherichia coli O157:H7 from a cattle feedlot, is morphologically very similar to the classic phage ViI of Salmonella enterica serovar Typhi. Until recently, little was known genetically or physiologically about the ViI-like phages, and none targeting E. coli have been described in the literature. The genome of CBA120 has been fully sequenced and is highly similar to those of both ViI and the Shigella phage AG3. The core set of structural and replication-related proteins of CBA120 are homologous to those from T-even phages, but generally are more closely related to those from T4-like phages of Vibrio, Aeromonas and cyanobacteria than those of the Enterobacteriaceae. The baseplate and method of adhesion to the host are, however, very different from those of either T4 or the cyanophages. None of the outer baseplate proteins are conserved. Instead of T4's long and short tail fibers, CBA120, like ViI, encodes tail spikes related to those normally seen on podoviruses. The 158 kb genome, like that of T4, is circularly permuted and terminally redundant, but unlike T4 CBA120 does not substitute hmdCyt for cytosine in its DNA. However, in contrast to other coliphages, CBA120 and related coliphages we have isolated cannot incorporate 3H-thymidine (3H-dThd) into their DNA. Protein sequence comparisons cluster the putative "thymidylate synthase" of CBA120, ViI and AG3 much more closely with those of Delftia phage φW-14, Bacillus subtilis phage SPO1, and Pseudomonas phage YuA, all known to produce and incorporate hydroxymethyluracil (hmdUra). PMID:21899740

Phage-Encoded Colanic Acid-Degrading Enzyme Permits Lytic Phage Infection of a Capsule-Forming Resistant Mutant Escherichia coli Strain

PubMed Central

Kim, Min Soo; Kim, Young Deuk; Hong, Sung Sik; Park, Kwangseo; Ko, Kwan Soo

2014-01-01

In this study, we isolated a bacteriophage T7-resistant mutant strain of Escherichia coli (named S3) and then proceeded to characterize it. The mutant bacterial colonies appeared to be mucoid. Microarray analysis revealed that genes related to colanic acid production were upregulated in the mutant. Increases in colanic acid production by the mutant bacteria were observed when l-fucose was measured biochemically, and protective capsule formation was observed under an electron microscope. We found a point mutation in the lon gene promoter in S3, the mutant bacterium. Overproduction of colanic acid was observed in some phage-resistant mutant bacteria after infection with other bacteriophages, T4 and lambda. Colanic acid overproduction was also observed in clinical isolates of E. coli upon phage infection. The overproduction of colanic acid resulted in the inhibition of bacteriophage adsorption to the host. Biofilm formation initially decreased shortly after infection but eventually increased after 48 h of incubation due to the emergence of the mutant bacteria. Bacteriophage PBECO4 was shown to infect the colanic acid-overproducing mutant strains of E. coli. We confirmed that the gene product of open reading frame 547 (ORF547) of PBECO4 harbored colanic acid-degrading enzymatic (CAE) activity. Treatment of the T7-resistant bacteria with both T7 and PBECO4 or its purified enzyme (CAE) led to successful T7 infection. Biofilm formation decreased with the mixed infection, too. This procedure, using a phage cocktail different from those exploiting solely receptor differences, represents a novel strategy for overcoming phage resistance in mutant bacteria. PMID:25416767

Dualities in the analysis of phage DNA packaging motors

PubMed Central

Serwer, Philip; Jiang, Wen

2012-01-01

The DNA packaging motors of double-stranded DNA phages are models for analysis of all multi-molecular motors and for analysis of several fundamental aspects of biology, including early evolution, relationship of in vivo to in vitro biochemistry and targets for anti-virals. Work on phage DNA packaging motors both has produced and is producing dualities in the interpretation of data obtained by use of both traditional techniques and the more recently developed procedures of single-molecule analysis. The dualities include (1) reductive vs. accretive evolution, (2) rotation vs. stasis of sub-assemblies of the motor, (3) thermal ratcheting vs. power stroking in generating force, (4) complete motor vs. spark plug role for the packaging ATPase, (5) use of previously isolated vs. new intermediates for analysis of the intermediate states of the motor and (6) a motor with one cycle vs. a motor with two cycles. We provide background for these dualities, some of which are under-emphasized in the literature. We suggest directions for future research. PMID:23532204

Efficient identification of tubby-binding proteins by an improved system of T7 phage display.

PubMed

Caberoy, Nora B; Zhou, Yixiong; Jiang, Xiaoyu; Alvarado, Gabriela; Li, Wei

2010-01-01

Mutation in the tubby gene causes adult-onset obesity, progressive retinal, and cochlear degeneration with unknown mechanism. In contrast, mutations in tubby-like protein 1 (Tulp1), whose C-terminus is highly homologous to tubby, only lead to retinal degeneration. We speculate that their diverse N-terminus may define their distinct disease profile. To elucidate the binding partners of tubby, we used tubby N-terminus (tubby-N) as bait to identify unknown binding proteins with open-reading-frame (ORF) phage display. T7 phage display was engineered with three improvements: high-quality ORF phage display cDNA library, specific phage elution by protease cleavage, and dual phage display for sensitive high throughput screening. The new system is capable of identifying unknown bait-binding proteins in as fast as approximately 4-7 days. While phage display with conventional cDNA libraries identifies high percentage of out-of-frame unnatural short peptides, all 28 tubby-N-binding clones identified by ORF phage display were ORFs. They encode 16 proteins, including 8 nuclear proteins. Fourteen proteins were analyzed by yeast two-hybrid assay and protein pull-down assay with ten of them independently verified. Comparative binding analyses revealed several proteins binding to both tubby and Tulp1 as well as one tubby-specific binding protein. These data suggest that tubby-N is capable of interacting with multiple nuclear and cytoplasmic protein binding partners. These results demonstrated that the newly-engineered ORF phage display is a powerful technology to identify unknown protein-protein interactions. (c) 2009 John Wiley & Sons, Ltd.

Longitudinal monitoring of Listeria monocytogenes and Listeria phages in seafood processing environments in Thailand.

PubMed

Vongkamjan, Kitiya; Benjakul, Soottawat; Kim Vu, Hue Thi; Vuddhakul, Varaporn

2017-09-01

Listeria monocytogenes is a foodborne pathogen commonly found in environments of seafood processing, thus presenting a challenge for eradication from seafood processing facilities. Monitoring the prevalence and subtype diversity of L. monocytogenes together with phages that are specific to Listeria spp. ("Listeria phages") will provide knowledge on the bacteria-phage ecology in food processing plants. In this work, a total of 595 samples were collected from raw material, finished seafood products and environmental samples from different sites of a seafood processing plant during 17 sampling visits in 1.5 years of study. L. monocytogenes and Listeria spp. (non-monocytogenes) were found in 22 (3.7%) and 43 (7.2%) samples, respectively, whereas 29 Listeria phages were isolated from 9 (1.5%) phage-positive samples. DNA fingerprint analysis of L. monocytogenes isolates revealed 11 Random Amplified Polymorphic DNA (RAPD) profiles, with two subtypes were frequently observed over time. Our data reveal a presence of Listeria phages within the same seafood processing environments where a diverse set of L. monocytogenes subtypes was also found. Although serotype 4b was observed at lower frequency, data indicate that isolates from this seafood processing plant belonged to both epidemiologically important serotypes 1/2a and 4b, which may suggest a potential public health risk. Phages (all showed a unique genome size of 65 ± 2 kb) were classified into 9 host range groups, representing both broad- and narrow-host range. While most L. monocytogenes isolates from this facility were susceptible to phages, five isolates showed resistance to 12-20 phages. Variations in phage host range among Listeria phages isolated from food processing plant may affect a presence of a diverse set of L. monocytogenes isolates derived from the same processing environment in Thailand. Copyright © 2017 Elsevier Ltd. All rights reserved.

PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates.

PubMed Central

Barnes, W M

1994-01-01

A target length limitation to PCR amplification of DNA has been identified and addressed. Concomitantly, the base-pair fidelity, the ability to use PCR products as primers, and the maximum yield of target fragment were increased. These improvements were achieved by the combination of a high level of an exonuclease-free, N-terminal deletion mutant of Taq DNA polymerase, Klentaq1, with a very low level of a thermostable DNA polymerase exhibiting a 3'-exonuclease activity (Pfu, Vent, or Deep Vent). At least 35 kb can be amplified to high yields from 1 ng of lambda DNA template. Images PMID:8134376

Frameshifting in the p6 cDNA phage display system.

PubMed

Govarts, Cindy; Somers, Klaartje; Stinissen, Piet; Somers, Veerle

2010-12-20

Phage display is a powerful technique that enables easy identification of targets for any type of ligand. Targets are displayed at the phage surface as a fusion protein to one of the phage coat proteins. By means of a repeated process of affinity selection on a ligand, specific enrichment of displayed targets will occur. In our studies using C-terminal display of cDNA fragments to phage coat protein p6, we noticed the occasional enrichment of targets that do not contain an open reading frame. This event has previously been described in other phage display studies using N-terminal display of targets to phage coat proteins and was due to uncommon translational events like frameshifting. The aim of this study was to examine if C-terminal display of targets to p6 is also subjected to frameshifting. To this end, an enriched target not containing an open reading frame was selected and an E-tag was coupled at the C-terminus in order to measure target display at the surface of the phage. The tagged construct was subsequently expressed in 3 different reading frames and display of both target and E-tag measured to detect the occurrence of frameshifting. As a result, we were able to demonstrate display of the target both in the 0 and in the +1 reading frame indicating that frameshifting can also take place when C-terminal fusion to minor coat protein p6 is applied.

Toward Understanding Phage:Host Interactions in the Rumen; Complete Genome Sequences of Lytic Phages Infecting Rumen Bacteria

PubMed Central

Gilbert, Rosalind A.; Kelly, William J.; Altermann, Eric; Leahy, Sinead C.; Minchin, Catherine; Ouwerkerk, Diane; Klieve, Athol V.

2017-01-01

The rumen is known to harbor dense populations of bacteriophages (phages) predicted to be capable of infecting a diverse range of rumen bacteria. While bacterial genome sequencing projects are revealing the presence of phages which can integrate their DNA into the genome of their host to form stable, lysogenic associations, little is known of the genetics of phages which utilize lytic replication. These phages infect and replicate within the host, culminating in host lysis, and the release of progeny phage particles. While lytic phages for rumen bacteria have been previously isolated, their genomes have remained largely uncharacterized. Here we report the first complete genome sequences of lytic phage isolates specifically infecting three genera of rumen bacteria: Bacteroides, Ruminococcus, and Streptococcus. All phages were classified within the viral order Caudovirales and include two phage morphotypes, representative of the Siphoviridae and Podoviridae families. The phage genomes displayed modular organization and conserved viral genes were identified which enabled further classification and determination of closest phage relatives. Co-examination of bacterial host genomes led to the identification of several genes responsible for modulating phage:host interactions, including CRISPR/Cas elements and restriction-modification phage defense systems. These findings provide new genetic information and insights into how lytic phages may interact with bacteria of the rumen microbiome. PMID:29259581

Protein and Antibody Engineering by Phage Display

PubMed Central

Frei, J.C.; Lai, J.R.

2017-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. PMID:27586328

Phage display on the base of filamentous bacteriophages: application for recombinant antibodies selection.

PubMed

Tikunova, N V; Morozova, V V

2009-10-01

The display of peptides and proteins on the surface of filamentous bacteriophage is a powerful methodology for selection of peptides and protein domains, including antibodies. An advantage of this methodology is the direct physical link between the phenotype and the genotype, as an analyzed polypeptide and its encoding DNA fragment exist in one phage particle. Development of phage display antibody libraries provides repertoires of phage particles exposing antibody fragments of great diversity. The biopanning procedure facilitates selection of antibodies with high affinity and specificity for almost any target. This review is an introduction to phage display methodology. It presents recombinant antibodies display in more details:, construction of phage libraries of antibody fragments and different strategies for the biopanning procedure.

Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage

PubMed Central

Brok-Volchanskaya, Vera S.; Kadyrov, Farid A.; Sivogrivov, Dmitry E.; Kolosov, Peter M.; Sokolov, Andrey S.; Shlyapnikov, Michael G.; Kryukov, Valentine M.; Granovsky, Igor E.

2008-01-01

Homing endonucleases initiate nonreciprocal transfer of DNA segments containing their own genes and the flanking sequences by cleaving the recipient DNA. Bacteriophage T4 segB gene, which is located in a cluster of tRNA genes, encodes a protein of unknown function, homologous to homing endonucleases of the GIY-YIG family. We demonstrate that SegB protein is a site-specific endonuclease, which produces mostly 3′ 2-nt protruding ends at its DNA cleavage site. Analysis of SegB cleavage sites suggests that SegB recognizes a 27-bp sequence. It contains 11-bp conserved sequence, which corresponds to a conserved motif of tRNA TψC stem-loop, whereas the remainder of the recognition site is rather degenerate. T4-related phages T2L, RB1 and RB3 contain tRNA gene regions that are homologous to that of phage T4 but lack segB gene and several tRNA genes. In co-infections of phages T4 and T2L, segB gene is inherited with nearly 100% of efficiency. The preferred inheritance depends absolutely on the segB gene integrity and is accompanied by the loss of the T2L tRNA gene region markers. We suggest that SegB is a homing endonuclease that functions to ensure spreading of its own gene and the surrounding tRNA genes among T4-related phages. PMID:18281701

[Construction of human phage antibody library and screening for human monoclonal antibodies of amylin].

PubMed

Gong, Qian; Li, Chang-ying; Chang, Ji-wu; Zhu, Tie-hong

2012-06-01

To screen monoclonal antibodies to amylin from a constructed human phage antibody library and identify their antigenic specificity and combining activities. The heavy chain Fd fragment and light chain of human immunoglobulin genes were amplified from peripheral blood lymphocytes of healthy donors using RT-PCR, and then inserted into phagemid pComb3XSS to generate a human phage antibody library. The insertion of light chain or heavy chain Fd genes were identified by PCR after the digestion of Sac I, Xba I, Xho Iand Spe I. One of positive clones was analyzed by DNA sequencing. The specific anti-amylin clones were screened from antibody library against human amylin antigens and then the positive clones were determined by Phage-ELISA analysis. A Fab phage antibody library with 0.8×10(8); members was constructed with the efficacy of about 70%. DNA sequence analysis indicated V(H); gene belonged to V(H);3 gene family and V(λ); gene belonged to the V(λ); gene family. Using human amylin as panning antigen, specific anti-amylin Fab antibodies were enriched by screening the library for three times. Phage-ELISA assay showed the positive clones had very good specificity to amylin antigen. The successful construction of a phage antibody library and the identification of anti-amylin Fab antibodies provide a basis for further study and preparation of human anti-amylin antibodies.

Identification and Characterization of Anti-Platelet Antibodies in Idiopathic Thrombocytopenic Purpura Patients

PubMed Central

Aghabeigi, N; Lindsey, N; Zamani, A; Shishaeyan, B

2012-01-01

Background: The autoimmune disease known as Idiopathic (immune thrombocytopenic purpura thrombocytopenic purpura (ITP) is clinically defined by a low numbers of platelets in the circulation blood. This study aimed to isolate autoantibodies made against the platelet glycoproteins using platelets from healthy volunteers, to determine their specificity and further elucidate their effects on platelet function. Methods: This study used a phage display system to recognize Fab anti-platelet antibodies. Anti-platelet After isolation, the anti-platelet Fab-expressing phage was characterized by ELISA and Western blotting. The Fab-bearing phage pool obtained from five rounds of panning was analysed in order to determine its anti-platelet reactivity. Of the phage colonies obtained, 100 colonies of different sizes were randomly selected for reaction with whole platelets, using M13 phage as a negative control. Results: Twelve colonies of them had strong reactions against the whole platelet preparation, but only four colonies showed substantial reactivity against the lysed platelet preparation (lysate). Three of the four colonies showed three bands representing proteins with different molecular weights. The fourth colony showed only a single band. The final experiment to characterise the protein isolated from the phage library was a DNA gel agarose test. Conclusion: Each colony showed a DNA band that corresponded with the molecular size marker for 5.4 kbase pairs, and this suggested the presence of heavy and light antibody chains in the phage. PMID:23113135

Molecular weights and subunit structure of LamB proteins.

PubMed

Nakae, T; Ishii, J N

1982-01-01

Phage lambda-receptor proteins of Escherichia coli, LamB proteins, form oligomeric aggregates to build transmembrane diffusion pores selective for maltose and maltodextrins. The molecular weights (MW) of functional oligomers as well as dissociated monomers were determined by sedimentation equilibrium analysis in homogeneous non-ionic surfactant and deuterium oxide and in 6 M guanidine-HCl, respectively. The MW of oligomers and monomers appeared as 135 600 and 45 900, respectively. Thus, functional Lamb proteins consisted of three identical subunits.

Bacteriophages as a reservoir of extended-spectrum β-lactamase and fluoroquinolone resistance genes in the environment.

PubMed

Marti, E; Variatza, E; Balcázar, J L

2014-07-01

Six antibiotic resistance genes (blaCTX-M , blaSHV , blaTEM , qnrA, qnrB and qnrS) were quantified by qPCR in both phage and bacterial DNA fractions of environmental water samples in order to determine the contribution of phages to the dissemination of antibiotic resistance genes (ARGs) in the environment. Although the highest copy numbers (p <0.05) of ARGs were detected in the bacterial DNA fraction, qnrS and blaSHV genes were found in the phage DNA from all samples analysed, reaching up to 4 log10 copy numbers/mL in hospital samples. These results indicate that bacteriophages are a potential reservoir of resistance genes and may act as efficient vehicles for horizontal gene transfer. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

Complete Genome Sequence of the Streptomyces Phage Nanodon.

PubMed

Erill, Ivan; Caruso, Steven M

2016-10-06

Streptomyces phage Nanodon is a temperate double-stranded DNA Siphoviridae belonging to cluster BD1. It was isolated from soil collected in Kilauea, HI, using Streptomyces griseus subsp. griseus as a host. Copyright © 2016 Erill et al.

Regulation of Bacteriophage T5 Development by ColI Factors

PubMed Central

Moyer, R. W.; Fu, A. S.; Szabo, C.

1972-01-01

The I-type colicinogenic factor ColIb transforms Escherichia coli from a permissive to a nonpermissive host for bacteriophage T5 reproduction by preventing complete expression of the phage genome. T5-infected ColIb+ cells synthesize only class I (early) phage protein and ribonucleic acid (RNA). Neither phage-specific class II proteins [associated with viral deoxyribonucleic acid (DNA) replication] nor class III proteins (phage structural components) are formed due to the failure of the infected ColIb+ cells to synthesize class II or class III phage-specific messenger RNA. Comparable studies with T5-infected cells colicinogenic for the related ColIa factor revealed no decrease in the yield of progeny phage although the presence of the ColIa factor leads to a significant reduction in the amount of phage-directed class III protein synthesis. Images PMID:4554465

Targeting GPR110 in HER2-Overexpressing Breast Cancers

DTIC Science & Technology

2015-10-01

lentiviral plasmids containing GPR110 cDNA using the pHAGE system, which includes the HA tag, under the control of inducible Tet-on promoter. The map of... pHAGE lentiviral plasmid is shown in Figure 3A. Using this, the BT474 and SKBR3 parental cells were stably infected with the lentiviral plasmid...in HER2+ breast cancer. Figure𔃽.’GPR110/overexpression’using’pHAGE’len:viral’mediated’infec:on’of’BT474’cells.’ A.#Map#of# pHAGE # len/viral

The Lambda Select cII Mutation Detection System.

PubMed

Besaratinia, Ahmad; Tommasi, Stella

2018-04-26

A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of these, transgenic mice and the Lambda (λ) Select cII Mutation Detection System have been employed for mutagenicity experiments by many research groups worldwide. Here, we describe a detailed protocol for the Lambda Select cII mutation assay, which can be applied to cultured cells of transgenic mice/rats or the corresponding animals treated with a chemical/physical agent of interest. The protocol consists of the following steps: (1) isolation of genomic DNA from the cells or organs/tissues of transgenic animals treated in vitro or in vivo, respectively, with a test compound; (2) recovery of the lambda shuttle vector carrying a mutational reporter gene (i.e., cII transgene) from the genomic DNA; (3) packaging of the rescued vectors into infectious bacteriophages; (4) infecting a host bacteria and culturing under selective conditions to allow propagation of the induced cII mutations; and (5) scoring the cII-mutants and DNA sequence analysis to determine the cII mutant frequency and mutation spectrum, respectively.

Bacteriophage T4 Infection of Stationary Phase E. coli: Life after Log from a Phage Perspective

PubMed Central

Bryan, Daniel; El-Shibiny, Ayman; Hobbs, Zack; Porter, Jillian; Kutter, Elizabeth M.

2016-01-01

Virtually all studies of phage infections investigate bacteria growing exponentially in rich media. In nature, however, phages largely encounter non-growing cells. Bacteria entering stationary phase often activate well-studied stress defense mechanisms that drastically alter the cell, facilitating its long-term survival. An understanding of phage-host interactions in such conditions is of major importance from both an ecological and therapeutic standpoint. Here, we show that bacteriophage T4 can efficiently bind to, infect and kill E. coli in stationary phase, both in the presence and absence of a functional stationary-phase sigma factor, and explore the response of T4-infected stationary phase cells to the addition of fresh nutrients 5 or 24 h after that infection. An unexpected new mode of response has been identified. “Hibernation” mode is a persistent but reversible dormant state in which the infected cells make at least some phage enzymes, but halt phage development until appropriate nutrients become available before producing phage particles. Our evidence indicates that the block in hibernation mode occurs after the middle-mode stage of phage development; host DNA breakdown and the incorporation of the released nucleotides into phage DNA indicate that the enzymes of the nucleotide synthesizing complex, under middle-mode control, have been made and assembled into a functional state. Once fresh glucose and amino acids become available, the standard lytic infection process rapidly resumes and concentrations of up to 1011 progeny phage (an average of about 40 phage per initially present cell) are produced. All evidence is consistent with the hibernation-mode control point lying between middle mode and late mode T4 gene expression. We have also observed a “scavenger” response, where the infecting phage takes advantage of whatever few nutrients are available to produce small quantities of progeny within 2 to 5 h after infection. The scavenger response seems able to produce no more than an average of one phage per originally available cell, and few if any further progeny are produced by cells in this mode even if fresh nutrients are made available later. PMID:27660625

Novel “Superspreader” Bacteriophages Promote Horizontal Gene Transfer by Transformation

PubMed Central

Bliskovsky, Valery V.; Malagon, Francisco; Baker, James D.; Prince, Jeffrey S.; Klaus, James S.; Adhya, Sankar L.

2017-01-01

ABSTRACT Bacteriophages infect an estimated 1023 to 1025 bacterial cells each second, many of which carry physiologically relevant plasmids (e.g., those encoding antibiotic resistance). However, even though phage-plasmid interactions occur on a massive scale and have potentially significant evolutionary, ecological, and biomedical implications, plasmid fate upon phage infection and lysis has not been investigated to date. Here we show that a subset of the natural lytic phage population, which we dub “superspreaders,” releases substantial amounts of intact, transformable plasmid DNA upon lysis, thereby promoting horizontal gene transfer by transformation. Two novel Escherichia coli phage superspreaders, SUSP1 and SUSP2, liberated four evolutionarily distinct plasmids with equal efficiency, including two close relatives of prominent antibiotic resistance vectors in natural environments. SUSP2 also mediated the extensive lateral transfer of antibiotic resistance in unbiased communities of soil bacteria from Maryland and Wyoming. Furthermore, the addition of SUSP2 to cocultures of kanamycin-resistant E. coli and kanamycin-sensitive Bacillus sp. bacteria resulted in roughly 1,000-fold more kanamycin-resistant Bacillus sp. bacteria than arose in phage-free controls. Unlike many other lytic phages, neither SUSP1 nor SUSP2 encodes homologs to known hydrolytic endonucleases, suggesting a simple potential mechanism underlying the superspreading phenotype. Consistent with this model, the deletion of endonuclease IV and the nucleoid-disrupting protein ndd from coliphage T4, a phage known to extensively degrade chromosomal DNA, significantly increased its ability to promote plasmid transformation. Taken together, our results suggest that phage superspreaders may play key roles in microbial evolution and ecology but should be avoided in phage therapy and other medical applications. PMID:28096488

Complete Genome Sequence of EtG, the First Phage Sequenced from Erwinia tracheiphila.

PubMed

Andrade-Domínguez, Andrés; Kolter, Roberto; Shapiro, Lori R

2018-02-22

Erwinia tracheiphila is the causal agent of bacterial wilt of cucurbits. Here, we report the genome sequence of the temperate phage EtG, which was isolated from an E. tracheiphila -infected cucumber plant. Phage EtG has a linear 30,413-bp double-stranded DNA genome with cohesive ends and 45 predicted open reading frames. Copyright © 2018 Andrade-Domínguez et al.

Current-voltage characteristics of double stranded versus single stranded DNA molecules

NASA Astrophysics Data System (ADS)

Hartzell, B.; Chen, Hong; Heremans, J. J.; McCord, B.; Soghomonian, V.

2004-03-01

Investigation of DNA conductivity has focused on the native, duplex structure, with controversial results. Here, we present the influence of the double-helical structure on charge transport through lambda DNA molecules. The current-voltage (I-V) characteristics of both disulfide-labeled double stranded DNA (dsDNA) and disulfide-labeled single stranded DNA (ssDNA) were measured. The ssDNA was formed from the dsDNA using two different methods for comparison purposes: a thermal/chemical denaturation and enzymatic digestion utilizing lambda exonuclease. Resulting I-V characteristics of both the double stranded and single stranded samples were close-to-linear when measured at room temperature. However, the ssDNA samples consistently gave conductivity values about two orders of magnitude smaller in amplitude. Our results suggest an integral relationship between the native structure of DNA with its stacked base pairs and the molecule's ability to support charge transport.(NSF NIRT 0103034)

Requirement for XLF/Cernunnos in alignment-based gap filling by DNA polymerases lambda and mu for nonhomologous end joining in human whole-cell extracts.

PubMed

Akopiants, Konstantin; Zhou, Rui-Zhe; Mohapatra, Susovan; Valerie, Kristoffer; Lees-Miller, Susan P; Lee, Kyung-Jong; Chen, David J; Revy, Patrick; de Villartay, Jean-Pierre; Povirk, Lawrence F

2009-07-01

XLF/Cernunnos is a core protein of the nonhomologous end-joining pathway of DNA double-strand break repair. To better define the role of Cernunnos in end joining, whole-cell extracts were prepared from Cernunnos-deficient human cells. These extracts effected little joining of DNA ends with cohesive 5' or 3' overhangs, and no joining at all of partially complementary 3' overhangs that required gap filling prior to ligation. Assays in which gap-filled but unligated intermediates were trapped using dideoxynucleotides revealed that there was no gap filling on aligned DSB ends in the Cernunnos-deficient extracts. Recombinant Cernunnos protein restored gap filling and end joining of partially complementary overhangs, and stimulated joining of cohesive ends more than twentyfold. XLF-dependent gap filling was nearly eliminated by immunodepletion of DNA polymerase lambda, but was restored by addition of either polymerase lambda or polymerase mu. Thus, Cernunnos is essential for gap filling by either polymerase during nonhomologous end joining, suggesting that it plays a major role in aligning the two DNA ends in the repair complex.

Cypermethrin and lambda-cyhalothrin induced alterations in nucleic acids and protein contents in a freshwater fish, Channa punctatus.

PubMed

Kumar, Amit; Sharma, Bechan; Pandey, Ravi Shankar

2008-12-01

In this study, a freshwater fish Channa punctatus was exposed to subacute concentrations of synthetic pyrethroid insecticides (cypermethrin and lambda-cyhalothrin) for 96 h to evaluate their impact on the levels of nucleic acids and protein in its different organs. Significant enhancement in the level of DNA was recorded in all tissues of the fish at high concentration of cypermethrin, whereas RNA and protein contents increased in tissues at all concentrations of cypermethrin tested. In contrast, lambda-cyhalothrin treatment caused an increase in the level of DNA only in liver and brain, whereas increase of RNA and protein varied to different levels in different tissues. Cypermethrin treatment induced RNA/DNA ratio in all fish organs tested, whereas lambda-cyhalothrin caused a sharp decrease in the ratio. Protein/DNA ratios were found to be tissue specific in treatments with both of the insecticides. The results clearly indicated that both of these pyrethroids exerted their effects in a similar manner in fish liver but differed in other tissues. These insecticides acted as potential biomodulators in C. punctatus, though following different routes. The results may be an indicator of aquatic pollution affecting freshwater fauna and flora and thus signaling the need for strict regulation on the indiscriminate input of pyrethroids from agricultural sites.

Synthesis of bacteriophage phiC DNA in dna mutants of Esherichia coli.

PubMed

Kodaira, K I; Taketo, A

1978-06-01

Host dna functions involved in the replication of microvirid phage phiC DNA were investigated in vivo. Although growth of this phage was markedly inhibited even at 35-37 degrees C even in dna+ host, conversion of the infecting single-stranded DNA into the double-stranded parental replicative form (stage I synthesis) occurred normally at 43 degrees C in dna+, dnaA, dnaB, dnaC(D), and dnaE cells. In dnaG mutant, the stage I synthesis was severely inhibited at 43 degrees C but not at 30 degrees C. The stage I replication of phiC DNA was clearly thermosensitive in dnaZ cells incubated in nutrient broth. In Tris-casamino acids-glucose medium, however, the dnaZ mutant sufficiently supported synthesis of the parental replicative form. At 43 degrees C, synthesis of the progeny replicative form DNA (stage II replication) was significantly inhibited even in dna+ cells and was nearly completely blocked in dnaB or dnaC(D) mutant. At 37 degrees C, the stage II replication proceeded normally in dna+ bacteria.

Resistance gene transfer: induction of transducing phage by sub-inhibitory concentrations of antimicrobials is not correlated to induction of lytic phage

PubMed Central

Stanczak-Mrozek, Kinga I.; Laing, Ken G.

2017-01-01

Objectives: Horizontal gene transfer of antimicrobial resistance (AMR) genes between clinical isolates via transduction is poorly understood. MRSA are opportunistic pathogens resistant to all classes of antimicrobial agents but currently no strains are fully drug resistant. AMR gene transfer between Staphylococcus aureus isolates is predominantly due to generalized transduction via endogenous bacteriophage, and recent studies have suggested transfer is elevated during host colonization. The aim was to investigate whether exposure to sub-MIC concentrations of antimicrobials triggers bacteriophage induction and/or increased efficiency of AMR gene transfer. Methods: Isolates from MRSA carriers were exposed to nine antimicrobials and supernatants were compared for lytic phage particles and ability to transfer an AMR gene. A new technology, droplet digital PCR, was used to measure the concentration of genes in phage particles. Results: All antibiotics tested induced lytic phage and AMR gene transduction, although the ratio of transducing particles to lytic particles differed substantially for each antibiotic. Mupirocin induced the highest ratio of transducing versus lytic particles. Gentamicin and novobiocin reduced UV-induced AMR transduction. The genes carried in phage particles correlated with AMR transfer or lytic particle activity, suggesting antimicrobials influence which DNA sequences are packaged into phage particles. Conclusions: Sub-inhibitory antibiotics induce AMR gene transfer between clinical MRSA, while combination therapy with an inhibiting antibiotic could potentially alter AMR gene packaging into phage particles, reducing AMR transfer. In a continually evolving environment, pathogens have an advantage if they can transfer DNA while lowering the risk of lytic death. PMID:28369562

A Novel Bacteriophage Targeting Cronobacter sakazakii Is a Potential Biocontrol Agent in Foods

PubMed Central

Lee, Ju-Hoon; Bai, Jaewoo; Shin, Hakdong; Kim, Yeran; Park, Bookyung; Heu, Sunggi

2015-01-01

Cronobacter sakazakii is an important pathogen that causes high mortality in infants. Due to its occasional antibiotic resistance, a bacteriophage approach might be an alternative effective method for the control of this pathogen. To develop a novel biocontrol agent using bacteriophages, the C. sakazakii-infecting phage CR5 was newly isolated and characterized. Interestingly, this phage exhibited efficient and relatively durable host lysis activity. In addition, a specific gene knockout study and subsequent complementation experiment revealed that this phage infected the host strain using the bacterial flagella. The complete genome sequence analysis of phage CR5 showed that its genome contains 223,989 bp of DNA, including 231 predicted open reading frames (ORFs), and it has a G+C content of 50.06%. The annotated ORFs were classified into six functional groups (structure, packaging, host lysis, DNA manipulation, transcription, and additional functions); no gene was found to be related to virulence or toxin or lysogen formation, but >80% of the predicted ORFs are unknown. In addition, a phage proteomic analysis using SDS-PAGE and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) revealed that seven phage structural proteins are indeed present, supporting the ORF predictions. To verify the potential of this phage as a biocontrol agent against C. sakazakii, it was added to infant formula milk contaminated with a C. sakazakii clinical isolate or food isolate, revealing complete growth inhibition of the isolates by the addition of phage CR5 when the multiplicity of infection (MOI) was 105. PMID:26497465

Phage as a Genetically Modifiable Supramacromolecule in Chemistry, Materials and Medicine.

PubMed

Cao, Binrui; Yang, Mingying; Mao, Chuanbin

2016-06-21

Filamentous bacteriophage (phage) is a genetically modifiable supramacromolecule. It can be pictured as a semiflexible nanofiber (∼900 nm long and ∼8 nm wide) made of a DNA core and a protein shell with the former genetically encoding the latter. Although phage bioengineering and phage display techniques were developed before the 1990s, these techniques have not been widely used for chemistry, materials, and biomedical research from the perspective of supramolecular chemistry until recently. Powered by our expertise in displaying a foreign peptide on its surface through engineering phage DNA, we have employed phage to identify target-specific peptides, construct novel organic-inorganic nanohybrids, develop biomaterials for disease treatment, and generate bioanalytical methods for disease diagnosis. Compared with conventional biomimetic chemistry, phage-based supramolecular chemistry represents a new frontier in chemistry, materials science, and medicine. In this Account, we introduce our recent successful efforts in phage-based supramolecular chemistry, by integrating the unique nanofiber-like phage structure and powerful peptide display techniques into the fields of chemistry, materials science, and medicine: (1) successfully synthesized and assembled silica, hydroxyapatite, and gold nanoparticles using phage templates to form novel functional materials; (2) chemically introduced azo units onto the phage to form photoresponsive functional azo-phage nanofibers via a diazotization reaction between aromatic amino groups and the tyrosine residues genetically displayed on phage surfaces; (3) assembled phage into 2D films for studying the effects of both biochemical (the peptide sequences displayed on the phages) and biophysical (the topographies of the phage films) cues on the proliferation and differentiation of mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) and identified peptides and topographies that can induce their osteogenic differentiation; (4) discovered that phage could induce angiogenesis and osteogenesis for MSC-based vascularized bone regeneration; (5) identified novel breast cancer cell-targeting and MSC-targeting peptides and used them to significantly improve the efficiency of targeted cancer therapy and MSC-based gene delivery, respectively; (6) employed engineered phage as a probe to achieve ultrasensitive detection of biomarkers from serum of human patients for disease diagnosis; and (7) constructed centimeter-scale 3D multilayered phage assemblies with the potential application as scaffolds for bone regeneration and functional device fabrication. Our findings demonstrated that phage is indeed a very powerful supramacromolecule suitable for not only developing novel nanostructures and biomaterials but also advancing important fields in biomedicine, including molecular targeting, cancer diagnosis and treatment, drug and gene delivery, stem cell fate direction, and tissue regeneration. Our successes in exploiting phage in chemistry, materials, and medicine suggest that phage itself is nontoxic at the cell level and can be safely used for detecting biomarkers in vitro. Moreover, although we have demonstrated successful in vivo tissue regeneration induced by phage, we believe future studies are needed to evaluate the in vivo biodistribution and potential risks of the phage-based biomaterials.

Phage as a Genetically Modifiable Supramacromolecule in Chemistry, Materials and Medicine

PubMed Central

Cao, Binrui; Yang, Mingying; Mao, Chuanbin

2016-01-01

CONSPECTUS Filamentous bacteriophage (phage) is a genetically modifiable supramacromolecule. It can be pictured as a semiflexible nanofiber (~900 nm long and ~8 nm wide) made of a DNA core and a protein shell with the former genetically encoding the latter. Although phage bioengineering and phage display techniques were developed before the 1990s, these techniques have not been widely used for chemistry, materials, and biomedical research from the perspective of supramolecular chemistry until recently. Powered by our expertise in displaying a foreign peptide on its surface through engineering phage DNA, we have employed phage to identify target-specific peptides, construct novel organic–inorganic nanohybrids, develop biomaterials for disease treatment, and generate bioanalytical methods for disease diagnosis. Compared with conventional biomimetic chemistry, phage-based supramolecular chemistry represents a new frontier in chemistry, materials science, and medicine. In this Account, we introduce our recent successful efforts in phage-based supramolecular chemistry, by integrating the unique nanofiber-like phage structure and powerful peptide display techniques into the fields of chemistry, materials science, and medicine: (1) successfully synthesized and assembled silica, hydroxyapatite, and gold nanoparticles using phage templates to form novel functional materials; (2) chemically introduced azo units onto the phage to form photoresponsive functional azo-phage nanofibers via a diazotization reaction between aromatic amino groups and the tyrosine residues genetically displayed on phage surfaces; (3) assembled phage into 2D films for studying the effects of both biochemical (the peptide sequences displayed on the phages) and biophysical (the topographies of the phage films) cues on the proliferation and differentiation of mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) and identified peptides and topographies that can induce their osteogenic differentiation; (4) discovered that phage could induce angiogenesis and osteogenesis for MSC-based vascularized bone regeneration; (5) identified novel breast cancer cell-targeting and MSC-targeting peptides and used them to significantly improve the efficiency of targeted cancer therapy and MSC-based gene delivery, respectively; (6) employed engineered phage as a probe to achieve ultrasensitive detection of biomarkers from serum of human patients for disease diagnosis; and (7) constructed centimeter-scale 3D multilayered phage assemblies with the potential application as scaffolds for bone regeneration and functional device fabrication. Our findings demonstrated that phage is indeed a very powerful supramacromolecule suitable for not only developing novel nanostructures and biomaterials but also advancing important fields in biomedicine, including molecular targeting, cancer diagnosis and treatment, drug and gene delivery, stem cell fate direction, and tissue regeneration. Our successes in exploiting phage in chemistry, materials, and medicine suggest that phage itself is nontoxic at the cell level and can be safely used for detecting biomarkers in vitro. Moreover, although we have demonstrated successful in vivo tissue regeneration induced by phage, we believe future studies are needed to evaluate the in vivo biodistribution and potential risks of the phage-based biomaterials. PMID:27153341

Two-stage continuous operation of recombinant Escherichia coli using the bacteriophage lambda Q- vector.

PubMed

Oh, Jeong Seok; Cho, Daechul; Park, Tai Hyun

2005-11-01

A two-stage continuous culture of Escherichia coli in combination with a bacteriophage lambda system was performed in order to overcome the intrinsic plasmid instability that is frequently observed in recombinant fermentation. A phage lambda vector with a Q(-) mutation was used to enhance the expression of the lambda system. The optimal values of the important operational variables such as the substrate concentration, the dilution rate, and the mean residence time on the expression of the cloned gene were determined in both batch and continuous cultures. For all culturing modes, the full induction of the cloned gene was observed 4 h after the temperature shift. In the two stage continuous culture, the overproduction reached their maxima at D=0.25 h(-1) with 1.5 S(0) of the medium supply. The maximum productivity of the total beta-galactosidase was 16.3x10(6) U l(-1) h(-1), which was approximately seven times higher than that in the single-copy lysogenic stage. The recombinant cells were stable in the lysogenic state for more than 260 h, while they were stable for 40 h in the lytic state. The instability that developed rapidly in the second tank is believed to be due to the accumulation of lysis proteins as a result of vector leakage during the operation.

Two-Stage Dynamics of In Vivo Bacteriophage Genome Ejection

NASA Astrophysics Data System (ADS)

Chen, Yi-Ju; Wu, David; Gelbart, William; Knobler, Charles M.; Phillips, Rob; Kegel, Willem K.

2018-04-01

Biopolymer translocation is a key step in viral infection processes. The transfer of information-encoding genomes allows viruses to reprogram the cell fate of their hosts. Constituting 96% of all known bacterial viruses [A. Fokine and M. G. Rossmann, Molecular architecture of tailed double-stranded DNA phages, Bacteriophage 4, e28281 (2014)], the tailed bacteriophages deliver their DNA into host cells via an "ejection" process, leaving their protein shells outside of the bacteria; a similar scenario occurs for mammalian viruses like herpes, where the DNA genome is ejected into the nucleus of host cells, while the viral capsid remains bound outside to a nuclear-pore complex. In light of previous experimental measurements of in vivo bacteriophage λ ejection, we analyze here the physical processes that give rise to the observed dynamics. We propose that, after an initial phase driven by self-repulsion of DNA in the capsid, the ejection is driven by anomalous diffusion of phage DNA in the crowded bacterial cytoplasm. We expect that this two-step mechanism is general for phages that operate by pressure-driven ejection, and we discuss predictions of our theory to be tested in future experiments.

States of phage T3/T7 capsids: buoyant density centrifugation and cryo-EM.

PubMed

Serwer, Philip; Wright, Elena T; Demeler, Borries; Jiang, Wen

2018-04-01

Mature double-stranded DNA bacteriophages have capsids with symmetrical shells that typically resist disruption, as they must to survive in the wild. However, flexibility and associated dynamism assist function. We describe biochemistry-oriented procedures used to find previously obscure flexibility for capsids of the related phages, T3 and T7. The primary procedures are hydration-based buoyant density ultracentrifugation and purified particle-based cryo-electron microscopy (cryo-EM). We review the buoyant density centrifugation in detail. The mature, stable T3/T7 capsid is a shell flexibility-derived conversion product of an initially assembled procapsid (capsid I). During DNA packaging, capsid I expands and loses a scaffolding protein to form capsid II. The following are observations made with capsid II. (1) The in vivo DNA packaging of wild type T3 generates capsid II that has a slight (1.4%), cryo-EM-detected hyper-expansion relative to the mature phage capsid. (2) DNA packaging in some altered conditions generates more extensive hyper-expansion of capsid II, initially detected by hydration-based preparative buoyant density centrifugation in Nycodenz density gradients. (3) Capsid contraction sometimes occurs, e.g., during quantized leakage of DNA from mature T3 capsids without a tail.

Genome sequences and characterization of the related Gordonia phages GTE5 and GRU1 and their use as potential biocontrol agents.

PubMed

Petrovski, Steve; Tillett, Daniel; Seviour, Robert J

2012-01-01

Activated sludge plants suffer frequently from the operational problem of stable foam formation on aerobic reactor surfaces, which can be difficult to prevent. Many foams are stabilized by mycolic acid-containing Actinobacteria, the mycolata. The in situ biocontrol of foaming using phages is an attractive strategy. We describe two polyvalent phages, GTE5 and GRU1, targeting Gordonia terrae and Gordonia rubrupertincta, respectively, isolated from activated sludge. Phage GRU1 also propagates on Nocardia nova. Both phages belong to the family Siphoviridae and have similar-size icosahedral heads that encapsulate double-stranded DNA genomes (∼65 kb). Their genome sequences are similar to each other but markedly different from those of other sequenced phages. Both are arranged in a modular fashion. These phages can reduce or eliminate foam formation by their host cells under laboratory conditions.

DNA packaging by the Bacillus subtilis defective bacteriophage PBSX.

PubMed Central

Anderson, L M; Bott, K F

1985-01-01

Defective bacteriophage PBSX, a resident of all Bacillus subtilis 168 chromosomes, packages fragments of DNA from all portions of the host chromosome when induced by mitomycin C. In this study, the physical process for DNA packaging of both chromosomal and plasmid DNAs was examined. Discrete 13-kilobase (kb) lengths of DNA were packaged by wild-type phage, and the process was DNase I resistant and probably occurred by a head-filling mechanism. Genetically engineered isogenic host strains having a chloramphenicol resistance determinant integrated as a genetic flag at two different regions of the chromosome were used to monitor the packaging of specific chromosomal regions. No dramatic selectivity for these regions could be documented. If the wild-type strain 168 contains autonomously replicating plasmids, especially pC194, the mitomycin C induces an increase in size of resident plasmid DNA, which is then packaged as 13-kb pieces into phage heads. In strain RB1144, which lacks substantial portions of the PBSX resident phage region, mitomycin C treatment did not affect the structure of resident plasmids. Induction of PBSX started rolling circle replication on plasmids, which then became packaged as 13-kb fragments. This alteration or cannibalization of plasmid replication resulting from mitomycin C treatment requires for its function some DNA within the prophage deletion of strain RB1144. Images PMID:3923209

The Crystal Structure of Bacteriophage HK97 gp6: Defining a Large Family of Head-Tail Connector Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cardarelli, Lia; Lam, Robert; Tuite, Ashleigh

2010-08-17

The final step in the morphogenesis of long-tailed double-stranded DNA bacteriophages is the joining of the DNA-filled head to the tail. The connector is a specialized structure of the head that serves as the interface for tail attachment and the point of egress for DNA from the head during infection. Here, we report the determination of a 2.1 {angstrom} crystal structure of gp6 of bacteriophage HK97. Through structural comparisons, functional studies, and bioinformatic analysis, gp6 has been determined to be a component of the connector of phage HK97 that is evolutionarily related to gp15, a well-characterized connector component of bacteriophagemore » SPP1. Whereas the structure of gp15 was solved in a monomeric form, gp6 crystallized as an oligomeric ring with the dimensions expected for a connector protein. Although this ring is composed of 13 subunits, which does not match the symmetry of the connector within the phage, sequence conservation and modeling of this structure into the cryo-electron microscopy density of the SPP1 connector indicate that this oligomeric structure represents the arrangement of gp6 subunits within the mature phage particle. Through sequence searches and genomic position analysis, we determined that gp6 is a member of a large family of connector proteins that are present in long-tailed phages. We have also identified gp7 of HK97 as a homologue of gp16 of phage SPP1, which is the second component of the connector of this phage. These proteins are members of another large protein family involved in connector assembly.« less

The Crystal Structure of Bacteriophage HK97 gp6: Defining a Large Family of Head-Tail Connector Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cardarelli, Lia; Lam, Robert; Tuite, Ashleigh

2011-11-23

The final step in the morphogenesis of long-tailed double-stranded DNA bacteriophages is the joining of the DNA-filled head to the tail. The connector is a specialized structure of the head that serves as the interface for tail attachment and the point of egress for DNA from the head during infection. Here, we report the determination of a 2.1 Å crystal structure of gp6 of bacteriophage HK97. Through structural comparisons, functional studies, and bioinformatic analysis, gp6 has been determined to be a component of the connector of phage HK97 that is evolutionarily related to gp15, a well-characterized connector component of bacteriophagemore » SPP1. Whereas the structure of gp15 was solved in a monomeric form, gp6 crystallized as an oligomeric ring with the dimensions expected for a connector protein. Although this ring is composed of 13 subunits, which does not match the symmetry of the connector within the phage, sequence conservation and modeling of this structure into the cryo-electron microscopy density of the SPP1 connector indicate that this oligomeric structure represents the arrangement of gp6 subunits within the mature phage particle. Through sequence searches and genomic position analysis, we determined that gp6 is a member of a large family of connector proteins that are present in long-tailed phages. We have also identified gp7 of HK97 as a homologue of gp16 of phage SPP1, which is the second component of the connector of this phage. These proteins are members of another large protein family involved in connector assembly.« less

Human scFv antibody fragments specific for hepatocellular carcinoma selected from a phage display library.

PubMed

Yu, Bing; Ni, Ming; Li, Wen-Han; Lei, Ping; Xing, Wei; Xiao, Dai-Wen; Huang, Yu; Tang, Zhen-Jie; Zhu, Hui-Fen; Shen, Guan-Xin

2005-07-14

To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library. A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DNA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in E.coli HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation. Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E.coli HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted M(r) value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells. The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.

The extracellular phage-host interactions involved in the bacteriophage LL-H infection of Lactobacillus delbrueckii ssp. lactis ATCC 15808

PubMed Central

Munsch-Alatossava, Patricia; Alatossava, Tapani

2013-01-01

The complete genome sequence of Lactobacillus bacteriophage LL-H was determined in 1996. Accordingly, LL-H has been used as a model phage for the infection of dairy Lactobacillus, specifically for thermophilic Lactobacillus delbrueckii ssp. lactis host strains, such as ATCC 15808. One of the major goals of phage LL-H research consisted of the characterization of the first phage-host interactions at the level of phage adsorption and phage DNA injection steps to determine effective and practical methods to minimize the risks associated with the appearance and attack of phages in the manufacture of yogurt, and Swiss or Italian hard type cheeses, which typically use thermophilic lactic acid bacteria starter cultures containing L. delbrueckii strains among others. This mini review article summarizes the present data concerning (i) the special features, particle structure, and components of phage LL-H and (ii) the structure and properties of lipoteichoic acids (LTAs), which are the phage LL-H receptor components of L. delbrueckii ssp. lactis host strains. Moreover, a model of the first, extracellular, phage-host interactions for the infection of L. delbrueckii ssp. lactis ATCC 15808 by phage LL-H is presented and further discussed. PMID:24400001

The extracellular phage-host interactions involved in the bacteriophage LL-H infection of Lactobacillus delbrueckii ssp. lactis ATCC 15808.

PubMed

Munsch-Alatossava, Patricia; Alatossava, Tapani

2013-12-24

The complete genome sequence of Lactobacillus bacteriophage LL-H was determined in 1996. Accordingly, LL-H has been used as a model phage for the infection of dairy Lactobacillus, specifically for thermophilic Lactobacillus delbrueckii ssp. lactis host strains, such as ATCC 15808. One of the major goals of phage LL-H research consisted of the characterization of the first phage-host interactions at the level of phage adsorption and phage DNA injection steps to determine effective and practical methods to minimize the risks associated with the appearance and attack of phages in the manufacture of yogurt, and Swiss or Italian hard type cheeses, which typically use thermophilic lactic acid bacteria starter cultures containing L. delbrueckii strains among others. This mini review article summarizes the present data concerning (i) the special features, particle structure, and components of phage LL-H and (ii) the structure and properties of lipoteichoic acids (LTAs), which are the phage LL-H receptor components of L. delbrueckii ssp. lactis host strains. Moreover, a model of the first, extracellular, phage-host interactions for the infection of L. delbrueckii ssp. lactis ATCC 15808 by phage LL-H is presented and further discussed.

Stochasticity in the Expression of LamB and its Affect on λ phage Infection

NASA Astrophysics Data System (ADS)

Chapman, Emily; Wu, Xiao-Lun

2006-03-01

λ phage binds to E. Coli's lamB protein and injects its DNA into the cell. The phage quickly replicates and after a latent period the bacteria bursts, emitting mature phages. We developed a mathematical model based on the known physical events that occur when a λ phage infects an E.Coli cell. The results of these models predict that the bacteria and phage populations become extinct unless the parameters of the model are very finely tuned, which is untrue in the nature. The lamB protein is part of the maltose regulon and can be repressed to minimal levels when grown in the absence of inducer. Therefore, a cell that is not expressing any lamB protein at that moment is resistant against phage infection. We studied the dynamic relationship between λ phage and E. Coli when the concentration of phage greatly outnumbers the concentration of bacteria. We study how the stochasticity of the expression of lamB affects the percentage of cells that the λ phage infects. We show that even in the case when the maltose regulon is fully induced a percentage of cells continue to persist against phage infection.

Antibiotic Resistance Genes in the Bacteriophage DNA Fraction of Environmental Samples

PubMed Central

Colomer-Lluch, Marta; Jofre, Juan; Muniesa, Maite

2011-01-01

Antibiotic resistance is an increasing global problem resulting from the pressure of antibiotic usage, greater mobility of the population, and industrialization. Many antibiotic resistance genes are believed to have originated in microorganisms in the environment, and to have been transferred to other bacteria through mobile genetic elements. Among others, β-lactam antibiotics show clinical efficacy and low toxicity, and they are thus widely used as antimicrobials. Resistance to β-lactam antibiotics is conferred by β-lactamase genes and penicillin-binding proteins, which are chromosomal- or plasmid-encoded, although there is little information available on the contribution of other mobile genetic elements, such as phages. This study is focused on three genes that confer resistance to β-lactam antibiotics, namely two β-lactamase genes (blaTEM and blaCTX-M9) and one encoding a penicillin-binding protein (mecA) in bacteriophage DNA isolated from environmental water samples. The three genes were quantified in the DNA isolated from bacteriophages collected from 30 urban sewage and river water samples, using quantitative PCR amplification. All three genes were detected in the DNA of phages from all the samples tested, in some cases reaching 104 gene copies (GC) of blaTEM or 102 GC of blaCTX-M and mecA. These values are consistent with the amount of fecal pollution in the sample, except for mecA, which showed a higher number of copies in river water samples than in urban sewage. The bla genes from phage DNA were transferred by electroporation to sensitive host bacteria, which became resistant to ampicillin. blaTEM and blaCTX were detected in the DNA of the resistant clones after transfection. This study indicates that phages are reservoirs of resistance genes in the environment. PMID:21390233

Protein and Antibody Engineering by Phage Display.

PubMed

Frei, J C; Lai, J R

2016-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. © 2016 Elsevier Inc. All rights reserved.

Effect of Escherichia coli DNA binding protein on the transcription of single-stranded phage M13 DNA by Escherichia coli RNA polymerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Niyogi, S.K.; Ratrie, H. III; Datta, A.K.

E. coli DNA binding protein strongly inhibits the transcription of single-stranded rather than double-stranded phage M13 DNA by E. coli RNA polymerase. This inhibition cannot be significantly overcome by increasing the concentration of RNA polymerase. Nor does the order of addition of binding protein affect its inhibitory property: inhibition is evident whether binding protein is added before or after the formation of the RNA polymerase--DNA complex. Inhibition is also observed if binding protein is added at various times after initiation of RNA synthesis. Maximal inhibition occurs at a binding protein-to-DNA ratio (w/w) of about 8:1. This corresponds to one bindingmore » protein molecule covering about 30 nucleotides, in good agreement with values obtained by physical measurements.« less

Recombination directionality factor gp3 binds ϕC31 integrase via the zinc domain, potentially affecting the trajectory of the coiled-coil motif

PubMed Central

Younger, Ellen; Fernando, Booshini D; Khaleel, Thanafez; Stark, W Marshall; Smith, Margaret C M

2018-01-01

Abstract To establish a prophage state, the genomic DNA of temperate bacteriophages normally becomes integrated into the genome of their host bacterium by integrase-mediated, site-specific DNA recombination. Serine integrases catalyse a single crossover between an attachment site in the host (attB) and a phage attachment site (attP) on the circularized phage genome to generate the integrated prophage DNA flanked by recombinant attachment sites, attL and attR. Exiting the prophage state and entry into the lytic growth cycle requires an additional phage-encoded protein, the recombination directionality factor or RDF, to mediate recombination between attL and attR and excision of the phage genome. The RDF is known to bind integrase and switch its activity from integration (attP x attB) to excision (attL x attR) but its precise mechanism is unclear. Here, we identify amino acid residues in the RDF, gp3, encoded by the Streptomyces phage ϕC31 and within the ϕC31 integrase itself that affect the gp3:Int interaction. We show that residue substitutions in integrase that reduce gp3 binding adversely affect both excision and integration reactions. The mutant integrase phenotypes are consistent with a model in which the RDF binds to a hinge region at the base of the coiled-coil motif in ϕC31 integrase. PMID:29228292

The internal head protein Gp16 controls DNA ejection from the bacteriophage T7 virion.

PubMed

Struthers-Schlinke, J S; Robins, W P; Kemp, P; Molineux, I J

2000-08-04

A wild-type T7 virion ejects about 850 bp of the 40 kb genome into the bacterial cell by a transcription-independent process. Internalization of the remainder of the genome normally requires transcription. Inhibition of transcription-independent DNA translocation beyond the leading 850 bp is not absolute but the time taken by a population of phage genomes in overcoming the block averages about 20 minutes at 30 degrees C. There are additional blocks to transcription-independent translocation and less than 20 % of infecting DNA molecules completely penetrate the cell cytoplasm after four hours of infection. Mutant virions containing an altered gene 16 protein either prevent the blocks to transcription-independent DNA translocation or effect rapid release from blocking sites and allow the entire phage DNA molecule to enter the cell at a constant rate of about 75 bp per second. This rate is likely the same at which the leading 850 bp is ejected into the cell from a wild-type virion. All mutations fall into two clusters contained within 380 bp of the 4 kb gene 16, suggesting that a 127 residue segment of gp16 controls DNA ejection from the phage particle. We suggest that this segment of gp16 acts as a clamp to prevent transcription-independent DNA translocation. Copyright 2000 Academic Press.

Multiplex PCR to detect bacteriophages from natural whey cultures of buffalo milk and characterisation of two phages active against Lactococcus lactis, ΦApr-1 and ΦApr-2.

PubMed

Aprea, Giuseppe; Mullan, William Michael; Murru, Nicoletta; Fitzgerald, Gerald; Buonanno, Marialuisa; Cortesi, Maria Luisa; Prencipe, Vincenza Annunziata; Migliorati, Giacomo

2017-09-30

This work investigated bacteriophage induced starter failures in artisanal buffalo Mozzarella production plants in Southern Italy. Two hundred and ten samples of whey starter cultures were screened for bacteriophage infection. Multiplex polymerase chain reaction (PCR) revealed phage infection in 28.56% of samples, all showing acidification problems during cheese making. Based on DNA sequences, bacteriophages for Lactococcus lactis (L. lactis), Lactobacillus delbruekii (L. delbruekii) and Streptococcus thermophilus (S. thermophilus) were detected. Two phages active against L. lactis, ΦApr-1 and ΦApr-2, were isolated and characterised. The genomes, approximately 31.4 kb and 31 kb for ΦApr-1 and ΦApr-2 respectively, consisted of double-stranded linear DNA with pac-type system. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‑PAGE) showed one major structural protein of approximately 32.5 kDa and several minor proteins. This is the first report of phage isolation in buffalo milk and of the use of multiplex PCR to screen and study the diversity of phages against Lactic Acid Bacteria (LAB) strains in artisanal Water Buffalo Mozzarella starters.

Bacteriophages of Gordonia spp. Display a Spectrum of Diversity and Genetic Relationships

PubMed Central

Pope, Welkin H.; Mavrich, Travis N.; Garlena, Rebecca A.; Guerrero-Bustamante, Carlos A.; Jacobs-Sera, Deborah; Montgomery, Matthew T.; Russell, Daniel A.; Warner, Marcie H.

2017-01-01

ABSTRACT The global bacteriophage population is large, dynamic, old, and highly diverse genetically. Many phages are tailed and contain double-stranded DNA, but these remain poorly characterized genomically. A collection of over 1,000 phages infecting Mycobacterium smegmatis reveals the diversity of phages of a common bacterial host, but their relationships to phages of phylogenetically proximal hosts are not known. Comparative sequence analysis of 79 phages isolated on Gordonia shows these also to be diverse and that the phages can be grouped into 14 clusters of related genomes, with an additional 14 phages that are “singletons” with no closely related genomes. One group of six phages is closely related to Cluster A mycobacteriophages, but the other Gordonia phages are distant relatives and share only 10% of their genes with the mycobacteriophages. The Gordonia phage genomes vary in genome length (17.1 to 103.4 kb), percentage of GC content (47 to 68.8%), and genome architecture and contain a variety of features not seen in other phage genomes. Like the mycobacteriophages, the highly mosaic Gordonia phages demonstrate a spectrum of genetic relationships. We show this is a general property of bacteriophages and suggest that any barriers to genetic exchange are soft and readily violable. PMID:28811342

Finding a needle in the virus metagenome haystack--micro-metagenome analysis captures a snapshot of the diversity of a bacteriophage armoire.

PubMed

Ray, Jessica; Dondrup, Michael; Modha, Sejal; Steen, Ida Helene; Sandaa, Ruth-Anne; Clokie, Martha

2012-01-01

Viruses are ubiquitous in the oceans and critical components of marine microbial communities, regulating nutrient transfer to higher trophic levels or to the dissolved organic pool through lysis of host cells. Hydrothermal vent systems are oases of biological activity in the deep oceans, for which knowledge of biodiversity and its impact on global ocean biogeochemical cycling is still in its infancy. In order to gain biological insight into viral communities present in hydrothermal vent systems, we developed a method based on deep-sequencing of pulsed field gel electrophoretic bands representing key viral fractions present in seawater within and surrounding a hydrothermal plume derived from Loki's Castle vent field at the Arctic Mid-Ocean Ridge. The reduction in virus community complexity afforded by this novel approach enabled the near-complete reconstruction of a lambda-like phage genome from the virus fraction of the plume. Phylogenetic examination of distinct gene regions in this lambdoid phage genome unveiled diversity at loci encoding superinfection exclusion- and integrase-like proteins. This suggests the importance of fine-tuning lyosgenic conversion as a viral survival strategy, and provides insights into the nature of host-virus and virus-virus interactions, within hydrothermal plumes. By reducing the complexity of the viral community through targeted sequencing of prominent dsDNA viral fractions, this method has selectively mimicked virus dominance approaching that hitherto achieved only through culturing, thus enabling bioinformatic analysis to locate a lambdoid viral "needle" within the greater viral community "haystack". Such targeted analyses have great potential for accelerating the extraction of biological knowledge from diverse and poorly understood environmental viral communities.

Biodiversity of Lactobacillus helveticus bacteriophages isolated from cheese whey starters.

PubMed

Zago, Miriam; Bonvini, Barbara; Rossetti, Lia; Meucci, Aurora; Giraffa, Giorgio; Carminati, Domenico

2015-05-01

Twenty-one Lactobacillus helveticus bacteriophages, 18 isolated from different cheese whey starters and three from CNRZ collection, were phenotypically and genetically characterised. A biodiversity between phages was evidenced both by host range and molecular (RAPD-PCR) typing. A more detailed characterisation of six phages showed similar structural protein profiles and a relevant genetic biodiversity, as shown by restriction enzyme analysis of total DNA. Latent period, burst time and burst size data evidenced that phages were active and virulent. Overall, data highlighted the biodiversity of Lb. helveticus phages isolated from cheese whey starters, which were confirmed to be one of the most common phage contamination source in dairy factories. More research is required to further unravel the ecological role of Lb. helveticus phages and to evaluate their impact on the dairy fermentation processes where whey starter cultures are used.

Serum free light chains, interferon-alpha, and interleukins in systemic lupus erythematosus.

PubMed

Jolly, M; Francis, S; Aggarwal, R; Mikolaitis, R A; Niewold, T B; Chubinskaya, S; Block, J A; Scanzello, C; Sequeira, W

2014-08-01

Interleukin-6 (IL-6), interleukin-10 (IL-10), interferon-alpha (IFN-α), and free light chains (FLCs: lambda, kappa) have all been noted to be of importance in systemic lupus erythematosus (SLE). Herein, we quantified and explored the relationship between these inflammatory mediators and disease activity in SLE; and stratified by their current anti-dsDNA antibody status. Seventy-seven SLE patients underwent assessment of disease activity using the SLE disease activity index (SLEDAI). Serum FLC (lambda, kappa, and total), IL-6, IL-10, and IFN-α were quantified. Demographics of disease characteristics were determined by chart reviews. Statistical analyses included Mann-Whitney test, chi square, and linear regression analyses. Mean (SD) age of the patients was 44.9 ± 12.7 years; SLEDAI (mean ± SD) was 3.4 ± 4.0. Serum lambda FLC levels had a moderate correlation (r = 0.46 with physician global assessment, 0.44 with SLEDAI) and the strongest correlation with disease activity as compared with other inflammatory mediators including current dsDNA antibody status. After adjusting for prednisone use, the correlation of lambda FLC with PGA (r = 0.48) and SLEDAI (r = 0.52) was better than of current dsDNA antibody status with PGA (r = 0.33) and adjusted SLEDAI (r = 0.24), respectively. IL-10 and IFN-α activity did not correlate with disease activity. Serum FLC and IL-6 levels could differentiate between active and inactive SLE patients. Serum lambda FLC and IL-6 levels differed significantly among patients with and without current dsDNA antibodies. Serum lambda FLC levels accounted for 31% of variance in SLEDAI scores. Serum FLC and IL-6 are potentially useful biomarkers of disease activity in SLE. Further studies, with larger study sample and longitudinal design, are indicated. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Isolation and Genome Characterization of the Virulent Staphylococcus aureus Bacteriophage SA97

PubMed Central

Chang, Yoonjee; Shin, Hakdong; Lee, Ju-Hoon; Park, Chul Jong; Paik, Soon-Young; Ryu, Sangryeol

2015-01-01

A novel bacteriophage that infects S. aureus, SA97, was isolated and characterized. The phage SA97 belongs to the Siphoviridae family, and the cell wall teichoic acid (WTA) was found to be a host receptor of the phage SA97. Genome analysis revealed that SA97 contains 40,592 bp of DNA encoding 54 predicted open reading frames (ORFs), and none of these genes were related to virulence or drug resistance. Although a few genes associated with lysogen formation were detected in the phage SA97 genome, the phage SA97 produced neither lysogen nor transductant in S. aureus. These results suggest that the phage SA97 may be a promising candidate for controlling S. aureus. PMID:26437428

Efficient engineering of a bacteriophage genome using the type I-E CRISPR-Cas system.

PubMed

Kiro, Ruth; Shitrit, Dror; Qimron, Udi

2014-01-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system has recently been used to engineer genomes of various organisms, but surprisingly, not those of bacteriophages (phages). Here we present a method to genetically engineer the Escherichia coli phage T7 using the type I-E CRISPR-Cas system. T7 phage genome is edited by homologous recombination with a DNA sequence flanked by sequences homologous to the desired location. Non-edited genomes are targeted by the CRISPR-Cas system, thus enabling isolation of the desired recombinant phages. This method broadens CRISPR Cas-based editing to phages and uses a CRISPR-Cas type other than type II. The method may be adjusted to genetically engineer any bacteriophage genome.

Genomic characterization of Ralstonia solanacearum phage phiRSB1, a T7-like wide-host-range phage.

PubMed

Kawasaki, Takeru; Shimizu, Mio; Satsuma, Hideki; Fujiwara, Akiko; Fujie, Makoto; Usami, Shoji; Yamada, Takashi

2009-01-01

PhiRSB1 is a wide-host-range, T7-like bacteriophage that infects and efficiently lyses the phytopathogenic bacterium Ralstonia solanacearum. The phiRSB1 genome comprises 43,079 bp of double-stranded DNA (61.7% G+C) with 325-bp terminal repeats and contains 47 open reading frames. Strong activity of tandem early promoters and wide specificity of phage promoters of phiRSB1 were demonstrated.

Capture and detection of T7 bacteriophages on a nanostructured interface.

PubMed

Han, Jin-Hee; Wang, Min S; Das, Jayanti; Sudheendra, L; Vonasek, Erica; Nitin, Nitin; Kennedy, Ian M

2014-04-09

A highly ordered array of T7 bacteriophages was created by the electrophoretic capture of phages onto a nanostructured array with wells that accommodated the phages. Electrophoresis of bacteriophages was achieved by applying a positive potential on an indium tin oxide electrode at the bottom of the nanowells. Nanoscale arrays of phages with different surface densities were obtained by changing the electric field applied to the bottom of the nanowells. The applied voltage was shown to be the critical factor in generating a well-ordered phage array. The number of wells occupied by a phage, and hence the concentration of phages in a sample solution, could be quantified by using a DNA intercalating dye that rapidly stains the T7 phage. The fluorescence signal was enhanced by the intrinsic photonic effect made available by the geometry of the platform. It was shown that the quantification of phages on the array was 6 orders of magnitude better than could be obtained with a fluorescent plate reader. The device opens up the possibility that phages can be detected directly without enrichment or culturing, and by detecting phages that specifically infect bacteria of interest, rapid pathogen detection becomes possible.

Capture and Detection of T7 Bacteriophages on a Nanostructured Interface

PubMed Central

2015-01-01

A highly ordered array of T7 bacteriophages was created by the electrophoretic capture of phages onto a nanostructured array with wells that accommodated the phages. Electrophoresis of bacteriophages was achieved by applying a positive potential on an indium tin oxide electrode at the bottom of the nanowells. Nanoscale arrays of phages with different surface densities were obtained by changing the electric field applied to the bottom of the nanowells. The applied voltage was shown to be the critical factor in generating a well-ordered phage array. The number of wells occupied by a phage, and hence the concentration of phages in a sample solution, could be quantified by using a DNA intercalating dye that rapidly stains the T7 phage. The fluorescence signal was enhanced by the intrinsic photonic effect made available by the geometry of the platform. It was shown that the quantification of phages on the array was 6 orders of magnitude better than could be obtained with a fluorescent plate reader. The device opens up the possibility that phages can be detected directly without enrichment or culturing, and by detecting phages that specifically infect bacteria of interest, rapid pathogen detection becomes possible. PMID:24650205

Antisense RNA: effect of ribosome binding sites, target location, size, and concentration on the translation of specific mRNA molecules.

PubMed

Daugherty, B L; Hotta, K; Kumar, C; Ahn, Y H; Zhu, J D; Pestka, S

1989-01-01

A series of plasmids were constructed to generate RNA complementary to the beta-galactosidase messenger RNA under control of the phage lambda PL promoter. These plasmids generate anti-lacZ mRNA bearing or lacking a synthetic ribosome binding site adjacent to the lambda PL promoter and/or the lacZ ribosome binding site in reverse orientation. Fragments of lacZ DNA from the 5' and/or the 3' region were used in these constructions. When these anti-mRNA molecules were produced in Escherichia coli 294, maximal inhibition of beta-galactosidase synthesis occurred when a functional ribosome binding site was present near the 5' end of the anti-mRNA and the anti-mRNA synthesized was complementary to the 5' region of the mRNA corresponding to the lacZ ribosome binding site and/or the 5'-coding sequence. Anti-mRNAs producing maximal inhibition of beta-galactosidase synthesis exhibited an anti-lacZ mRNA:normal lacZ mRNA ratio of 100:1 or higher. Those showing lower levels of inhibition exhibited much lower anti-lacZ mRNA:normal lacZ mRNA ratios. A functional ribosome binding site at the 5'-end was found to decrease the decay rate of the anti-lacZ mRNAs. In addition, the incorporation of a transcription terminator just downstream of the antisense segment provided for more efficient inhibition of lacZ mRNA translation due to synthesis of smaller and more abundant anti-lacZ mRNAs. The optimal constructions produced undetectable levels of beta-galactosidase synthesis.

Models of life: epigenetics, diversity and cycles.

PubMed

Sneppen, Kim

2017-04-01

This review emphasizes aspects of biology that can be understood through repeated applications of simple causal rules. The selected topics include perspectives on gene regulation, phage lambda development, epigenetics, microbial ecology, as well as model approaches to diversity and to punctuated equilibrium in evolution. Two outstanding features are repeatedly described. One is the minimal number of rules to sustain specific states of complex systems for a long time. The other is the collapse of such states and the subsequent dynamical cycle of situations that restitute the system to a potentially new metastable state.

Models of life: epigenetics, diversity and cycles

NASA Astrophysics Data System (ADS)

Sneppen, Kim

2017-04-01

This review emphasizes aspects of biology that can be understood through repeated applications of simple causal rules. The selected topics include perspectives on gene regulation, phage lambda development, epigenetics, microbial ecology, as well as model approaches to diversity and to punctuated equilibrium in evolution. Two outstanding features are repeatedly described. One is the minimal number of rules to sustain specific states of complex systems for a long time. The other is the collapse of such states and the subsequent dynamical cycle of situations that restitute the system to a potentially new metastable state.

Study of the effect of simulated space environment on nucleoprotein and DNA thin films

NASA Astrophysics Data System (ADS)

Fekete, A.; Módos, K.; Hegedüs, M.; Rontó, Gy.; Kovács, G.; Bérces, A.; Kargl, G.; Kömle, N. I.; Lammer, H.

2002-11-01

The main goal of PUR experiment (phage and uracil response) is to examine and quantify the effect of specific space conditions on nucleic acid models. To achieve this an improved method was elaborated for the preparation of DNA and bacteriophage thin films. The homogeneity of the films was controlled by UV spectroscopy and microscopy. To provide experimental evidence for the hypothesis that interplanetary transfer of life is possible, phage T7 and isolated T7 DNA thin films have been exposed to selected space conditions: intense UVC radiation (λ = 254 nm) and high vacuum (10-5 mbar). The effects of DNA hydration, conformation and packing on UV radiation damage were examined. Characteristic changes in the absorption spectrum, in the electrophoretic pattern of DNA and the decrease of the amount of PCR products have been detected indicating the photodamage of isolated and intraphage DNA.

Simulation experiments of the effect of space environment on bacteriophage and DNA thin films

NASA Astrophysics Data System (ADS)

Fekete, A.; Rontó, Gy.; Hegedüs, M.; Módos, K.; Bérces, A.; Kovács, G.; Lammer, H.; Panitz, C.

2004-01-01

The main goal of PUR experiment (phage and uracil response) is to examine and quantify the effect of specific space conditions on nucleic acid models. To achieve this an improved method was elaborated for the preparation of DNA and bacteriophage thin films. The homogeneity of the films was controlled by UV spectroscopy and microscopy. To provide experimental evidence for the hypothesis that interplanetary transfer of the genetic material is possible, phage T7 and isolated T7 DNA thin films have been exposed to selected space conditions: intense UVC radiation ( λ=254 nm) and high vacuum (10 -4 Pa). The effects of DNA hydration, conformation and packing on UV radiation damage were examined. Characteristic changes in the absorption spectrum, in the electrophoretic pattern of DNA and the decrease of the amount of PCR products have been detected indicating the photodamage of isolated and intraphage DNA.

Examining a DNA Replication Requirement for Bacteriophage λ Red- and Rac Prophage RecET-Promoted Recombination in Escherichia coli.

PubMed

Thomason, Lynn C; Costantino, Nina; Court, Donald L

2016-09-13

Recombineering, in vivo genetic engineering with bacteriophage homologous recombination systems, is a powerful technique for making genetic modifications in bacteria. Two systems widely used in Escherichia coli are the Red system from phage λ and RecET from the defective Rac prophage. We investigated the in vivo dependence of recombineering on DNA replication of the recombining substrate using plasmid targets. For λ Red recombination, when DNA replication of a circular target plasmid is prevented, recombination with single-stranded DNA oligonucleotides is greatly reduced compared to that under replicating conditions. For RecET recombination, when DNA replication of the targeted plasmid is prevented, the recombination frequency is also reduced, to a level identical to that seen for the Red system in the absence of replication. The very low level of oligonucleotide recombination observed in the absence of any phage recombination functions is the same in the presence or absence of DNA replication. In contrast, both the Red and RecET systems recombine a nonreplicating linear dimer plasmid with high efficiency to yield a circular monomer. Therefore, the DNA replication requirement is substrate dependent. Our data are consistent with recombination by both the Red and RecET systems occurring predominately by single-strand annealing rather than by strand invasion. Bacteriophage homologous recombination systems are widely used for in vivo genetic engineering in bacteria. Single- or double-stranded linear DNA substrates containing short flanking homologies to chromosome targets are used to generate precise and accurate genetic modifications when introduced into bacteria expressing phage recombinases. Understanding the molecular mechanism of these recombination systems will facilitate improvements in the technology. Here, two phage-specific systems are shown to require exposure of complementary single-strand homologous targets for efficient recombination; these single-strand regions may be created during DNA replication or by single-strand exonuclease digestion of linear duplex DNA. Previously, in vitro studies reported that these recombinases promote the single-strand annealing of two complementary DNAs and also strand invasion of a single DNA strand into duplex DNA to create a three-stranded region. Here, in vivo experiments show that recombinase-mediated annealing of complementary single-stranded DNA is the predominant recombination pathway in E. coli. Copyright © 2016 Thomason et al.

UV irradiation experiments under simulated martian surface conditions: Bio-effects on glycine, phage T7 and isolated T7 DNA

NASA Astrophysics Data System (ADS)

Bérces, Attila; ten Kate, I. L.; Fekete, A.; Hegedus, M.; Garry, J. R. C.; Lammer, Helmut; Ehrenfreund, Pascale; Peeters, Zan; Kovacs, G.; Ronto, G.

Mars is considered as a main target for astrobiologically relevant exploration programmes. In order to explain the non-detection of organic material to a detection level of several parts per billion (ppb) by the Viking landers, several hypotheses have been suggested, including degradation processes occurring on the martian surface and in the martian soil and subsurface. UV exposure experiments have been performed in which thin layers of glycine ( 300 nm), and aqueous suspensions of phage T7 and isolated T7 DNA were irradiated with a Deuterium lamp and for comparison with a Xenon arc lamp, modified to simulate the solar irradiation on the surface of Mars (MarsUV). The glycine sample was subjected to 24 hours of irradiation with MarsUV. The results of this glycine experiment show a destruction rate comparable to the results of previous experiments in which thin layers of glycine were irradiated with a deuterium lamp (ten Kate et al., 2005, 2006). After exposure of different doses of simulated Martian UV radiation a decrease of the biological activity of phages and characteristic changes in the UV absorption spectrum have been detected, indicating the UV damage of isolated and intraphage T7 DNA. The results of our experiments show that intraphage DNA is 4 times more sensitive to simulated martian UV and deuterium lamp radiation than isolated T7 DNA. This result indicates the significant role that phage proteins play in the UV damage. The effect of simulated martian radiation is smaller than the biological defects observed after the exposure with a deuterium lamp for both cases, in intraphage and isolated DNA, despite of the 100 times larger intensity of the MarsUV lamp. The detected spectral differences are about ten times smaller; the biological activity is about 3 - 4 times smaller, indicating that the shorter wavelength UV radiation from the deuterium lamp is more effective in inducing DNA damage, irrespective of being intraphage or isolated.

Double-stranded DNA organization in bacteriophage heads: an alternative toroid-based model.

PubMed Central

Hud, N V

1995-01-01

Studies of the organization of double-stranded DNA within bacteriophage heads during the past four decades have produced a wealth of data. However, despite the presentation of numerous models, the true organization of DNA within phage heads remains unresolved. The observations of toroidal DNA structures in electron micrographs of phage lysates have long been cited as support for the organization of DNA in a spool-like fashion. This particular model, like all other models, has not been found to be consistent will all available data. Recently we proposed that DNA within toroidal condensates produced in vitro is organized in a manner significantly different from that suggested by the spool model. This new toroid model has allowed the development of an alternative model for DNA organization within bacteriophage heads that is consistent with a wide range of biophysical data. Here we propose that bacteriophage DNA is packaged in a toroid that is folded into a highly compact structure. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 PMID:8534805

Identifying Bacterial Immune Evasion Proteins Using Phage Display.

PubMed

Fevre, Cindy; Scheepmaker, Lisette; Haas, Pieter-Jan

2017-01-01

Methods aimed at identification of immune evasion proteins are mainly rely on in silico prediction of sequence, structural homology to known evasion proteins or use a proteomics driven approach. Although proven successful these methods are limited by a low efficiency and or lack of functional identification. Here we describe a high-throughput genomic strategy to functionally identify bacterial immune evasion proteins using phage display technology. Genomic bacterial DNA is randomly fragmented and ligated into a phage display vector that is used to create a phage display library expressing bacterial secreted and membrane bound proteins. This library is used to select displayed bacterial secretome proteins that interact with host immune components.

Conformational changes leading to T7 DNA delivery upon interaction with the bacterial receptor.

PubMed

González-García, Verónica A; Pulido-Cid, Mar; Garcia-Doval, Carmela; Bocanegra, Rebeca; van Raaij, Mark J; Martín-Benito, Jaime; Cuervo, Ana; Carrascosa, José L

2015-04-17

The majority of bacteriophages protect their genetic material by packaging the nucleic acid in concentric layers to an almost crystalline concentration inside protein shells (capsid). This highly condensed genome also has to be efficiently injected into the host bacterium in a process named ejection. Most phages use a specialized complex (often a tail) to deliver the genome without disrupting cell integrity. Bacteriophage T7 belongs to the Podoviridae family and has a short, non-contractile tail formed by a tubular structure surrounded by fibers. Here we characterize the kinetics and structure of bacteriophage T7 DNA delivery process. We show that T7 recognizes lipopolysaccharides (LPS) from Escherichia coli rough strains through the fibers. Rough LPS acts as the main phage receptor and drives DNA ejection in vitro. The structural characterization of the phage tail after ejection using cryo-electron microscopy (cryo-EM) and single particle reconstruction methods revealed the major conformational changes needed for DNA delivery at low resolution. Interaction with the receptor causes fiber tilting and opening of the internal tail channel by untwisting the nozzle domain, allowing release of DNA and probably of the internal head proteins. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Free Shiga toxin 1-encoding bacteriophages are less prevalent than Shiga toxin 2 phages in extraintestinal environments.

PubMed

Grau-Leal, Ferran; Quirós, Pablo; Martínez-Castillo, Alexandre; Muniesa, Maite

2015-11-01

Stx bacteriophages are involved in the pathogenicity of Stx-producing Escherichia coli. Induction of the Stx phage lytic cycle increases Stx expression and releases Stx phages that reach extracellular environments. Stx phage family comprises different phages that harbour any stx subtype. Stx2 is closely related with severe disease and therefore previous studies focused on free Stx2 phages in extraintestinal environments. To provide similar information regarding Stx1 phages, we evaluate free Stx1 phages in 357 samples of human and animal wastewater, faeces, river water, soil, sludge and food. Our method, based on quantification of stx1 in the DNA from the viral fraction, was validated using electron microscopy counting of phages and infectivity. The overall prevalence of Stx1 phages was very low: 7.6% of positive samples and values below 3 × 10(3) GC (gene copies) ml(-1) . These results contrast starkly with the abundance of Stx2 phages in the samples (68.4%). This environmental scarcity of free Stx1 phages is attributed to their lower rates of induction and the fact that Stx1 does not require phage induction to be expressed because it possesses an independent promoter. The implications of the low prevalence of free Stx1 phages for the emergence of new pathogenic strains in the environment are discussed. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

The genome of the Lactobacillus sanfranciscensis temperate phage EV3

PubMed Central

2013-01-01

Background Bacteriophages infection modulates microbial consortia and transduction is one of the most important mechanism involved in the bacterial evolution. However, phage contamination brings food fermentations to a halt causing economic setbacks. The number of phage genome sequences of lactic acid bacteria especially of lactobacilli is still limited. We analysed the genome of a temperate phage active on Lactobacillus sanfranciscensis, the predominant strain in type I sourdough fermentations. Results Sequencing of the DNA of EV3 phage revealed a genome of 34,834 bp and a G + C content of 36.45%. Of the 43 open reading frames (ORFs) identified, all but eight shared homology with other phages of lactobacilli. A similar genomic organization and mosaic pattern of identities align EV3 with the closely related Lactobacillus vaginalis ATCC 49540 prophage. Four unknown ORFs that had no homologies in the databases or predicted functions were identified. Notably, EV3 encodes a putative dextranase. Conclusions EV3 is the first L. sanfranciscensis phage that has been completely sequenced so far. PMID:24308641

Studies with infections fragments of phage DNA. Final report, January 1, 1970--June 30, 1976

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schachtele, C. F.

The minute, virulent and structurally intricate Bacillus subtilis bacteriophage phi 29 was utilized to study in vivo viral development. Purified strands of phi 29 DNA were used to analyze transcription of the viral genome. Early mRNA hybridizes to the light DNA strand which controls DNA replication and other early functions. Late mRNA hybridizes to the heavy DNA strand which codes for phage structural proteins. The temporal sequence of specific viral protein synthesis was analyzed by gel electrophoresis and was shown to directly correlate with the RNA transcription pattern. The genes carried by phi 29 have been marked with ts andmore » sus mutations and mapped by appropriate crosses yielding a linear map of 17 cistrons. Fragments of the phi 29 DNA were shown to retain their biological activity and marker rescue studies indicated that gene transfer could be performed with pieces having a molecular weight of less than 1 million daltons. Mutant infection under nonpermissive conditions and the analysis of precursor structures has allowed the formation of a tentative morphogenetic pathway leading to the formation of infectious particles. Work with phi 29 has established this virus as an advantageous model system for studying a variety of problems in molecular biology and approximately a dozen laboratories in the country and abroad are working with this phage.« less

Specific growth rate and multiplicity of infection affect high-cell-density fermentation with bacteriophage M13 for ssDNA production.

PubMed

Kick, Benjamin; Hensler, Samantha; Praetorius, Florian; Dietz, Hendrik; Weuster-Botz, Dirk

2017-04-01

The bacteriophage M13 has found frequent applications in nanobiotechnology due to its chemically and genetically tunable protein surface and its ability to self-assemble into colloidal membranes. Additionally, its single-stranded (ss) genome is commonly used as scaffold for DNA origami. Despite the manifold uses of M13, upstream production methods for phage and scaffold ssDNA are underexamined with respect to future industrial usage. Here, the high-cell-density phage production with Escherichia coli as host organism was studied in respect of medium composition, infection time, multiplicity of infection, and specific growth rate. The specific growth rate and the multiplicity of infection were identified as the crucial state variables that influence phage amplification rate on one hand and the concentration of produced ssDNA on the other hand. Using a growth rate of 0.15 h -1 and a multiplicity of infection of 0.05 pfu cfu -1 in the fed-batch production process, the concentration of pure isolated M13 ssDNA usable for scaffolded DNA origami could be enhanced by 54% to 590 mg L -1 . Thus, our results help enabling M13 production for industrial uses in nanobiotechnology. Biotechnol. Bioeng. 2017;114: 777-784. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Role of DNA-DNA Interactions on the Structure and Thermodynamics of Bacteriophages Lambda and P4

PubMed Central

Petrov, Anton S.; Harvey, Stephen C.

2010-01-01

Electrostatic interactions play an important role in both packaging of DNA inside bacteriophages and its release into bacterial cells. While at physiological conditions DNA strands repel each other, the presence of polyvalent cations such as spermine and spermidine in solutions leads to the formation of DNA condensates. In this study, we discuss packaging of DNA into bacteriophages P4 and Lambda under repulsive and attractive conditions using a coarse-grained model of DNA and capsids. Packaging under repulsive conditions leads to the appearance of the coaxial spooling conformations; DNA occupies all available space inside the capsid. Under the attractive potential both packed systems reveal toroidal conformations, leaving the central part of the capsids empty. We also present a detailed thermodynamic analysis of packaging and show that the forces required to pack the genomes in the presence of polyamines are significantly lower than those observed under repulsive conditions. The analysis reveals that in both the repulsive and attractive regimes the entropic penalty of DNA confinement has a significant non-negligible contribution into the total energy of packaging. Additionally we report the results of simulations of DNA condensation inside partially packed Lambda. We found that at low densities DNA behaves as free unconfined polymer and condenses into the toroidal structures; at higher densities rearrangement of the genome into toroids becomes hindered, and condensation results in the formation of non-equilibrium structures. In all cases packaging in a specific conformation occurs as a result of interplay between bending stresses experienced by the confined polymer and interactions between the strands. PMID:21074621

Phylogenetic and Complementation Analysis of a Single-Stranded DNA Binding Protein Family from Lactococcal Phages Indicates a Non-Bacterial Origin

PubMed Central

Mariadassou, Mahendra; Bardowski, Jacek K.; Bidnenko, Elena

2011-01-01

Background The single-stranded-nucleic acid binding (SSB) protein superfamily includes proteins encoded by different organisms from Bacteria and their phages to Eukaryotes. SSB proteins share common structural characteristics and have been suggested to descend from an ancestor polypeptide. However, as other proteins involved in DNA replication, bacterial SSB proteins are clearly different from those found in Archaea and Eukaryotes. It was proposed that the corresponding genes in the phage genomes were transferred from the bacterial hosts. Recently new SSB proteins encoded by the virulent lactococcal bacteriophages (Orf14bIL67-like proteins) have been identified and characterized structurally and biochemically. Methodology/Principal Findings This study focused on the determination of phylogenetic relationships between Orf14bIL67-like proteins and other SSBs. We have performed a large scale phylogenetic analysis and pairwise sequence comparisons of SSB proteins from different phyla. The results show that, in remarkable contrast to other phage SSBs, the Orf14bIL67–like proteins form a distinct, self-contained and well supported phylogenetic group connected to the archaeal SSBs. Functional studies demonstrated that, despite the structural and amino acid sequence differences from bacterial SSBs, Orf14bIL67 protein complements the conditional lethal ssb-1 mutation of Escherichia coli. Conclusions/Significance Here we identified for the first time a group of phages encoded SSBs which are clearly distinct from their bacterial counterparts. All methods supported the recognition of these phage proteins as a new family within the SSB superfamily. Our findings suggest that unlike other phages, the virulent lactococcal phages carry ssb genes that were not acquired from their hosts, but transferred from an archaeal genome. This represents a unique example of a horizontal gene transfer between Archaea and bacterial phages. PMID:22073223

Naturally resident and exogenously applied T4-like and T5-like bacteriophages can reduce Escherichia coli O157:H7 levels in sheep guts

PubMed Central

Raya, Raul R; Oot, Rebecca A; Moore-Maley, Ben; Wieland, Serena; Callaway, Todd R; Kutter, Elizabeth M

2011-01-01

In preparing sheep for an in vivo Escherichia coli O157:H7 eradication trial, we found that 20/39 members of a single flock were naturally colonized by O157:H7-infecting phages. Characterization showed these were all one phage type (subsequently named CEV2) infecting 15/16 O157:H7, 7/72 ECOR and common lab strains. Further characterization by PFGE (genome∼120 kb), restriction enzyme digest (DNA appears unmodified), receptor studies (FhuA but not TonB is required for infection) and sequencing (>95% nucleotide identity) showed it is a close relative of the classically studied coliphage T5. Unlike T5, CEV2 infects O157:H7 in vitro, both aerobically and anaerobically, rapidly adsorbing and killing, but resistant mutants regrew within 24 h. When used together with T4-like CEV1 (MOI ∼2 per phage), bacterial killing was longer lasting. CEV2 did not reproduce when co-infecting the same cell as CEV1, presumably succumbing to CEV1's ability to shut off transcription of cytosine-containing DNA. In vivo sheep trials to remove resident O157:H7 showed that a cocktail of CEV2 and CEV1 (∼1011 total PFU) applied once orally was more effective (>99.9% reduction) than CEV1 alone (∼99%) compared to the untreated phage-free control. Those sheep naturally carrying CEV2, receiving no additional phage treatment, had the lowest O157:H7 levels (∼99.99% reduction). These data suggest that phage cocktails are more effective than individual phage in removing O157:H7 that have taken residence if the phage work in concert with one another and that naturally resident O157:H7-infecting phages may prevent O157:H7 gut colonization and be one explanation for the transient O157:H7 colonization in ruminants. PMID:21687531

Genomic characteristics of vB_PpaP_PP74, a T7-like Autographivirinae bacteriophage infecting a potato pathogen of the newly proposed species Pectobacterium parmentieri.

PubMed

Kabanova, Anastasia; Shneider, Mikhail; Bugaeva, Eugenia; Ha, Vo Thi Ngoc; Miroshnikov, Kirill; Korzhenkov, Aleksei; Kulikov, Eugene; Toschakov, Stepan; Ignatov, Alexander; Miroshnikov, Konstantin

2018-06-01

Bacteriophage vB_PpaP_PP74 (PP74) is a novel virulent phage that infects members of the species Pectobacterium parmentieri, a newly established species of soft-rot-causing bacteria in the family Pectobacteriaceae, derived from potato-specific Pectobacterium wasabiae. vB_PpaP_PP74 was identified as a member of the family Podoviridae by transmission electron microscopy. The phage has a 39,790-bp dsDNA genome containing 50 open reading frames (ORFs). Because of the absence of genes encoding toxins or lysogeny factors, PP74 may be considered a candidate phage for pathogen biocontrol applications. The genome layout is similar to genomes of T7-like phages within the subfamily Autographivirinae, and therefore, functions can be attributed to most of ORFs. However, the closest nucleotide sequence homologs of phage PP74 are unclassified Escherichia phages. Based on phylogenetic analysis, vB_PpaP_PP74 is a sensu lato T7-like phage, but it forms a distant subgenus group together with homologous enterobacterial phages.

Sequences of heavy and light chain variable regions from four bovine immunoglobulins.

PubMed

Armour, K L; Tempest, P R; Fawcett, P H; Fernie, M L; King, S I; White, P; Taylor, G; Harris, W J

1994-12-01

Oligodeoxyribonucleotide primers based on the 5' ends of bovine IgG1/2 and lambda constant (C) region genes, together with primers encoding conserved amino acids at the N-terminus of mature variable (V) regions from other species, have been used in cDNA and polymerase chain reactions (PCRs) to amplify heavy and light chain V region cDNA from bovine heterohybridomas. The amino acid sequences of VH and V lambda from four bovine immunoglobulins of different specificities are presented.

Molecular Mechanisms of Cytopathogenicity of Primate Lymphotropic Retroviruses: Relevance to Treatment and Vaccine for AIDS

DTIC Science & Technology

1989-03-10

fragment of the HIV-1 genome was isolated from XBH10 and inserted into an M13 phage vector. Mutations were introduced by use of 25-mer oligonucleotides which... M13 by Eco RI and religated into the corresponding position of pHXB2gpt. The mutant AS was prepared directly from pHXB2gpt by digestion with Nde I and...detect immunocomplexes. Molecular Cloning and Sequencing of Proviral DNA A X phage library was constructed from the genomic DNA isolated from Hut78 cells

Identification of Tumor Rejection Antigens for Breast Cancer Using a Mouse Tumor Rejection Model

DTIC Science & Technology

2009-05-01

plaques were randomly picked and PCR with T3 and T7 primer was done to validate the cDNA insert. The inserts ranged from 500 to 3,500 bp. A...modifications. Briefly, 5 103 phage clones were plated with XL-Blue on NZY agar plates. After 4 hours of incubation at 37jC, isopropyl-L-thio-h-D...using XLOLR cells and ExAssist helper phage (Stratagene). Plasmid DNA was prepared using a FastPlasmid kit (Eppendorf, Hamburg, Germany). The nucleotide

Total enantioselectivity in the DNA binding of the dinuclear ruthenium(II) complex [[Ru(Me2bpy)2]2(mu-bpm)]4+ [bpm = 2,2'-bipyrimidine; Me2bpy = 4,4'-dimethyl-2,2'-bipyridine].

PubMed

Smith, Jayden A; Collins, J Grant; Patterson, Bradley T; Keene, F Richard

2004-05-07

The binding of the three stereoisomers (DeltaDelta-, LambdaLambda- and DeltaLambda-) of the dinuclear ruthenium(II) complex [[Ru(Me2bpy)2]2(mu-bpm)]4+ [Me2bpy = 4,4'-dimethyl-2,2'-bipyridine; bpm = 2,2'-bipyrimidine] to a tridecanucleotide containing a single adenine bulge has been studied by 1H NMR spectroscopy. The addition of the DeltaDelta-isomer to d(CCGAGAATTCCGG)2 induced significant chemical shift changes for the base and sugar resonances of the residues at the bulge site (G3A4G5/C11C10), whereas small shifts were observed upon addition of the enantiomeric LambdaLambda-form. NOESY spectra of the tridecanucleotide bound with the DeltaDelta-isomer revealed intermolecular NOE's between the metal complex and the nucleotide residues at the bulge site, while only weak NOE's were observed to terminal residues to the LambdaLambda-form. Competitive binding studies were performed where both enantiomers were simultaneously added to the tridecanucleotide, and for all ratios of the two stereoisomers the DeltaDelta-isomer remained selectively bound at the bulge site with the LambdaLambda-enantiomer localised at the terminal regions of the tridecanucleotide. The meso-diastereoisomer (DeltaLambda) was found to bind to the tridecanucleotide with characteristics intermediate between the DeltaDelta- and LambdaLambda-enantiomers of the rac form. Two distinct sets of metal complex resonances were observed, with one set having essentially the same shift as the free metal complex, whilst the other set of resonances exhibited significant shifts. The NOE data indicated that the meso-diastereoisomer does not bind as selectively as the DeltaDelta-isomer, with NOE's observed to a greater number of nucleotide residues compared to the DeltaDelta-form. This study provides a rare example of total enantioselectivity in the binding of an inert transition metal complex to DNA, produced by the shape recognition of both ruthenium(II) centres.

Twelve previously unknown phage genera are ubiquitous in global oceans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holmfeldt, Karin; Solonenko, Natalie; Shah, Manesh B

Viruses are fundamental to ecosystems ranging from oceans to humans, yet our ability to study them is bottlenecked by the lack of ecologically relevant isolates, resulting in unknowns dominating culture-independent surveys. Here we present genomes from 31 phages infecting multiple strains of the aquatic bacterium Cellulophaga baltica (Bacteroidetes) to provide data for an underrepresented and environmentally abundant bacterial lineage. Comparative genomics delineated 12 phage groups that (i) each represent a new genus, and (ii) represent one novel and four wellknown viral families. This diversity contrasts the few well-studied marine phage systems, but parallels the diversity of phages infecting human-associated bacteria.more » Although all 12 Cellulophaga phages represent new genera, the podoviruses and icosahedral, nontailed ssDNA phages were exceptional, with genomes up to twice as large as those previously observed for each phage type. Structural novelty was also substantial, requiring experimental phage proteomics to identify 83% of the structural proteins. The presence of uncommon nucleotide metabolism genes in four genera likely underscores the importance of scavenging nutrient-rich molecules as previously seen for phages in marine environments. Metagenomic recruitment analyses suggest that these particular Cellulophaga phages are rare and may represent a first glimpse into the phage side of the rare biosphere. However, these analyses also revealed that these phage genera are widespread, occurring in 94% of 137 investigated metagenomes. Together, this diverse and novel collection of phages identifies a small but ubiquitous fraction of unknown marine viral diversity and provides numerous environmentally relevant phage host systems for experimental hypothesis testing.« less

Twelve previously unknown phage genera are ubiquitous in global oceans.

PubMed

Holmfeldt, Karin; Solonenko, Natalie; Shah, Manesh; Corrier, Kristen; Riemann, Lasse; Verberkmoes, Nathan C; Sullivan, Matthew B

2013-07-30

Viruses are fundamental to ecosystems ranging from oceans to humans, yet our ability to study them is bottlenecked by the lack of ecologically relevant isolates, resulting in "unknowns" dominating culture-independent surveys. Here we present genomes from 31 phages infecting multiple strains of the aquatic bacterium Cellulophaga baltica (Bacteroidetes) to provide data for an underrepresented and environmentally abundant bacterial lineage. Comparative genomics delineated 12 phage groups that (i) each represent a new genus, and (ii) represent one novel and four well-known viral families. This diversity contrasts the few well-studied marine phage systems, but parallels the diversity of phages infecting human-associated bacteria. Although all 12 Cellulophaga phages represent new genera, the podoviruses and icosahedral, nontailed ssDNA phages were exceptional, with genomes up to twice as large as those previously observed for each phage type. Structural novelty was also substantial, requiring experimental phage proteomics to identify 83% of the structural proteins. The presence of uncommon nucleotide metabolism genes in four genera likely underscores the importance of scavenging nutrient-rich molecules as previously seen for phages in marine environments. Metagenomic recruitment analyses suggest that these particular Cellulophaga phages are rare and may represent a first glimpse into the phage side of the rare biosphere. However, these analyses also revealed that these phage genera are widespread, occurring in 94% of 137 investigated metagenomes. Together, this diverse and novel collection of phages identifies a small but ubiquitous fraction of unknown marine viral diversity and provides numerous environmentally relevant phage-host systems for experimental hypothesis testing.

Generation and analysis of the improved human HAL9/10 antibody phage display libraries.

PubMed

Kügler, Jonas; Wilke, Sonja; Meier, Doris; Tomszak, Florian; Frenzel, André; Schirrmann, Thomas; Dübel, Stefan; Garritsen, Henk; Hock, Björn; Toleikis, Lars; Schütte, Mark; Hust, Michael

2015-02-19

Antibody phage display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries - in theory - antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody phage display library. The naive antibody gene libraries HAL9 and HAL10, with a combined theoretical diversity of 1.5×10(10) independent clones, were constructed from 98 healthy donors using improved phage display vectors. In detail, most common phagemids employed for antibody phage display are using a combined His/Myc tag for detection and purification. We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display. For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate. Deletion of a phenylalanine at the end of the CL linker sequence in our new phagemid design increased scFv production rate and frequency of selected kappa antibodies significantly. The HAL libraries and 834 antibodies selected against 121 targets were analyzed regarding the used germline V-genes, used V-gene combinations and CDR-H3/-L3 length and composition. The amino acid diversity and distribution in the CDR-H3 of the initial library was retrieved in the CDR-H3 of selected antibodies showing that all CDR-H3 amino acids occurring in the human antibody repertoire can be functionally used and is not biased by E. coli expression or phage selection. Further, the data underline the importance of CDR length variations. The highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv phagemid vector design. Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.

Examining the Interactome of Huperzine A by Magnetic Biopanning

PubMed Central

Guo, Wei; Liu, Shupeng; Peng, Jinliang; Wei, Xiaohui; Sun, Ye; Qiu, Yangsheng; Gao, Guangwei; Wang, Peng; Xu, Yuhong

2012-01-01

Huperzine A is a bioactive compound derived from traditional Chinese medicine plant Qian Ceng Ta (Huperzia serrata), and was found to have multiple neuroprotective effects. In addition to being a potent acetylcholinesterase inhibitor, it was thought to act through other mechanisms such as antioxidation, antiapoptosis, etc. However, the molecular targets involved with these mechanisms were not identified. In this study, we attempted to exam the interactome of Huperzine A using a cDNA phage display library and also mammalian brain tissue extracts. The drugs were chemically linked on the surface of magnetic particles and the interactive phages or proteins were collected and analyzed. Among the various cDNA expressing phages selected, one was identified to encode the mitochondria NADH dehydrogenase subunit 1. Specific bindings between the drug and the target phages and target proteins were confirmed. Another enriched phage clone was identified as mitochondria ATP synthase, which was also panned out from the proteome of mouse brain tissue lysate. These data indicated the possible involvement of mitochondrial respiratory chain matrix enzymes in Huperzine A's pharmacological effects. Such involvement had been suggested by previous studies based on enzyme activity changes. Our data supported the new mechanism. Overall we demonstrated the feasibility of using magnetic biopanning as a simple and viable method for investigating the complex molecular mechanisms of bioactive molecules. PMID:22615909

Drug delivery vectors based on filamentous bacteriophages and phage-mimetic nanoparticles.

PubMed

Ju, Zhigang; Sun, Wei

2017-11-01

With the development of nanomedicine, a mass of nanocarriers have been exploited and utilized for targeted drug delivery, including liposomes, polymers, nanoparticles, viruses, and stem cells. Due to huge surface bearing capacity and flexible genetic engineering property, filamentous bacteriophage and phage-mimetic nanoparticles are attracting more and more attentions. As a rod-like bio-nanofiber without tropism to mammalian cells, filamentous phage can be easily loaded with drugs and directly delivered to the lesion location. In particular, chemical drugs can be conjugated on phage surface by chemical modification, and gene drugs can also be inserted into the genome of phage by recombinant DNA technology. Meanwhile, specific peptides/proteins displayed on the phage surface are able to conjugate with nanoparticles which will endow them specific-targeting and huge drug-loading capacity. Additionally, phage peptides/proteins can directly self-assemble into phage-mimetic nanoparticles which may be applied for self-navigating drug delivery nanovehicles. In this review, we summarize the production of phage particles, the identification of targeting peptides, and the recent applications of filamentous bacteriophages as well as their protein/peptide for targeting drug delivery in vitro and in vivo. The improvement of our understanding of filamentous bacteriophage and phage-mimetic nanoparticles will supply new tools for biotechnological approaches.

Simulation of the M13 life cycle II: Investigation of the control mechanisms of M13 infection and establishment of the carrier state.

PubMed

Smeal, Steven W; Schmitt, Margaret A; Pereira, Ronnie Rodrigues; Prasad, Ashok; Fisk, John D

2017-01-01

Bacteriophage M13 is a true parasite of bacteria, able to co-opt the infected cell and control the production of progeny across many cellular generations. Here, our genetically-structured simulation of M13 is applied to quantitatively dissect the interplay between the host cellular environment and the controlling interactions governing the phage life cycle during the initial establishment of infection and across multiple cell generations. Multiple simulations suggest that phage-encoded feedback interactions constrain the utilization of host DNA polymerase, RNA polymerase and ribosomes. The simulation reveals the importance of p5 translational attenuation in controlling the production of phage double-stranded DNA and suggests an underappreciated role for p5 translational self-attenuation in resource allocation. The control elements active in a single generation are sufficient to reproduce the experimentally-observed multigenerational curing of the phage infection. Understanding the subtleties of regulation will be important for maximally exploiting M13 particles as scaffolds for nanoscale devices. Copyright © 2016 Elsevier Inc. All rights reserved.

Exploitation of rolling circle amplification for the construction of large phage-display antibody libraries.

PubMed

Shahsavarian, Melody A; Le Minoux, Damien; Matti, Kalyankumar M; Kaveri, Srini; Lacroix-Desmazes, Sébastien; Boquet, Didier; Friboulet, Alain; Avalle, Bérangère; Padiolleau-Lefèvre, Séverine

2014-05-01

Phage display antibody libraries have proven to have a significant role in the discovery of therapeutic antibodies and polypeptides with desired biological and physicochemical properties. Obtaining a large and diverse phage display antibody library, however, is always a challenging task. Various steps of this technique can still undergo optimization in order to obtain an efficient library. In the construction of a single chain fragment variable (scFv) phage display library, the cloning of the scFv fragments into a phagemid vector is of crucial importance. An efficient restriction enzyme digestion of the scFv DNA leads to its proper ligation with the phagemid followed by its successful cloning and expression. Here, we are reporting a different approach to enhance the efficiency of the restriction enzyme digestion step. We have exploited rolling circle amplification (RCA) to produce a long strand of DNA with tandem repeats of scFv sequences, which is found to be highly susceptible to restriction digestion. With this important modification, we are able to construct a large phage display antibody library of naive SJL/J mice. The size of the library is estimated as ~10(8) clones. The number of clones containing a scFv fragment is estimated at 90%. Hence, the present results could considerably aid the utilization of the phage-display technique in order to get an efficiently large antibody library. Copyright © 2014 Elsevier B.V. All rights reserved.

An efficient method for variable region assembly in the construction of scFv phage display libraries using independent strand amplification

PubMed Central

Sotelo, Pablo H.; Collazo, Noberto; Zuñiga, Roberto; Gutiérrez-González, Matías; Catalán, Diego; Ribeiro, Carolina Hager; Aguillón, Juan Carlos; Molina, María Carmen

2012-01-01

Phage display library technology is a common method to produce human antibodies. In this technique, the immunoglobulin variable regions are displayed in a bacteriophage in a way that each filamentous virus displays the product of a single antibody gene on its surface. From the collection of different phages, it is possible to isolate the virus that recognizes specific targets. The most common form in which to display antibody variable regions in the phage is the single chain variable fragment format (scFv), which requires assembly of the heavy and light immunoglobulin variable regions in a single gene. In this work, we describe a simple and efficient method for the assembly of immunoglobulin heavy and light chain variable regions in a scFv format. This procedure involves a two-step reaction: (1) DNA amplification to produce the single strand form of the heavy or light chain gene required for the fusion; and (2) mixture of both single strand products followed by an assembly reaction to construct a complete scFv gene. Using this method, we produced 6-fold more scFv encoding DNA than the commonly used splicing by overlap extension PCR (SOE-PCR) approach. The scFv gene produced by this method also proved to be efficient in generating a diverse scFv phage display library. From this scFv library, we obtained phages that bound several non-related antigens, including recombinant proteins and rotavirus particles. PMID:22692130

Control of Recombination Directionality by the Listeria Phage A118 Protein Gp44 and the Coiled-Coil Motif of Its Serine Integrase.

PubMed

Mandali, Sridhar; Gupta, Kushol; Dawson, Anthony R; Van Duyne, Gregory D; Johnson, Reid C

2017-06-01

The serine integrase of phage A118 catalyzes integrative recombination between attP on the phage and a specific attB locus on the chromosome of Listeria monocytogenes , but it is unable to promote excisive recombination between the hybrid attL and attR sites found on the integrated prophage without assistance by a recombination directionality factor (RDF). We have identified and characterized the phage-encoded RDF Gp44, which activates the A118 integrase for excision and inhibits integration. Gp44 binds to the C-terminal DNA binding domain of integrase, and we have localized the primary binding site to be within the mobile coiled-coil (CC) motif but distinct from the distal tip of the CC that is required for recombination. This interaction is sufficient to inhibit integration, but a second interaction involving the N-terminal end of Gp44 is also required to activate excision. We provide evidence that these two contacts modulate the trajectory of the CC motifs as they extend out from the integrase core in a manner dependent upon the identities of the four att sites. Our results support a model whereby Gp44 shapes the Int-bound complexes to control which att sites can synapse and recombine. IMPORTANCE Serine integrases mediate directional recombination between bacteriophage and bacterial chromosomes. These highly regulated site-specific recombination reactions are integral to the life cycle of temperate phage and, in the case of Listeria monocytogenes lysogenized by A118 family phage, are an essential virulence determinant. Serine integrases are also utilized as tools for genetic engineering and synthetic biology because of their exquisite unidirectional control of the DNA exchange reaction. Here, we identify and characterize the recombination directionality factor (RDF) that activates excision and inhibits integration reactions by the phage A118 integrase. We provide evidence that the A118 RDF binds to and modulates the trajectory of the long coiled-coil motif that extends from the large carboxyl-terminal DNA binding domain and is postulated to control the early steps of recombination site synapsis. Copyright © 2017 American Society for Microbiology.

Sequence analysis of malacoherpesvirus proteins: Pan-herpesvirus capsid module and replication enzymes with an ancient connection to "Megavirales".

PubMed

Mushegian, Arcady; Karin, Eli Levy; Pupko, Tal

2018-01-01

The order Herpesvirales includes animal viruses with large double-strand DNA genomes replicating in the nucleus. The main capsid protein in the best-studied family Herpesviridae contains a domain with HK97-like fold related to bacteriophage head proteins, and several virion maturation factors are also homologous between phages and herpesviruses. The origin of herpesvirus DNA replication proteins is less well understood. While analyzing the genomes of herpesviruses in the family Malacohepresviridae, we identified nearly 30 families of proteins conserved in other herpesviruses, including several phage-related domains in morphogenetic proteins. Herpesvirus DNA replication factors have complex evolutionary history: some are related to cellular proteins, but others are closer to homologs from large nucleocytoplasmic DNA viruses. Phylogenetic analyses suggest that the core replication machinery of herpesviruses may have been recruited from the same pool as in the case of other large DNA viruses of eukaryotes. Published by Elsevier Inc.

Inhibition of histone deacetylation and DNA methylation improves gene expression mediated by the adeno-associated virus/phage in cancer cells.

PubMed

Kia, Azadeh; Yata, Teerapong; Hajji, Nabil; Hajitou, Amin

2013-10-22

Bacteriophage (phage), viruses that infect bacteria only, have become promising vectors for targeted systemic delivery of genes to cancer, although, with poor efficiency. We previously designed an improved phage vector by incorporating cis genetic elements of adeno-associated virus (AAV). This novel AAV/phage hybrid (AAVP) specifically targeted systemic delivery of therapeutic genes into tumors. To advance the AAVP vector, we recently introduced the stress-inducible Grp78 tumor specific promoter and found that this dual tumor-targeted AAVP provides persistent gene expression, over time, in cancer cells compared to silenced gene expression from the CMV promoter in the parental AAVP. Herein, we investigated the effect of histone deacetylation and DNA methylation on AAVP-mediated gene expression in cancer cells and explored the effect of cell confluence state on AAVP gene expression efficacy. Using a combination of AAVP expressing the GFP reporter gene, flow cytometry, inhibitors of histone deacetylation, and DNA methylation, we have demonstrated that histone deacetylation and DNA methylation are associated with silencing of gene expression from the CMV promoter in the parental AAVP. Importantly, inhibitors of histone deacetylases boost gene expression in cancer cells from the Grp78 promoter in the dual tumor-targeted AAVP. However, cell confluence had no effect on AAVP-guided gene expression. Our findings prove that combination of histone deacetylase inhibitor drugs with the Grp78 promoter is an effective approach to improve AAVP-mediated gene expression in cancer cells and should be considered for AAVP-based clinical cancer gene therapy.

Specificity of the weak binding between the phage SPO1 transcription-inhibitory protein, TF1, and SPO1 DNA.

PubMed

Johnson, G G; Geiduschek, E P

1977-04-05

The interaction of the phage SPO1 protein transcription factor 1 (TF1), with DNA has been analyzed by membrane filter binding and by sedimentation methods. Substantially specific binding of TF1 to helical SPO1 DNA can be demonstrated by nitrocellulose filter-binding assays at relatively low ionic strength (0.08). However, TF1-DNA complexes dissociate and reequilibrate relatively rapidly and this makes filter-binding assays unsuitable for quantitative measurements of binding equilibra. Accordingly, the sedimentation properties of TF1-DNA complexes have been explored and a short-column centrifugation assay has been elaborated for quantitative measurements. Preferential binding of TF1 to the hydroxymethyluracil-containing SPO1 DNA has also been demonstrated by short-column centrifugation. TF1 binds relatively weakly and somewhat cooperatively to SPO1 DNA at many sites; TF1-DNA complexes dissociate and reequilibrate rapidly. At 20 degrees C in 0.01 M phosphate, pH 7.5, 0.15 KC1, one molecule of TF1 can bind to approximately every 60 nucleotide pairs of SPO1 DNA.

Effect of Osmotic Shock and Low Salt Concentration on Survival and Density of Bacteriophages T4B and T4Bo1

PubMed Central

Leibo, Stanley P.; Mazur, Peter

1966-01-01

Measurements of survival and buoyant densities of bacteriophages T4B, T4Bo1, and T4D have demonstrated the following: (a) After suspension in a concentrated salt solution, T4B and T4D are sensitive both to osmotic shock and to subsequent exposure to low monovalent salt concentrations. (b) Sensitivity of T4B to dilution from a concentrated salt solution is dependent on dilution rate, that of T4D is less dependent, and that of T4Bo1 is independent. (c) Sensitivity of all three phages to low salt concentrations depends on initial salt concentrations to a variable extent. (d) Density gradient profiles indicate that nearly half of osmotically shocked T4B retain their DNA. Similar analysis demonstrates that few, if any, T4Bo1 lose DNA when subjected to a treatment causing 90% loss of infectivity. (e) The effective buoyant densities of T4B and T4Bo1 depend significantly on the dilution treatments to which the phages are subjected prior to centrifugation in CsCl gradients. These data are explicable in terms of the different relative permeabilities of the phages to water and solutes, and of alterations in the counterion distribution surrounding the DNA within the phage heads. PMID:5972376

A Newly Isolated Bacteriophage, PBES 02, Infecting Cronobacter sakazakii.

PubMed

Lee, Hyung Ju; Kim, Wan Il; Kwon, Young Chan; Cha, Kyung Eun; Kim, Minjin; Myung, Heejoon

2016-09-28

A novel bacteriophage, PBES 02, infecting Cronobacter sakazakii was isolated and characterized. It has a spherical head of 90 nm in diameter and a tail of 130 nm in length, and belongs to Myoviridae as observed under a transmission electron microscope. The major virion protein appears to be 38 kilodaltons (kDa) in size. The latent period of PBES 02 is 30 min and the burst size is 250. Infectivity of the phage remained intact after exposure to temperatures ranging from 4°C to 55°C for 1 h. It was also stable after exposure to pHs ranging from 6 to 10 for 1 h. The phage effectively removed contaminating Cronobacter sakazakii from broth infant formula. PBES 02 has a double-stranded DNA genome of 149,732 bases. Its GC ratio is 50.7%. Sequence analysis revealed that PBES 02 has 299 open reading frames (ORFs) and 14 tRNA genes. Thirty-nine ORFs were annotated, including 24 related to replication and regulation functions, 10 related to structural proteins, and 5 related to DNA packaging. The genome of PBES 02 is closely related to that of two other C. sakazakii phages, CR3 and CR8. Comparison of DNA sequences of genes encoding the major capsid protein revealed a wide geographical distribution of related phages over Asia, Europe, and America.

Vibrio cholerae typing phage N4: genome sequence and its relatedness to T7 viral supergroup.

PubMed

Das, Mayukh; Nandy, R K; Bhowmick, Tushar Suvra; Yamasaki, S; Ghosh, A; Nair, G B; Sarkar, B L

2012-01-01

In countries where cholera is endemic, Vibrio cholerae O1 bacteriophages have been detected in sewage water. These have been used to serve not only as strain markers, but also for the typing of V. cholerae strains. Vibriophage N4 (ATCC 51352-B1) occupies a unique position in the new phage-typing scheme and can infect a larger number of V. cholerae O1 biotype El Tor strains. Here we characterized the complete genome sequence of this typing vibriophage. The complete DNA sequence of the N4 genome was determined by using a shotgun sequencing approach. Complete genome sequence explored that phage N4 is comprised of one circular, double-stranded chromosome of 38,497 bp with an overall GC content of 42.8%. A total of 47 open reading frames were identified and functions could be assigned to 30 of them. Further, a close relationship with another vibriophage, VP4, and the enterobacteriophage T7 could be established. DNA-DNA hybridization among V. cholerae O1 and O139 phages revealed homology among O1 vibriophages at their genomic level. This study indicates two evolutionary distinctive branches of the possible phylogenetic origin of O1 and O139 vibriophages and provides an unveiled collection of information on viral gene products of typing vibriophages. Copyright © 2011 S. Karger AG, Basel.

Genome-wide characterization of vibrio phage ϕpp2 with unique arrangements of the mob-like genes

PubMed Central

2012-01-01

Background Vibrio parahaemolyticus is associated with gastroenteritis, wound infections, and septicemia in human and animals. Phages can control the population of the pathogen. So far, the only one reported genome among giant vibriophages is KVP40: 244,835 bp with 26% coding regions that have T4 homologs. Putative homing endonucleases (HE) were found in Vibrio phage KVP40 bearing one segD and Vibrio cholerae phage ICP1 carrying one mobC/E and one segG. Results A newly isolated Vibrio phage ϕpp2, which was specific to the hosts of V. parahaemolyticus and V. alginolyticus, featured a long nonenveloped head of ~90 × 150 nm and tail of ~110 nm. The phage can survive at 50°C for more than one hour. The genome of the phage ϕpp2 was sequenced to be 246,421 bp, which is 1587 bp larger than KVP40. 383 protein-encoding genes (PEGs) and 30 tRNAs were found in the phage ϕpp2. Between the genomes of ϕpp2 and KVP40, 254 genes including 29 PEGs for viral structure were of high similarity, whereas 17 PEGs of KVP40 and 21 PEGs of ϕpp2 were unmatched. In both genomes, the capsid and tail genes have been identified, as well as the extensive representation of the DNA replication, recombination, and repair enzymes. In addition to the three giant indels of 1098, 1143 and 3330 nt, ϕpp2 possessed unique proteins involved in potassium channel, gp2 (DNA end protector), tRNA nucleotidyltransferase, and mob-type HEs, which were not reported in KVP40. The ϕpp2 PEG274, with strong promoters and translational initiation, was identified to be a mobE type, flanked by NrdA and NrdB/C homologs. Coincidently, several pairs of HE-flanking homologs with empty center were found in the phages of Vibrio phages ϕpp2 and KVP40, as well as in Aeromonas phages (Aeh1 and Ae65), and cyanophage P-SSM2. Conclusions Vibrio phage ϕpp2 was characterized by morphology, growth, and genomics with three giant indels and different types of HEs. The gene analysis on the required elements for transcription and translation suggested that the ϕpp2 PEG274 was an active mobE gene. The phage was signified to be a new species of T4-related, differing from KVP40. PMID:22676552

A minimal kinetic model for a viral DNA packaging machine.

PubMed

Yang, Qin; Catalano, Carlos Enrique

2004-01-20

Terminase enzymes are common to both eukaryotic and prokaryotic double-stranded DNA viruses. These enzymes possess ATPase and nuclease activities that work in concert to "package" a viral genome into an empty procapsid, and it is likely that terminase enzymes from disparate viruses utilize a common packaging mechanism. Bacteriophage lambda terminase possesses a site-specific nuclease activity, a so-called helicase activity, a DNA translocase activity, and multiple ATPase catalytic sites that function to package viral DNA. Allosteric interactions between the multiple catalytic sites have been reported. This study probes these catalytic interactions using enzyme kinetic, photoaffinity labeling, and vanadate inhibition studies. The ensemble of data forms the basis for a minimal kinetic model for lambda terminase. The model incorporates an ADP-driven conformational reorganization of the terminase subunits assembled on viral DNA, which is central to the activation of a catalytically competent packaging machine. The proposed model provides a unifying mechanism for allosteric interaction between the multiple catalytic sites of the holoenzyme and explains much of the kinetic data in the literature. Given that similar packaging mechanisms have been proposed for viruses as dissimilar as lambda and the herpes viruses, the model may find general utility in our global understanding of the enzymology of virus assembly.

Complete genomic sequence of the Lactobacillus temperate phage LF1.

PubMed

Yoon, Bo Hyun; Chang, Hyo Ihl

2011-10-01

Bacteriophage LF1, a newly isolated temperate phage from a mitomycin-C-induced lysate of wild type Lactobacillus fermentum, was found to contain a double-strand DNA of 42,606 base pairs (bp) with a G+C content of 45%. Bioinformatic analysis of the phage genome revealed 57 putative open reading frames (ORFs). The predicted protein products of ORFs were determined and described. According to morphological analysis by transmission electron microscopy (TEM), LF1 has an isometric head and a non-contractile tail, indicating that it belongs to the family Siphoviridae. The temperate phage LF1 has a good genetic mosaic relationship with ΦPYB5 in the packaging module. To our knowledge, this is first report of genomic sequencing and characterization of temperate phage LF1 from wild-type L. fermentum isolated from Kimchi in Korea.

Bacteriophages of Gordonia spp. Display a Spectrum of Diversity and Genetic Relationships.

PubMed

Pope, Welkin H; Mavrich, Travis N; Garlena, Rebecca A; Guerrero-Bustamante, Carlos A; Jacobs-Sera, Deborah; Montgomery, Matthew T; Russell, Daniel A; Warner, Marcie H; Hatfull, Graham F

2017-08-15

The global bacteriophage population is large, dynamic, old, and highly diverse genetically. Many phages are tailed and contain double-stranded DNA, but these remain poorly characterized genomically. A collection of over 1,000 phages infecting Mycobacterium smegmatis reveals the diversity of phages of a common bacterial host, but their relationships to phages of phylogenetically proximal hosts are not known. Comparative sequence analysis of 79 phages isolated on Gordonia shows these also to be diverse and that the phages can be grouped into 14 clusters of related genomes, with an additional 14 phages that are "singletons" with no closely related genomes. One group of six phages is closely related to Cluster A mycobacteriophages, but the other Gordonia phages are distant relatives and share only 10% of their genes with the mycobacteriophages. The Gordonia phage genomes vary in genome length (17.1 to 103.4 kb), percentage of GC content (47 to 68.8%), and genome architecture and contain a variety of features not seen in other phage genomes. Like the mycobacteriophages, the highly mosaic Gordonia phages demonstrate a spectrum of genetic relationships. We show this is a general property of bacteriophages and suggest that any barriers to genetic exchange are soft and readily violable. IMPORTANCE Despite the numerical dominance of bacteriophages in the biosphere, there is a dearth of complete genomic sequences. Current genomic information reveals that phages are highly diverse genomically and have mosaic architectures formed by extensive horizontal genetic exchange. Comparative analysis of 79 phages of Gordonia shows them to not only be highly diverse, but to present a spectrum of relatedness. Most are distantly related to phages of the phylogenetically proximal host Mycobacterium smegmatis , although one group of Gordonia phages is more closely related to mycobacteriophages than to the other Gordonia phages. Phage genome sequence space remains largely unexplored, but further isolation and genomic comparison of phages targeted at related groups of hosts promise to reveal pathways of bacteriophage evolution. Copyright © 2017 Pope et al.

Selective amplification of an mRNA and related pseudogene for a human ADP-ribosylation factor, a guanine nucleotide-dependent protein activator of cholera toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Monaco, L.; Murtagh, J.J.; Newman, K.B.

1990-03-01

ADP-ribosylation factors (ARFs) are {approx}20-kDa proteins that act as GTP-dependent allosteric activators of cholera toxin. With deoxyinosine-containing degenerate oligonucleotide primers corresponding to conserved GTP-binding domains in ARFs, the polymerase chain reaction (PCR) was used to amplify simultaneously from human DNA portions of three ARF genes that include codons for 102 amino acids, with intervening sequences. Amplification products that differed in size because of differences in intron sizes were separated by agarose gel electrophoresis. One amplified DNA contained no introns and had a sequence different from those of known AFRs. Based on this sequence, selective oligonucleotide probes were prepared and usedmore » to isolate clone {Psi}ARF 4, a putative ARF pseudogene, from a human genomic library in {lambda} phage EMBL3. Reverse transcription-PCR was then used to clone from human poly(A){sup +} RNA the cDNA corresponding to the expressed homolog of {Psi}ARF 4, referred to as human ARF 4. It appears that {Psi}ARF 4 arose during human evolution by integration of processed ARF 4 mRNA into the genome. Human ARF 4 differs from previously identified mammalian ARFs 1, 2, and 3. Hybridization of ARF 4-specific oligonucleotide probes with human, bovine, and rat RNA revealed a single 1.8-kilobase mRNA, which was clearly distinguished from the 1.9-kilobase mRNA for ARF 1 in these tissues. The PCR provides a powerful tool for investigating diversity in this and other multigene families, especially with primers targeted at domains believed to have functional significance.« less

A novel helper phage enabling construction of genome-scale ORF-enriched phage display libraries.

PubMed

Gupta, Amita; Shrivastava, Nimisha; Grover, Payal; Singh, Ajay; Mathur, Kapil; Verma, Vaishali; Kaur, Charanpreet; Chaudhary, Vijay K

2013-01-01

Phagemid-based expression of cloned genes fused to the gIIIP coding sequence and rescue using helper phages, such as VCSM13, has been used extensively for constructing large antibody phage display libraries. However, for randomly primed cDNA and gene fragment libraries, this system encounters reading frame problems wherein only one of 18 phages display the translated foreign peptide/protein fused to phagemid-encoded gIIIP. The elimination of phages carrying out-of-frame inserts is vital in order to improve the quality of phage display libraries. In this study, we designed a novel helper phage, AGM13, which carries trypsin-sensitive sites within the linker regions of gIIIP. This renders the phage highly sensitive to trypsin digestion, which abolishes its infectivity. For open reading frame (ORF) selection, the phagemid-borne phages are rescued using AGM13, so that clones with in-frame inserts express fusion proteins with phagemid-encoded trypsin-resistant gIIIP, which becomes incorporated into the phages along with a few copies of AGM13-encoded trypsin-sensitive gIIIP. In contrast, clones with out-of-frame inserts produce phages carrying only AGM13-encoded trypsin-sensitive gIIIP. Trypsin treatment of the phage population renders the phages with out-of-frame inserts non-infectious, whereas phages carrying in-frame inserts remain fully infectious and can hence be enriched by infection. This strategy was applied efficiently at a genome scale to generate an ORF-enriched whole genome fragment library from Mycobacterium tuberculosis, in which nearly 100% of the clones carried in-frame inserts after selection. The ORF-enriched libraries were successfully used for identification of linear and conformational epitopes for monoclonal antibodies specific to mycobacterial proteins.

Structure, mechanics, and binding mode heterogeneity of LEDGF/p75-DNA nucleoprotein complexes revealed by scanning force microscopy

NASA Astrophysics Data System (ADS)

Vanderlinden, Willem; Lipfert, Jan; Demeulemeester, Jonas; Debyser, Zeger; de Feyter, Steven

2014-04-01

LEDGF/p75 is a transcriptional coactivator implicated in the pathogenesis of AIDS and leukemia. In these contexts, LEDGF/p75 acts as a cofactor by tethering protein cargo to transcriptionally active regions in the human genome. Our study - based on scanning force microscopy (SFM) imaging - is the first to provide structural information on the interaction of LEDGF/p75 with DNA. Two novel approaches that allow obtaining insights into the DNA conformation inside nucleoprotein complexes revealed (1) that LEDGF/p75 can bind at least in three different binding modes, (2) how DNA topology and protein dimerization affect these binding modes, and (3) geometrical and mechanical aspects of the nucleoprotein complexes. These structural and mechanical details will help us to better understand the cellular mechanisms of LEDGF/p75 as a transcriptional coactivator and as a cofactor in disease.LEDGF/p75 is a transcriptional coactivator implicated in the pathogenesis of AIDS and leukemia. In these contexts, LEDGF/p75 acts as a cofactor by tethering protein cargo to transcriptionally active regions in the human genome. Our study - based on scanning force microscopy (SFM) imaging - is the first to provide structural information on the interaction of LEDGF/p75 with DNA. Two novel approaches that allow obtaining insights into the DNA conformation inside nucleoprotein complexes revealed (1) that LEDGF/p75 can bind at least in three different binding modes, (2) how DNA topology and protein dimerization affect these binding modes, and (3) geometrical and mechanical aspects of the nucleoprotein complexes. These structural and mechanical details will help us to better understand the cellular mechanisms of LEDGF/p75 as a transcriptional coactivator and as a cofactor in disease. Electronic supplementary information (ESI) available: SFM topographs of phage lambda DNA in situ, in the absence and presence of LEDGF/p75; model-independent tests for DNA chain equilibration in 2D; SFM topographs of plasmid DNA substrates I-IV in the absence of LEDGF/p75; proof-of-principle of bend angle determination on supercoiled plasmid DNA-EcoRV binding to cognate and non-cognate sites in pBR322 plasmid DNA. See DOI: 10.1039/c4nr00022f

An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor

PubMed Central

Zhao, Wei; Jardine, Paul J.

2015-01-01

ABSTRACT During assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the packaging motor in the Bacillus subtilis bacteriophage ϕ29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activity in vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the packaging process. DNA packaging using restriction digests of ϕ29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA packaging, selectively packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, packaging both left- and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although ϕ29 differs from other double-stranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages. IMPORTANCE During virus assembly, genome packaging involves the delivery of newly synthesized viral nucleic acid into a protein shell. In the double-stranded DNA phages and herpesviruses, this is accomplished by a powerful molecular motor that translocates the viral DNA into a preformed viral shell. A key event in DNA packaging is recognition of the viral DNA among other nucleic acids in the host cell. Commonly, a DNA-binding protein mediates the interaction of viral DNA with the motor/head shell. Here we show that for the bacteriophage ϕ29, this essential step of genome recognition is mediated by a viral genome-encoded RNA rather than a protein. A domain of the prohead RNA (pRNA) imparts specificity and stringency to the motor by ensuring the correct orientation of DNA packaging and restricting initiation to a single event. Since this assembly step is unique to the virus, DNA packaging is a novel target for the development of antiviral drugs. PMID:26423956

An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor.

PubMed

Zhao, Wei; Jardine, Paul J; Grimes, Shelley

2015-12-01

During assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the packaging motor in the Bacillus subtilis bacteriophage ϕ29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activity in vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the packaging process. DNA packaging using restriction digests of ϕ29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA packaging, selectively packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, packaging both left- and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although ϕ29 differs from other double-stranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages. During virus assembly, genome packaging involves the delivery of newly synthesized viral nucleic acid into a protein shell. In the double-stranded DNA phages and herpesviruses, this is accomplished by a powerful molecular motor that translocates the viral DNA into a preformed viral shell. A key event in DNA packaging is recognition of the viral DNA among other nucleic acids in the host cell. Commonly, a DNA-binding protein mediates the interaction of viral DNA with the motor/head shell. Here we show that for the bacteriophage ϕ29, this essential step of genome recognition is mediated by a viral genome-encoded RNA rather than a protein. A domain of the prohead RNA (pRNA) imparts specificity and stringency to the motor by ensuring the correct orientation of DNA packaging and restricting initiation to a single event. Since this assembly step is unique to the virus, DNA packaging is a novel target for the development of antiviral drugs. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The in-feed antibiotic carbadox induces phage gene transcription in the swine gut microbiome

USDA-ARS?s Scientific Manuscript database

Carbadox is a quinoxaline-di-N-oxide antibiotic fed to over 40 percent of young pigs in the U.S. and has been shown to induce phage DNA transduction in vitro; however, the effects of carbadox on swine microbiome functions are poorly understood. We investigated the in vivo effects of carbadox on swin...

Narrow-Host-Range Bacteriophages That Infect Rhizobium etli Associate with Distinct Genomic Types

PubMed Central

Santamaría, Rosa Isela; Bustos, Patricia; Sepúlveda-Robles, Omar; Lozano, Luis; Rodríguez, César; Fernández, José Luis; Juárez, Soledad; Kameyama, Luis; Guarneros, Gabriel; Dávila, Guillermo

2014-01-01

In this work, we isolated and characterized 14 bacteriophages that infect Rhizobium etli. They were obtained from rhizosphere soil of bean plants from agricultural lands in Mexico using an enrichment method. The host range of these phages was narrow but variable within a collection of 48 R. etli strains. We obtained the complete genome sequence of nine phages. Four phages were resistant to several restriction enzymes and in vivo cloning, probably due to nucleotide modifications. The genome size of the sequenced phages varied from 43 kb to 115 kb, with a median size of ∼45 to 50 kb. A large proportion of open reading frames of these phage genomes (65 to 70%) consisted of hypothetical and orphan genes. The remainder encoded proteins needed for phage morphogenesis and DNA synthesis and processing, among other functions, and a minor percentage represented genes of bacterial origin. We classified these phages into four genomic types on the basis of their genomic similarity, gene content, and host range. Since there are no reports of similar sequences, we propose that these bacteriophages correspond to novel species. PMID:24185856

Novel bacteriophages containing a genome of another bacteriophage within their genomes.

PubMed

Swanson, Maud M; Reavy, Brian; Makarova, Kira S; Cock, Peter J; Hopkins, David W; Torrance, Lesley; Koonin, Eugene V; Taliansky, Michael

2012-01-01

A novel bacteriophage infecting Staphylococus pasteuri was isolated during a screen for phages in Antarctic soils. The phage named SpaA1 is morphologically similar to phages of the family Siphoviridae. The 42,784 bp genome of SpaA1 is a linear, double-stranded DNA molecule with 3' protruding cohesive ends. The SpaA1 genome encompasses 63 predicted protein-coding genes which cluster within three regions of the genome, each of apparently different origin, in a mosaic pattern. In two of these regions, the gene sets resemble those in prophages of Bacillus thuringiensis kurstaki str. T03a001 (genes involved in DNA replication/transcription, cell entry and exit) and B. cereus AH676 (additional regulatory and recombination genes), respectively. The third region represents an almost complete genome (except for the short terminal segments) of a distinct bacteriophage, MZTP02. Nearly the same gene module was identified in prophages of B. thuringiensis serovar monterrey BGSC 4AJ1 and B. cereus Rock4-2. These findings suggest that MZTP02 can be shuttled between genomes of other bacteriophages and prophages, leading to the formation of chimeric genomes. The presence of a complete phage genome in the genome of other phages apparently has not been described previously and might represent a 'fast track' route of virus evolution and horizontal gene transfer. Another phage (BceA1) nearly identical in sequence to SpaA1, and also including the almost complete MZTP02 genome within its own genome, was isolated from a bacterium of the B. cereus/B. thuringiensis group. Remarkably, both SpaA1 and BceA1 phages can infect B. cereus and B. thuringiensis, but only one of them, SpaA1, can infect S. pasteuri. This finding is best compatible with a scenario in which MZTP02 was originally contained in BceA1 infecting Bacillus spp, the common hosts for these two phages, followed by emergence of SpaA1 infecting S. pasteuri.

Influence of RNase E deficiency on the production of stx2-bearing phages and Shiga toxin in an RNase E-inducible strain of enterohaemorrhagic Escherichia coli (EHEC) O157:H7.

PubMed

Thuraisamy, Thujitha; Lodato, Patricia B

2018-05-01

In enterohaemorrhagic Escherichia coli (EHEC), stx1 or stx2 genes encode Shiga toxin (Stx1 or Stx2, respectively) and are carried by prophages. The production and release of both stx phages and toxin occur upon initiation of the phage lytic cycle. Phages can further disseminate stx genes by infecting naïve bacteria in the intestine. Here, the effect of RNase E deficiency on these two virulence traits was investigated. Cultures of the EHEC strains TEA028-rne containing low versus normal RNase E levels or the parental strain (TEA028) were treated with mitomycin C (MMC) to induce the phage lytic cycle. Phages and Stx2 titres were quantified by the double-agar assay and the receptor ELISA technique, respectively. RNase E deficiency in MMC-treated cells significantly reduced the yield of infectious stx2 phages. Delayed cell lysis and the appearance of encapsidated phage DNA copies suggest a slow onset of the lytic cycle. However, these observations do not entirely explain the decrease of phage yields. stx1 phages were not detected under normal or deficient RNase E levels. After an initial delay, high levels of toxin were finally produced in MMC-treated cultures. RNase E scarcity reduces stx2 phage production but not toxin. Normal concentrations of RNase E are likely required for correct phage morphogenesis. Our future work will address the mechanism of RNase E action on phage morphogenesis.

Suppressor Analysis of the Fusogenic Lambda Spanins.

PubMed

Cahill, Jesse; Rajaure, Manoj; Holt, Ashley; Moreland, Russell; O'Leary, Chandler; Kulkarni, Aneesha; Sloan, Jordan; Young, Ry

2017-07-15

The final step of lysis in phage λ infections of Escherichia coli is mediated by the spanins Rz and Rz1. These proteins form a complex that bridges the cell envelope and that has been proposed to cause fusion of the inner and outer membranes. Accordingly, mutations that block spanin function are found within coiled-coil domains and the proline-rich region, motifs essential in other fusion systems. To gain insight into spanin function, pseudorevertant alleles that restored plaque formation for lysis-defective mutants of Rz and Rz1 were selected. Most second-site suppressors clustered within a coiled-coil domain of Rz near the outer leaflet of the cytoplasmic membrane and were not allele specific. Suppressors largely encoded polar insertions within the hydrophobic core of the coiled-coil interface. Such suppressor changes resulted in decreased proteolytic stability of the Rz double mutants in vivo Unlike the wild type, in which lysis occurs while the cells retain a rod shape, revertant alleles with second-site suppressor mutations supported lysis events that were preceded by spherical cell formation. This suggests that destabilization of the membrane-proximal coiled coil restores function for defective spanin alleles by increasing the conformational freedom of the complex at the cost of its normal, all-or-nothing functionality. IMPORTANCE Caudovirales encode cell envelope-spanning proteins called spanins, which are thought to fuse the inner and outer membranes during phage lysis. Recent genetic analysis identified the functional domains of the lambda spanins, which are similar to class I viral fusion proteins. While the pre- and postfusion structures of model fusion systems have been well characterized, the intermediate structure(s) formed during the fusion reaction remains elusive. Genetic analysis would be expected to identify functional connections between intermediates. Since most membrane fusion systems are not genetically tractable, only few such investigations have been reported. Here, we report a suppressor analysis of lambda spanin function. To our knowledge this is the first suppression analysis of a class I-like complex and also the first such analysis of a prokaryote membrane fusion system. Copyright © 2017 American Society for Microbiology.

Phage M13 for the treatment of Alzheimer and Parkinson disease.

PubMed

Messing, Joachim

2016-06-01

The studies of microbes have been instrumental in combatting infectious diseases, but they have also led to great insights into basic biological mechanism like DNA replication, transcription, and translation of mRNA. In particular, the studies of bacterial viruses, also called bacteriophage, have been quite useful to study specific cellular processes because of the ease to isolate their DNA, mRNA, and proteins. Here, I review the recent discovery of how properties of the filamentous phage M13 emerge as a novel approach to combat neurodegenerative diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Bioinformatic analysis of phage AB3, a phiKMV-like virus infecting Acinetobacter baumannii.

PubMed

Zhang, J; Liu, X; Li, X-J

2015-01-16

The phages of Acinetobacter baumannii has drawn increasing attention because of the multi-drug resistance of A. baumanni. The aim of this study was to sequence Acinetobacter baumannii phage AB3 and conduct bioinformatic analysis to lay a foundation for genome remodeling and phage therapy. We isolated and sequenced A. baumannii phage AB3 and attempted to annotate and analyze its genome. The results showed that the genome is a double-stranded DNA with a total length of 31,185 base pairs (bp) and 97 open reading frames greater than 100 bp. The genome includes 28 predicted genes, of which 24 are homologous to phage AB1. The entire coding sequence is located on the negative strand, representing 90.8% of the total length. The G+C mol% was 39.18%, without areas of high G+C content over 200 bp in length. No GC island, tRNA gene, or repeated sequence was identified. Gene lengths were 120-3099 bp, with an average of 1011 bp. Six genes were found to be greater than 2000 bp in length. Genomic alignment and phylogenetic analysis of the RNA polymerase gene showed that similar to phage AB1, phage AB3 is a phiKMV-like virus in the T7 phage family.

Trinucleotide cassettes increase diversity of T7 phage-displayed peptide library.

PubMed

Krumpe, Lauren R H; Schumacher, Kathryn M; McMahon, James B; Makowski, Lee; Mori, Toshiyuki

2007-10-05

Amino acid sequence diversity is introduced into a phage-displayed peptide library by randomizing library oligonucleotide DNA. We recently evaluated the diversity of peptide libraries displayed on T7 lytic phage and M13 filamentous phage and showed that T7 phage can display a more diverse amino acid sequence repertoire due to differing processes of viral morphogenesis. In this study, we evaluated and compared the diversity of a 12-mer T7 phage-displayed peptide library randomized using codon-corrected trinucleotide cassettes with a T7 and an M13 12-mer phage-displayed peptide library constructed using the degenerate codon randomization method. We herein demonstrate that the combination of trinucleotide cassette amino acid codon randomization and T7 phage display construction methods resulted in a significant enhancement to the functional diversity of a 12-mer peptide library. This novel library exhibited superior amino acid uniformity and order-of-magnitude increases in amino acid sequence diversity as compared to degenerate codon randomized peptide libraries. Comparative analyses of the biophysical characteristics of the 12-mer peptide libraries revealed the trinucleotide cassette-randomized library to be a unique resource. The combination of T7 phage display and trinucleotide cassette randomization resulted in a novel resource for the potential isolation of binding peptides for new and previously studied molecular targets.

BVPaP-3, a T7-like lytic phage of Pseudomonas aeruginosa: its isolation and characterisation.

PubMed

Ahiwale, Sangeeta; Prakash, Divya; Gajbhiye, Milind; Jagdale, Smita; Patil, Nita; Kapadnis, Balu

2012-04-01

The increasing emergence of antibiotic-resistant bacteria has produced a growing interest among scientists in bacteriophages as alternative antimicrobial agents. This article reports a lytic phage against an antibiotic-resistant strain of Pseudomonas aeruginosa. Phage BVPaP-3 is a member of the Podoviridae family and morphologically similar to the T7-like phage gh-1. The phage has a hexagonal head of 58-59 nm in diameter and a short tail of 10 × 8 nm. It is stable at a wide range of pH (6-10) and temperatures (4-40°C). Its optimal growth temperature is 37°C and the adsorption rate constant is 1.19 × 10(-9). Latent and eclipse periods are 20 and 15 min, respectively, and the burst size is 44 after 35 min at 37°C. The phage has a DNA size of 41.31 kb and a proteome of 11 proteins. The major protein is 33 kDa in size.

An Evolutionary/Biochemical Connection Between Promoter- and Primer-Dependent Polymerases Revealed by Selective Evolution of Ligands by Exponential Enrichment (SELEX).

PubMed

Fenstermacher, Katherine J; Achuthan, Vasudevan; Schneider, Thomas D; DeStefano, Jeffrey J

2018-01-16

DNA polymerases (DNAPs) recognize 3' recessed termini on duplex DNA and carry out nucleotide catalysis. Unlike promoter-specific RNA polymerases (RNAPs), no sequence specificity is required for binding or initiation of catalysis. Despite this, previous results indicate that viral reverse transcriptases bind much more tightly to DNA primers that mimic the polypurine tract. In the current report, primer sequences that bind with high affinity to Taq and Klenow polymerases were identified using a modified Selective Evolution of Ligands by Exponential Enrichment (SELEX) approach. Two Taq -specific primers that bound ∼10 (Taq1) and over 100 (Taq2) times more stably than controls to Taq were identified. Taq1 contained 8 nucleotides (5' -CACTAAAG-3') that matched the phage T3 RNAP "core" promoter. Both primers dramatically outcompeted primers with similar binding thermodynamics in PCR reactions. Similarly, exonuclease minus Klenow polymerase also selected a high affinity primer that contained a related core promoter sequence from phage T7 RNAP (5' -ACTATAG-3'). For both Taq and Klenow, even small modifications to the sequence resulted in large losses in binding affinity suggesting that binding was highly sequence-specific. The results are discussed in the context of possible effects on multi-primer (multiplex) PCR assays, molecular information theory, and the evolution of RNAPs and DNAPs. Importance This work further demonstrates that primer-dependent DNA polymerases can have strong sequence biases leading to dramatically tighter binding to specific sequences. These may be related to biological function, or be a consequences of the structural architecture of the enzyme. New sequence specificity for Taq and Klenow polymerases were uncovered and among them were sequences that contained the core promoter elements from T3 and T7 phage RNA polymerase promoters. This suggests the intriguing possibility that phage RNA polymerases exploited intrinsic binding affinities of ancestral DNA polymerases to develop their promotors. Conversely, DNA polymerases could have evolved from related RNA polymerases and retained the intrinsic binding preference despite there being no clear function for such a preference in DNA biology. Copyright © 2018 American Society for Microbiology.

Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries.

PubMed

Huang, Renhua; Fang, Pete; Kay, Brian K

2012-09-01

Site-directed mutagenesis is routinely performed in protein engineering experiments. One method, termed Kunkel mutagenesis, is frequently used for constructing libraries of peptide or protein variants in M13 bacteriophage, followed by affinity selection of phage particles. To make this method more efficient, the following two modifications were introduced: culture was incubated at 25°C for phage replication, which yielded two- to sevenfold more single-stranded DNA template compared to growth at 37°C, and restriction endonuclease recognition sites were used to remove non-recombinants. With both of the improvements, we could construct primary libraries of high complexity and that were 99-100% recombinant. Finally, with a third modification to the standard protocol of Kunkel mutagenesis, two secondary (mutagenic) libraries of a fibronectin type III (FN3) monobody were constructed with DNA segments that were amplified by error-prone and asymmetric PCR. Two advantages of this modification are that it bypasses the lengthy steps of restriction enzyme digestion and ligation, and that the pool of phage clones, recovered after affinity selection, can be used directly to generate a secondary library. Screening one of the two mutagenic libraries yielded variants that bound two- to fourfold tighter to human Pak1 kinase than the starting clone. The protocols described in this study should accelerate the discovery of phage-displayed recombinant affinity reagents. Copyright © 2012 Elsevier Inc. All rights reserved.

Evaluation of Phage Display Discovered Peptides as Ligands for Prostate-Specific Membrane Antigen (PSMA)

PubMed Central

Edwards, W. Barry

2013-01-01

The aim of this study was to identify potential ligands of PSMA suitable for further development as novel PSMA-targeted peptides using phage display technology. The human PSMA protein was immobilized as a target followed by incubation with a 15-mer phage display random peptide library. After one round of prescreening and two rounds of screening, high-stringency screening at the third round of panning was performed to identify the highest affinity binders. Phages which had a specific binding activity to PSMA in human prostate cancer cells were isolated and the DNA corresponding to the 15-mers were sequenced to provide three consensus sequences: GDHSPFT, SHFSVGS and EVPRLSLLAVFL as well as other sequences that did not display consensus. Two of the peptide sequences deduced from DNA sequencing of binding phages, SHSFSVGSGDHSPFT and GRFLTGGTGRLLRIS were labeled with 5-carboxyfluorescein and shown to bind and co-internalize with PSMA on human prostate cancer cells by fluorescence microscopy. The high stringency requirements yielded peptides with affinities KD∼1 µM or greater which are suitable starting points for affinity maturation. While these values were less than anticipated, the high stringency did yield peptide sequences that apparently bound to different surfaces on PSMA. These peptide sequences could be the basis for further development of peptides for prostate cancer tumor imaging and therapy. PMID:23935860

Mapping the Tail Fiber as the Receptor Binding Protein Responsible for Differential Host Specificity of Pseudomonas aeruginosa Bacteriophages PaP1 and JG004

PubMed Central

Le, Shuai; He, Xuesong; Tan, Yinling; Huang, Guangtao; Zhang, Lin; Lux, Renate; Shi, Wenyuan; Hu, Fuquan

2013-01-01

The first step in bacteriophage infection is recognition and binding to the host receptor, which is mediated by the phage receptor binding protein (RBP). Different RBPs can lead to differential host specificity. In many bacteriophages, such as Escherichia coli and Lactococcal phages, RBPs have been identified as the tail fiber or protruding baseplate proteins. However, the tail fiber-dependent host specificity in Pseudomonas aeruginosa phages has not been well studied. This study aimed to identify and investigate the binding specificity of the RBP of P. aeruginosa phages PaP1 and JG004. These two phages share high DNA sequence homology but exhibit different host specificities. A spontaneous mutant phage was isolated and exhibited broader host range compared with the parental phage JG004. Sequencing of its putative tail fiber and baseplate region indicated a single point mutation in ORF84 (a putative tail fiber gene), which resulted in the replacement of a positively charged lysine (K) by an uncharged asparagine (N). We further demonstrated that the replacement of the tail fiber gene (ORF69) of PaP1 with the corresponding gene from phage JG004 resulted in a recombinant phage that displayed altered host specificity. Our study revealed the tail fiber-dependent host specificity in P. aeruginosa phages and provided an effective tool for its alteration. These contributions may have potential value in phage therapy. PMID:23874674

Phage-inducible chromosomal islands are ubiquitous within the bacterial universe.

PubMed

Fillol-Salom, Alfred; Martínez-Rubio, Roser; Abdulrahman, Rezheen F; Chen, John; Davies, Robert; Penadés, José R

2018-06-06

Phage-inducible chromosomal islands (PICIs) are a recently discovered family of pathogenicity islands that contribute substantively to horizontal gene transfer, host adaptation and virulence in Gram-positive cocci. Here we report that similar elements also occur widely in Gram-negative bacteria. As with the PICIs from Gram-positive cocci, their uniqueness is defined by a constellation of features: unique and specific attachment sites, exclusive PICI genes, a phage-dependent mechanism of induction, conserved replication origin organization, convergent mechanisms of phage interference, and specific packaging of PICI DNA into phage-like infectious particles, resulting in very high transfer frequencies. We suggest that the PICIs represent two or more distinct lineages, have spread widely throughout the bacterial world, and have diverged much more slowly than their host organisms or their prophage cousins. Overall, these findings represent the discovery of a universal class of mobile genetic elements.

Isolation and Characterization of a Shewanella Phage–Host System from the Gut of the Tunicate, Ciona intestinalis

PubMed Central

Leigh, Brittany; Karrer, Charlotte; Cannon, John P.; Breitbart, Mya; Dishaw, Larry J.

2017-01-01

Outnumbering all other biological entities on earth, bacteriophages (phages) play critical roles in structuring microbial communities through bacterial infection and subsequent lysis, as well as through horizontal gene transfer. While numerous studies have examined the effects of phages on free-living bacterial cells, much less is known regarding the role of phage infection in host-associated biofilms, which help to stabilize adherent microbial communities. Here we report the cultivation and characterization of a novel strain of Shewanella fidelis from the gut of the marine tunicate Ciona intestinalis, inducible prophages from the S. fidelis genome, and a strain-specific lytic phage recovered from surrounding seawater. In vitro biofilm assays demonstrated that lytic phage infection affects biofilm formation in a process likely influenced by the accumulation and integration of the extracellular DNA released during cell lysis, similar to the mechanism that has been previously shown for prophage induction. PMID:28327522

Genomic and molecular analysis of phage CMP1 from Clavibacter michiganensis subspecies michiganensis

PubMed Central

Wittmann, Johannes; Gartemann, Karl-Heinz; Eichenlaub, Rudolf

2011-01-01

Bacteriophage CMP1 is a member of the Siphoviridae family that infects specifically the plant-pathogen Clavibacter michiganensis subsp. michiganensis. The linear double- stranded DNA is terminally redundant and not circularly permuted. The complete nucleotide sequence of the bacteriophage CMP1 genome consists of 58,652 bp including the terminal redundant ends of 791 bp. The G+C content of the phage (57%) is significantly lower than that of its host (72.66%). 74 potential open reading frames were identified and annotated by different bioinformatic tools. Two large clusters which encode the early and the late functions could be identified which are divergently transcribed. There are only a few hypothetical gene products with conserved domains and significant similarity to sequences from the databases. Functional analyses confirmed the activity of four gene products, an endonuclease, an exonuclease, a single-stranded DNA binding protein and a thymidylate synthase. Partial genomic sequences of CN77, a phage of Clavibacter michiganensis subsp. nebraskensis, revealed a similar genome structure and significant similarities on the level of deduced amino acid sequences. An endolysin with peptidase activity has been identified for both phages, which may be good tools for disease control of tomato plants against Clavibacter infections. PMID:21687530

Genomic and molecular analysis of phage CMP1 from Clavibacter michiganensis subspecies michiganensis.

PubMed

Wittmann, Johannes; Gartemann, Karl-Heinz; Eichenlaub, Rudolf; Dreiseikelmann, Brigitte

2011-01-01

Bacteriophage CMP1 is a member of the Siphoviridae family that infects specifically the plant-pathogen Clavibacter michiganensis subsp. michiganensis. The linear double- stranded DNA is terminally redundant and not circularly permuted. The complete nucleotide sequence of the bacteriophage CMP1 genome consists of 58,652 bp including the terminal redundant ends of 791 bp. The G+C content of the phage (57%) is significantly lower than that of its host (72.66%). 74 potential open reading frames were identified and annotated by different bioinformatic tools. Two large clusters which encode the early and the late functions could be identified which are divergently transcribed. There are only a few hypothetical gene products with conserved domains and significant similarity to sequences from the databases. Functional analyses confirmed the activity of four gene products, an endonuclease, an exonuclease, a single-stranded DNA binding protein and a thymidylate synthase. Partial genomic sequences of CN77, a phage of Clavibacter michiganensis subsp. nebraskensis, revealed a similar genome structure and significant similarities on the level of deduced amino acid sequences. An endolysin with peptidase activity has been identified for both phages, which may be good tools for disease control of tomato plants against Clavibacter infections.

Structure of a headful DNA-packaging bacterial virus at 2.9 Å resolution by electron cryo-microscopy

PubMed Central

Zhao, Haiyan; Li, Kunpeng; Lynn, Anna Y.; Aron, Keith E.; Yu, Guimei; Jiang, Wen; Tang, Liang

2017-01-01

The enormous prevalence of tailed DNA bacteriophages on this planet is enabled by highly efficient self-assembly of hundreds of protein subunits into highly stable capsids. These capsids can stand with an internal pressure as high as ∼50 atmospheres as a result of the phage DNA-packaging process. Here we report the complete atomic model of the headful DNA-packaging bacteriophage Sf6 at 2.9 Å resolution determined by electron cryo-microscopy. The structure reveals the DNA-inflated, tensed state of a robust protein shell assembled via noncovalent interactions. Remarkable global conformational polymorphism of capsid proteins, a network formed by extended N arms, mortise-and-tenon–like intercapsomer joints, and abundant β-sheet–like mainchain:mainchain intermolecular interactions, confers significant strength yet also flexibility required for capsid assembly and DNA packaging. Differential formations of the hexon and penton are mediated by a drastic α–helix-to-β–strand structural transition. The assembly scheme revealed here may be common among tailed DNA phages and herpesviruses. PMID:28320961

Simulation experiments of the effect of space environment on bacteriophage and DNA thin films

NASA Astrophysics Data System (ADS)

Fekete, A.; Rontó, G.; Hegedûs, M.; Módos, K.; Bérces, A.; Kovács, G.; Lammer, H.

PUR experiment (phage and uracil response) is part of the ROSE consortium selected for the first mission on the ISS and its main goal to examine and quantify the effect of specific space parameters such as VUV, UV radiation, dehydration effects, non-oxidative environments etc. related to space vacuum conditions on nucleic acid models. An improved method for the preparation of DNA thin films (NaDNA and LiDNA) was elaborated and the homogeneity of the films were controlled by spectroscopy and phase contrast microscopy. The complete recovery of the amount of DNA from the thin film was found after dissolution. Electrophoresis of the dissolved DNA indicated an intact DNA structure, while successful PCR amplification an intact function of the molecule, so they are likely candidates for the flight on the EXPOSE facility. A new method for preparation of bacteriophage T7 thin layer has been developed, the quality was controlled by spectroscopy and microscopy. After dissolution almost 90% of the viability of the phage particles remained, and the intactness of DNA structure was checked by PCR. DNA and phage thin films were produced in sandwich form as well, and stored in an atmosphere containing a mixture of N2 and H2 , by quality control of the samples no change has been found. They were tested under simulated space conditions at IWF space simulation facility in Graz. DNA thin films and bacteriophage T7 thin layers at different r.h. values have been irradiated in sandwich form in normal atmospheric conditions by using a low pressure Mercury lamp and high power (300W) Deuterium lamp containing short wavelength ( < 240 nm) UVC components simulating theextraterrestrial solar radiation. Characteristic change in the absorption spectrum and the decrease of the amount of PCR products have been detected indicating the photodamage of isolated and intraphage DNA.

Selective inhibition by harmane of the apurinic apyrimidinic endonuclease activity of phage T4-induced UV endonuclease.

PubMed

Warner, H R; Persson, M L; Bensen, R J; Mosbaugh, D W; Linn, S

1981-11-25

1-Methyl-9H-pyrido-[3,4-b]indole (harmane) inhibits the apurinic/apyrimidinic (AP) endonuclease activity of the UV endonuclease induced by phage T4, whereas it stimulates the pyrimidine dimer-DNA glycosylase activity of that enzyme. E. coli endonuclease IV, E. coli endonuclease VI (the AP endonuclease activity associated with E. coli exonuclease III), and E. coli uracil-DNA glycosylase were not inhibited by harmane. Human fibroblast AP endonucleases I and II also were only slightly inhibited. Therefore, harmane is neither a general inhibitor of AP endonucleases, nor a general inhibitor of Class I AP endonucleases which incise DNA on the 3'-side of AP sites. However, E. coli endonuclease III and its associated dihydroxythymine-DNA glycosylase activity were both inhibited by harmane. This observation suggests that harmane may inhibit only AP endonucleases which have associated glycosylase activities.

Selective inhibition by harmane of the apurinic apyrimidinic endonuclease activity of phage T4-induced UV endonuclease.

PubMed Central

Warner, H R; Persson, M L; Bensen, R J; Mosbaugh, D W; Linn, S

1981-01-01

1-Methyl-9H-pyrido-[3,4-b]indole (harmane) inhibits the apurinic/apyrimidinic (AP) endonuclease activity of the UV endonuclease induced by phage T4, whereas it stimulates the pyrimidine dimer-DNA glycosylase activity of that enzyme. E. coli endonuclease IV, E. coli endonuclease VI (the AP endonuclease activity associated with E. coli exonuclease III), and E. coli uracil-DNA glycosylase were not inhibited by harmane. Human fibroblast AP endonucleases I and II also were only slightly inhibited. Therefore, harmane is neither a general inhibitor of AP endonucleases, nor a general inhibitor of Class I AP endonucleases which incise DNA on the 3'-side of AP sites. However, E. coli endonuclease III and its associated dihydroxythymine-DNA glycosylase activity were both inhibited by harmane. This observation suggests that harmane may inhibit only AP endonucleases which have associated glycosylase activities. PMID:6273822

Magic-angle spinning NMR of intact bacteriophages: Insights into the capsid, DNA and their interface

NASA Astrophysics Data System (ADS)

Abramov, Gili; Morag, Omry; Goldbourt, Amir

2015-04-01

Bacteriophages are viruses that infect bacteria. They are complex macromolecular assemblies, which are composed of multiple protein subunits that protect genomic material and deliver it to specific hosts. Various biophysical techniques have been used to characterize their structure in order to unravel phage morphogenesis. Yet, most bacteriophages are non-crystalline and have very high molecular weights, in the order of tens of MegaDaltons. Therefore, complete atomic-resolution characterization on such systems that encompass both capsid and DNA is scarce. In this perspective article we demonstrate how magic-angle spinning solid-state NMR has and is used to characterize in detail bacteriophage viruses, including filamentous and icosahedral phage. We discuss the process of sample preparation, spectral assignment of both capsid and DNA and the use of chemical shifts and dipolar couplings to probe the capsid-DNA interface, describe capsid structure and dynamics and extract structural differences between viruses.

Structure, Assembly, and DNA Packaging of the Bacteriophage T4 Head

PubMed Central

Black, Lindsay W.; Rao, Venigalla B.

2014-01-01

The bacteriophage T4 head is an elongated icosahedron packed with 172 kb of linear double-stranded DNA and numerous proteins. The capsid is built from three essential proteins: gp23*, which forms the hexagonal capsid lattice; gp24*, which forms pentamers at 11 of the 12 vertices; and gp20, which forms the unique dodecameric portal vertex through which DNA enters during packaging and exits during infection. Intensive work over more than half a century has led to a deep understanding of the phage T4 head. The atomic structure of gp24 has been determined. A structural model built for gp23 using its similarity to gp24 showed that the phage T4 major capsid protein has the same fold as numerous other icosahedral bacteriophages. However, phage T4 displays an unusual membrane and portal initiated assembly of a shape determining self-sufficient scaffolding core. Folding of gp23 requires the assistance of two chaperones, the Escherichia coli chaperone GroEL acting with the phage-coded gp23-specific cochaperone, gp31. The capsid also contains two nonessential outer capsid proteins, Hoc and Soc, which decorate the capsid surface. Through binding to adjacent gp23 subunits, Soc reinforces the capsid structure. Hoc and Soc have been used extensively in bipartite peptide display libraries and to display pathogen antigens, including those from human immunodeficiency virus (HIV), Neisseria meningitides, Bacillus anthracis, and foot and mouth disease virus. The structure of Ip1*, one of a number of multiple (>100) copy proteins packed and injected with DNA from the full head, shows it to be an inhibitor of one specific restriction endonuclease specifically targeting glycosylated hydroxymethyl cytosine DNA. Extensive mutagenesis, combined with atomic structures of the DNA packaging/terminase proteins gp16 and gp17, elucidated the ATPase and nuclease functional motifs involved in DNA translocation and headful DNA cutting. The cryoelectron microscopy structure of the T4 packaging machine showed a pentameric motor assembled with gp17 subunits on the portal vertex. Single molecule optical tweezers and fluorescence studies showed that the T4 motor packages DNA at the highest rate known and can package multiple segments. Förster resonance energy transfer–fluorescence correlation spectroscopy studies indicate that DNA gets compressed in the stalled motor and that the terminase-to-portal distance changes during translocation. Current evidence suggests a linear two-component (large terminase plus portal) translocation motor in which electrostatic forces generated by ATP hydrolysis drive DNA translocation by alternating the motor between tensed and relaxed states. PMID:22420853

Characterization of group H streptococcal temperate bacteriophage phi 227.

PubMed

Nugent, K M; Cole, R M

1977-03-01

phi 227, a temperate phage from a group H streptococcus (Streptococcus sanguis), was propagated vegetatively in group H strain Wicky 4-EryR, and its characteristics were determined. A procedure dependent on multiplicity of infection, incubation time, and treatment of crude lysates with diatomaceous earth was found to optimize phage yield, resulting in titers of 1 X 10(10) to 2 X 10(10) PFU/ml. Without prior treatment with diatomaceous earth, subsequent purification procedures (methanol, ammonium sulfate, polyethylene glycol) gave recoveries of less than 1% of crude lysate titers. Adsorption of phi227 to host cells was relatively unaffected by the medium, but calcium (not substituted by magnesium) was required for formation of infectious centers. The phage receptor was present on purified cell walls, resisted trypsin and heat, and was removed ty hydrochloric acid, trichloracetic acid, and hot formamide: however, formamide-extracted material failed to inactivate phage, and the nature of the receptor is unknown. Single-step growth experiments showed a latent period of 39 min and a burst size of 100 PFU/infectious center; results were unaffected by omission of supplemental Ca2+, by supplementation with Mg2, addition of glucose, or changes of pH between 6.35 and 8.0; but increased temperature (40 to 43 degrees C) shortened the latent period and decreased the burst size. The latent period was prolonged in genetically competent host cells and in chemically defined medium; and in the latter, the burst size was smaller. Phage replication was sensitive to those metabolic inhibitors which inhibited the host streptococcus: these included rifampin, fluorodeoxyuridine, hydroxyurea, dihydrostreptomycin, and 6-P-hydroxyphenylazouracil. The data suggest that phi227 does not code for a rifampin-resistant RNA polymerase. However, in a rifampin-resistant host strain, phage replication and lysogen formation were both decreased suggesting that altered host core polymerase had less affinity for (some) promotors on the phi227 template. In transfection, a Ca2+-dependent stabilization step that was inhibited by Mg2+ was demonstrated; transformation was not affected by either Ca2+ or Mg2+, and the site and nature of the stabilization are unknown. More than one molecule of DNA was required for plaque formation. Biophysical characterization showed a type B phage of buoyant density (CsCl) 1.50, containing five proteins and 54.8% DNA. The duplex linear DNA had a molecular weight (calculated from contour length) of 23.2 X 10(6) and a guanine plus cytosine content (calculated from melting point) of 42.3 mol%. Similar characterizations of streptococcal phages, including biophysical data, have not been previously available.

RNA-dependent RNA targeting by CRISPR-Cas9

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strutt, Steven C.; Torrez, Rachel M.; Kaya, Emine

Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo.more » We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. In conclusion, these results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications.« less

RNA-dependent RNA targeting by CRISPR-Cas9

DOE PAGES

Strutt, Steven C.; Torrez, Rachel M.; Kaya, Emine; ...

2018-01-05

Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo.more » We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. In conclusion, these results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications.« less

RNA-dependent RNA targeting by CRISPR-Cas9

PubMed Central

Strutt, Steven C; Torrez, Rachel M; Kaya, Emine; Negrete, Oscar A

2018-01-01

Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo. We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. These results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications. PMID:29303478

Preparation of metagenomic libraries from naturally occurring marine viruses.

PubMed

Solonenko, Sergei A; Sullivan, Matthew B

2013-01-01

Microbes are now well recognized as major drivers of the biogeochemical cycling that fuels the Earth, and their viruses (phages) are known to be abundant and important in microbial mortality, horizontal gene transfer, and modulating microbial metabolic output. Investigation of environmental phages has been frustrated by an inability to culture the vast majority of naturally occurring diversity coupled with the lack of robust, quantitative, culture-independent methods for studying this uncultured majority. However, for double-stranded DNA phages, a quantitative viral metagenomic sample-to-sequence workflow now exists. Here, we review these advances with special emphasis on the technical details of preparing DNA sequencing libraries for metagenomic sequencing from environmentally relevant low-input DNA samples. Library preparation steps broadly involve manipulating the sample DNA by fragmentation, end repair and adaptor ligation, size fractionation, and amplification. One critical area of future research and development is parallel advances for alternate nucleic acid types such as single-stranded DNA and RNA viruses that are also abundant in nature. Combinations of recent advances in fragmentation (e.g., acoustic shearing and tagmentation), ligation reactions (adaptor-to-template ratio reference table availability), size fractionation (non-gel-sizing), and amplification (linear amplification for deep sequencing and linker amplification protocols) enhance our ability to generate quantitatively representative metagenomic datasets from low-input DNA samples. Such datasets are already providing new insights into the role of viruses in marine systems and will continue to do so as new environments are explored and synergies and paradigms emerge from large-scale comparative analyses. © 2013 Elsevier Inc. All rights reserved.

In vitro selection of zinc fingers with altered DNA-binding specificity.

PubMed

Jamieson, A C; Kim, S H; Wells, J A

1994-05-17

We have used random mutagenesis and phage display to alter the DNA-binding specificity of Zif268, a transcription factor that contains three zinc finger domains. Four residues in the helix of finger 1 of Zif268 that potentially mediate DNA binding were identified from an X-ray structure of the Zif268-DNA complex. A library was constructed in which these residues were randomly mutated and the Zif268 variants were fused to a truncated version of the gene III coat protein on the surface of M13 filamentous phage particles. The phage displayed the mutant proteins in a monovalent fashion and were sorted by repeated binding and elution from affinity matrices containing different DNA sequences. When the matrix contained the natural nine base pair operator sequence 5'-GCG-TGG-GCG-3', native-like zinc fingers were isolated. New finger 1 variants were found by sorting with two different operators in which the singly modified triplets, GTG and TCG, replaced the native finger 1 triplet, GCG. Overall, the selected finger 1 variants contained a preponderance of polar residues at the four sites. Interestingly, the net charge of the four residues in any selected finger never derived more that one unit from neutrality despite the fact that about half the variants contained three or four charged residues over the four sites. Measurements of the dissociation constants for two of these purified finger 1 variants by gel-shift assay showed their specificities to vary over a 10-fold range, with the greatest affinity being for the DNA binding site for which they were sorted.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage recombination systems and biotechnical applications.

PubMed

Nafissi, Nafiseh; Slavcev, Roderick

2014-04-01

Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses' life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, λ, and N15, the integrase from the Streptomyces phage, ΦC31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed.

A target-unrelated peptide in an M13 phage display library traced to an advantageous mutation in the gene II ribosome-binding site.

PubMed

Brammer, Leighanne A; Bolduc, Benjamin; Kass, Jessica L; Felice, Kristin M; Noren, Christopher J; Hall, Marilena Fitzsimons

2008-02-01

Screening of the commercially available Ph.D.-7 phage-displayed heptapeptide library for peptides that bind immobilized Zn2+ resulted in the repeated selection of the peptide HAIYPRH, although binding assays indicated that HAIYPRH is not a zinc-binding peptide. HAIYPRH has also been selected in several other laboratories using completely different targets, and its ubiquity suggests that it is a target-unrelated peptide. We demonstrated that phage displaying HAIYPRH are enriched after serial amplification of the library without exposure to target. The amplification of phage displaying HAIYPRH was found to be dramatically faster than that of the library itself. DNA sequencing uncovered a mutation in the Shine-Dalgarno (SD) sequence for gIIp, a protein involved in phage replication, imparting to the SD sequence better complementarity to the 16S ribosomal RNA (rRNA). Introducing this mutation into phage lacking a displayed peptide resulted in accelerated propagation, whereas phage displaying HAIYPRH with a wild-type SD sequence were found to amplify normally. The SD mutation may alter gIIp expression and, consequently, the rate of propagation of phage. In the Ph.D.-7 library, the mutation is coincident with the displayed peptide HAIYPRH, accounting for the target-unrelated selection of this peptide in multiple reported panning experiments.

A general insert label for peptide display on chimeric filamentous bacteriophages.

PubMed

Kaplan, Gilad; Gershoni, Jonathan M

2012-01-01

The foreign insert intended to be displayed via recombinant phage proteins can have a negative effect on protein expression and phage assembly. A typical example is the case of display of peptides longer than 6 amino acid residues on the major coat protein, protein VIII of the filamentous bacteriophages M13 and fd. A solution to this problem has been the use of "two-gene systems" generating chimeric phages that concomitantly express wild-type protein VIII along with recombinant protein VIII. Although the two-gene systems are much more permissive in regard to insert length and composition, some cases can still adversely affect phage assembly. Although these phages genotypically contain the desired DNA of the insert, they appear to be phenotypically wild type. To avoid false-negative results when using chimeric phages in binding studies, it is necessary to confirm that the observed lack of phage recognition is not due to faulty assembly and display of the intended insert. Here we describe a strategy for generating antibodies that specifically recognize recombinant protein VIII regardless of the nature of its foreign insert. These antibodies can be used as a general monitor of the display of recombinant protein VIII into phage particles. Copyright © 2011 Elsevier Inc. All rights reserved.

Evidence for the presence of restriction/modification systems in Lactobacillus delbrueckii.

PubMed

Suárez, Viviana; Zago, Miriam; Giraffa, Giorgio; Reinheimer, Jorge; Quiberoni, Andrea

2009-11-01

The bacteriophages Cb1/204 and Cb1/342 were obtained by induction from the commercial strain Lactobacillus delbrueckii subsp. lactis Cb1, and propagated on Lactobacillus delbrueckii subsp. lactis 204 (Lb.l 204) and Lactobacillus delbrueckii subsp. bulgaricus 342 (Lb.b 342), respectively. By cross sensitivity, it was possible to detect a delay in the lysis of Lb.l 204 with Cb1/342 phage, while the adsorption rate was high (99.5%). Modified and unmodified phages were isolated using phage Cb1/342 and strain Lb.l 204. The EOP (Efficiency of Plaquing) values for the four phages (Cb1/204, Cb1/342, Cb1/342modified and Cb1/342unmodified) suggested that an R/M system modified the original temperate phage, and the BglII-DNA restriction patterns of these phages might point out the presence of a Type II R/M system. Also, the existence of a Type I R/M system was demonstrated by PCR and nucleotide sequence, being the percentages of alignment homology with Type I R/M systems reported previously higher than 95%. In this study it was possible to demonstrate that the native phage resistant mechanisms and the occurrence of prophages in commercial host strains, contribute strongly to diversify the phage population in a factory environment.

New sub-family of lysozyme-like proteins shows no catalytic activity: crystallographic and biochemical study of STM3605 protein from Salmonella Typhimurium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Michalska, Karolina; Brown, Roslyn N.; Li, Hui

Phage viruses that infect prokaryotes integrate their genome into the host chromosome; thus, microbial genomes typically contain genetic remnants of both recent and ancient phage infections. Often phage genes occur in clusters of atypical G+C content that reflect integration of the foreign DNA. However, some phage genes occur in isolation without other phage gene neighbors, probably resulting from horizontal gene transfer. In these cases, the phage gene product is unlikely to function as a component of a mature phage particle, and instead may have been co-opted by the host for its own benefit. The product of one such gene frommore » Salmonella enterica serovar Typhimurium, STM3605, encodes a protein with modest sequence similarity to phage-like lysozyme (N-acetylmuramidase) but appears to lack essential catalytic residues that are strictly conserved in all lysozymes. Close homologs in other bacteria share this characteristic. The structure of the STM3605 protein was characterized by X-ray crystallography, and functional assays showed that it is a stable, folded protein whose structure closely resembles lysozyme. However, this protein is unlikely to hydrolyze peptidoglycan. Instead, STM3605 is presumed to have evolved an alternative function because it shows some lytic activity and partitions to micelles.« less

A dual host vector for Fab phage display and expression of native IgG in mammalian cells.

PubMed

Tesar, Devin; Hötzel, Isidro

2013-10-01

A significant bottleneck in antibody discovery by phage display is the transfer of immunoglobulin variable regions from phage clones to vectors that express immunoglobulin G (IgG) in mammalian cells for screening. Here, we describe a novel phagemid vector for Fab phage display that allows expression of native IgG in mammalian cells without sub-cloning. The vector uses an optimized mammalian signal sequence that drives robust expression of Fab fragments fused to an M13 phage coat protein in Escherichia coli and IgG expression in mammalian cells. To allow the expression of Fab fragments fused to a phage coat protein in E.coli and full-length IgG in mammalian cells from the same vector without sub-cloning, the sequence encoding the phage coat protein was embedded in an optimized synthetic intron within the immunoglobulin heavy chain gene. This intron is removed from transcripts in mammalian cells by RNA splicing. Using this vector, we constructed a synthetic Fab phage display library with diversity in the heavy chain only and selected for clones binding different antigens. Co-transfection of mammalian cells with DNA from individual phage clones and a plasmid expressing the invariant light chain resulted in the expression of native IgG that was used to assay affinity, ligand blocking activity and specificity.

Isolation and characterisation of lytic bacteriophages of Klebsiella pneumoniae and Klebsiella oxytoca.

PubMed

Karumidze, Natia; Kusradze, Ia; Rigvava, Sophio; Goderdzishvili, Marine; Rajakumar, Kumar; Alavidze, Zemphira

2013-03-01

Klebsiella bacteria have emerged as an increasingly important cause of community-acquired nosocomial infections. Extensive use of broad-spectrum antibiotics in hospitalised patients has led to both increased carriage of Klebsiella and the development of multidrug-resistant strains that frequently produce extended-spectrum β-lactamases and/or other defences against antibiotics. Many of these strains are highly virulent and exhibit a strong propensity to spread. In this study, six lytic Klebsiella bacteriophages were isolated from sewage-contaminated river water in Georgia and characterised as phage therapy candidates. Two of the phages were investigated in greater detail. Biological properties, including phage morphology, nucleic acid composition, host range, growth phenotype, and thermal and pH stability were studied for all six phages. Limited sample sequencing was performed to define the phylogeny of the K. pneumoniae- and K. oxytoca-specific bacteriophages vB_Klp_5 and vB_Klox_2, respectively. Both of the latter phages had large burst sizes, efficient rates of adsorption and were stable under different adverse conditions. Phages reported in this study are double-stranded DNA bacterial viruses belonging to the families Podoviridae and Siphoviridae. One or more of the six phages was capable of efficiently lysing ~63 % of Klebsiella strains comprising a collection of 123 clinical isolates from Georgia and the United Kingdom. These phages exhibit a number of properties indicative of potential utility in phage therapy cocktails.

Novel N4 Bacteriophages Prevail in the Cold Biosphere.

PubMed

Zhan, Yuanchao; Buchan, Alison; Chen, Feng

2015-08-01

Coliphage N4 is a lytic bacteriophage discovered nearly half a century ago, and it was considered to be a "genetic orphan" until very recently, when several additional N4-like phages were discovered to infect nonenteric bacterial hosts. Interest in this genus of phages is stimulated by their unique genetic features and propagation strategies. To better understand the ecology of N4-like phages, we investigated the diversity and geographic patterns of N4-like phages by examining 56 Chesapeake Bay viral communities, using a PCR-clone library approach targeting a diagnostic N4-like DNA polymerase gene. Many new lineages of N4-like phages were found in the bay, and their genotypes shift from the lower to the upper bay. Interestingly, signature sequences of N4-like phages were recovered only from winter month samples, when water temperatures were below 4°C. An analysis of existing metagenomic libraries from various aquatic environments supports the hypothesis that N4-like phages are most prolific in colder waters. In particular, a high number of N4-like phages were detected in Organic Lake, Antarctica, a cold and hypersaline system. The prevalence of N4-like phages in the cold biosphere suggests these viruses possess yet-to-be-determined mechanisms that facilitate lytic infections under cold conditions. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Vibrio Phage KVP40 Encodes a Functional NAD+ Salvage Pathway.

PubMed

Lee, Jae Yun; Li, Zhiqun; Miller, Eric S

2017-05-01

The genome of T4-type Vibrio bacteriophage KVP40 has five genes predicted to encode proteins of pyridine nucleotide metabolism, of which two, nadV and natV , would suffice for an NAD + salvage pathway. NadV is an apparent nicotinamide phosphoribosyltransferase (NAmPRTase), and NatV is an apparent bifunctional nicotinamide mononucleotide adenylyltransferase (NMNATase) and nicotinamide-adenine dinucleotide pyrophosphatase (Nudix hydrolase). Genes encoding the predicted salvage pathway were cloned and expressed in Escherichia coli , the proteins were purified, and their enzymatic properties were examined. KVP40 NadV NAmPRTase is active in vitro , and a clone complements a Salmonella mutant defective in both the bacterial de novo and salvage pathways. Similar to other NAmPRTases, the KVP40 enzyme displayed ATPase activity indicative of energy coupling in the reaction mechanism. The NatV NMNATase activity was measured in a coupled reaction system demonstrating NAD + biosynthesis from nicotinamide, phosphoribosyl pyrophosphate, and ATP. The NatV Nudix hydrolase domain was also shown to be active, with preferred substrates of ADP-ribose, NAD + , and NADH. Expression analysis using reverse transcription-quantitative PCR (qRT-PCR) and enzyme assays of infected Vibrio parahaemolyticus cells demonstrated nadV and natV transcription during the early and delayed-early periods of infection when other KVP40 genes of nucleotide precursor metabolism are expressed. The distribution and phylogeny of NadV and NatV proteins among several large double-stranded DNA (dsDNA) myophages, and also those from some very large siphophages, suggest broad relevance of pyridine nucleotide scavenging in virus-infected cells. NAD + biosynthesis presents another important metabolic resource control point by large, rapidly replicating dsDNA bacteriophages. IMPORTANCE T4-type bacteriophages enhance DNA precursor synthesis through reductive reactions that use NADH/NADPH as the electron donor and NAD + for ADP-ribosylation of proteins involved in transcribing and translating the phage genome. We show here that phage KVP40 encodes a functional pyridine nucleotide scavenging pathway that is expressed during the metabolic period of the infection cycle. The pathway is conserved in other large, dsDNA phages in which the two genes, nadV and natV , share an evolutionary history in their respective phage-host group. Copyright © 2017 American Society for Microbiology.

Decreasing Enterobacter sakazakii (Cronobacter spp.) food contamination level with bacteriophages: prospects and problems

PubMed Central

Zuber, Sophie; Boissin‐Delaporte, Catherine; Michot, Lise; Iversen, Carol; Diep, Benjamin; Brüssow, Harald; Breeuwer, Pieter

2008-01-01

Summary Enterobacter sakazakii (Cronobacter spp.) is an opportunistic pathogen, which can cause rare, but life‐threatening infections in neonates and infants through feeding of a contaminated milk formula. We isolated 67 phages from environmental samples and tested their lytic host range on a representative collection of 40 E. sakazakii strains. A cocktail of five phages prevented the outgrowth of 35 out of 40 test strains in artificially contaminated infant formula. Two E. sakazakii phages represented prolate head Myoviridae. Molecular tests identified them as close relatives of Escherichia coli phage T4. The remaining three phages represented isometric head Myoviridae with large genome size of 140 and 200 kb, respectively, which belonged to two different DNA hybridization groups. A high dose of 108 pfu ml−1 of phage could effectively sterilize a broth contaminated with both high and low pathogen counts (106 and 102 cfu ml−1). In contrast, broth inoculated with 104 phage and 102 bacteria per ml first showed normal bacterial growth until reaching a cell titre of 105 cfu ml−1. Only when crossing this threshold, phage replication started, but it could not reduce the contamination level below 100 cfu ml−1. Phages could be produced with titres of 1010 pfu ml−1 in broth culture, but they were not stable upon freeze‐drying. Addition of trehalose or milk formula stabilized the phage preparation, which then showed excellent storage stability even at elevated temperature. PMID:21261874

Prophagic DNA Fragments in Streptococcus agalactiae Strains and Association with Neonatal Meningitis

PubMed Central

van der Mee-Marquet, Nathalie; Domelier, Anne-Sophie; Mereghetti, Laurent; Lanotte, Philippe; Rosenau, Agnès; van Leeuwen, Willem; Quentin, Roland

2006-01-01

We identified—by randomly amplified polymorphic DNA (RAPD) analysis at the population level followed by DNA differential display, cloning, and sequencing—three prophage DNA fragments (F5, F7, and F10) in Streptococcus agalactiae that displayed significant sequence similarity to the DNA of S. agalactiae and Streptococcus pyogenes. The F5 sequence aligned with a prophagic gene encoding the large subunit of a terminase, F7 aligned with a phage-associated cell wall hydrolase and a phage-associated lysin, and F10 aligned with a transcriptional regulator (ArpU family) and a phage-associated endonuclease. We first determined the prevalence of F5, F7, and F10 by PCR in a collection of 109 strains isolated in the 1980s and divided into two populations: one with a high risk of causing meningitis (HR group) and the other with a lower risk of causing meningitis (LR group). These fragments were significantly more prevalent in the HR group than in the LR group (P < 0.001). Our findings suggest that lysogeny has increased the ability of some S. agalactiae strains to invade the neonatal brain endothelium. We then determined the prevalence of F5, F7, and F10 by PCR in a collection of 40 strains recently isolated from neonatal meningitis cases for comparison with the cerebrospinal fluid (CSF) strains isolated in the 1980s. The prevalence of the three prophage DNA fragments was similar in these two populations isolated 15 years apart. We suggest that the prophage DNA fragments identified have remained stable in many CSF S. agalactiae strains, possibly due to their importance in virulence or fitness. PMID:16517893

Phage Life Cycles Behind Bacterial Biodiversity.

PubMed

Olszak, Tomasz; Latka, Agnieszka; Roszniowski, Bartosz; Valvano, Miguel A; Drulis-Kawa, Zuzanna

2017-11-24

Bacteriophages (phages or bacterial viruses) are the most abundant biological entities in our planet; their influence reaches far beyond the microorganisms they parasitize. Phages are present in every environment and shape up every bacterial population in both active and passive ways. They participate in the circulation of organic matter and drive the evolution of microorganisms by horizontal gene transfer at unprecedented scales. The mass flow of genetic information in the microbial world influences the biosphere and poses challenges for science and medicine. The genetic flow, however, depends on the fate of the viral DNA injected into the bacterial cell. The archetypal notion of phages only engaging in predatorprey relationships is slowly fading. Because of their varied development cycles, environmental conditions, and the diversity of microorganisms they parasitize, phages form a dense and highly complex web of dependencies, which has important consequences for life on Earth. The sophisticated phage-bacteria interplay includes both aggressive action (bacterial lysis) and "diplomatic negotiations" (prophage domestication). Here, we review the most important mechanisms of interactions between phages and bacteria and their evolutionary consequences influencing their biodiversity. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Targeted delivery of siRNA into breast cancer cells via phage fusion proteins.

PubMed

Bedi, Deepa; Gillespie, James W; Petrenko, Vasily A; Ebner, Andreas; Leitner, Michael; Hinterdorfer, Peter; Petrenko, Valery A

2013-02-04

Nucleic acids, including antisense oligonucleotides, small interfering RNA (siRNA), aptamers, and rybozymes, emerged as versatile therapeutics due to their ability to interfere in a well-planned manner with the flow of genetic information from DNA to protein. However, a systemic use of NAs is hindered by their instability in physiological liquids and inability of intracellular accumulation in the site of action. We first evaluated the potential of cancer specific phage fusion proteins as targeting ligands that provide encapsulation, protection, and navigation of siRNA to the target cell. The tumor-specific proteins were isolated from phages that were affinity selected from a landscape phage library against target breast cancer cells. It was found that fusion phage coat protein fpVIII displaying cancer-targeting peptides can effectively encapsulate siRNAs and deliver them into the cells leading to specific silencing of the model gene GAPDH. Complexes of siRNA and phage protein form nanoparticles (nanophages), which were characterized by atomic force microscopy and ELISA, and their stability was demonstrated by resistance of encapsulated siRNA to degradation by serum nucleases. The phage protein/siRNA complexes can make a new type of highly selective, stable, active, and physiologically acceptable cancer nanomedicine.

Phages in the Human Body.

PubMed

Navarro, Ferran; Muniesa, Maite

2017-01-01

Bacteriophages, viruses that infect bacteria, have re-emerged as powerful regulators of bacterial populations in natural ecosystems. Phages invade the human body, just as they do other natural environments, to such an extent that they are the most numerous group in the human virome. This was only revealed in recent metagenomic studies, despite the fact that the presence of phages in the human body was reported decades ago. The influence of the presence of phages in humans has yet to be evaluated; but as in marine environments, a clear role in the regulation of bacterial populations could be envisaged, that might have an impact on human health. Moreover, phages are excellent vehicles of genetic transfer, and they contribute to the evolution of bacterial cells in the human body by spreading and acquiring DNA horizontally. The abundance of phages in the human body does not pass unnoticed and the immune system reacts to them, although it is not clear to what extent. Finally, the presence of phages in human samples, which most of the time is not considered, can influence and bias microbiological and molecular results; and, in view of the evidences, some studies suggest that more attention needs to be paid to their interference.

Immunocontraception: Filamentous Bacteriophage as a Platform for Vaccine Development.

PubMed

Samoylova, Tatiana I; Braden, Timothy D; Spencer, Jennifer A; Bartol, Frank F

2017-11-20

Population control of domestic, wild, invasive, and captive animal species is a global issue of importance to public health, animal welfare and the economy. There is pressing need for effective, safe, and inexpensive contraceptive technologies to address this problem. Contraceptive vaccines, designed to stimulate the immune system in order to block critical reproductive events and suppress fertility, may provide a solution. Filamentous bacteriophages can be used as platforms for development of such vaccines. In this review authors highlight structural and immunogenic properties of filamentous phages, and discuss applications of phage-peptide vaccines for advancement of immunocontraception technology in animals. Phages can be engineered to display fusion (non-phage) peptides as coat proteins. Such modifications can be accomplished via genetic manipulation of phage DNA, or by chemical conjugation of synthetic peptides to phage surface proteins. Phage fusions with antigenic determinants induce humoral as well as cell-mediated immune responses in animals, making them attractive as vaccines. Additional advantages of the phage platform include environmental stability, low cost, and safety for immunized animals and those administering the vaccines. Filamentous phages are viable platforms for vaccine development that can be engineered with molecular and organismal specificity. Phage-based vaccines can be produced in abundance at low cost, are environmentally stable, and are immunogenic when administered via multiple routes. These features are essential for a contraceptive vaccine to be operationally practical in animal applications. Adaptability of the phage platform also makes it attractive for design of human immunocontraceptive agents. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Characterization of novel virulent broad-host-range phages of Xylella fastidiosa and Xanthomonas.

PubMed

Ahern, Stephen J; Das, Mayukh; Bhowmick, Tushar Suvra; Young, Ry; Gonzalez, Carlos F

2014-01-01

The xylem-limited bacterium Xylella fastidiosa is the causal agent of several plant diseases, most notably Pierce's disease of grape and citrus variegated chlorosis. We report the isolation and characterization of the first virulent phages for X. fastidiosa, siphophages Sano and Salvo and podophages Prado and Paz, with a host range that includes Xanthomonas spp. Phages propagated on homologous hosts had observed adsorption rate constants of ~4 × 10(-12) ml cell(-1) min(-1) for X. fastidiosa strain Temecula 1 and ~5 × 10(-10) to 7 × 10(-10) ml cell(-1) min(-1) for Xanthomonas strain EC-12. Sano and Salvo exhibit >80% nucleotide identity to each other in aligned regions and are syntenic to phage BcepNazgul. We propose that phage BcepNazgul is the founding member of a novel phage type, to which Sano and Salvo belong. The lysis genes of the Nazgul-like phage type include a gene that encodes an outer membrane lipoprotein endolysin and also spanin gene families that provide insight into the evolution of the lysis pathway for phages of Gram-negative hosts. Prado and Paz, although exhibiting no significant DNA homology to each other, are new members of the phiKMV-like phage type, based on the position of the single-subunit RNA polymerase gene. The four phages are type IV pilus dependent for infection of both X. fastidiosa and Xanthomonas. The phages may be useful as agents for an effective and environmentally responsible strategy for the control of diseases caused by X. fastidiosa.

Immunocontraception: Filamentous Bacteriophage as a Platform for Vaccine Development

PubMed Central

Samoylova, Tatiana I.; Braden, Timothy D.; Spencer, Jennifer A.; Bartol, Frank F.

2017-01-01

Background: Population control of domestic, wild, invasive, and captive animal species is a global issue of importance to public health, animal welfare and the economy. There is pressing need for effective, safe, and inexpensive contraceptive technologies to ad-dress this problem. Contraceptive vaccines, designed to stimulate the immune system in order to block critical reproductive events and suppress fertility, may provide a solution. Fil-amentous bacteriophages can be used as platforms for development of such vaccines. Objective: In this review authors highlight structural and immunogenic properties of fila-mentous phages, and discuss applications of phage-peptide vaccines for advancement of immunocontraception technology in animals. Results: Phages can be engineered to display fusion (non-phage) peptides as coat proteins. Such modifications can be accomplished via genetic manipulation of phage DNA, or by chemical conjugation of synthetic peptides to phage surface proteins. Phage fusions with antigenic determinants induce humoral as well as cell-mediated immune responses in ani-mals, making them attractive as vaccines. Additional advantages of the phage platform include environmental stability, low cost, and safety for immunized animals and those ad-ministering the vaccines. Conclusion: Filamentous phages are viable platforms for vaccine development that can be engineered with molecular and organismal specificity. Phage-based vaccines can be pro-duced in abundance at low cost, are environmentally stable, and are immunogenic when administered via multiple routes. These features are essential for a contraceptive vaccine to be operationally practical in animal applications. Adaptability of the phage platform also makes it attractive for design of human immunocontraceptive agents. PMID:28901276

Biological characterization of v. Cholerae-specific bacteriophages isolated from water sources in Georgia.

PubMed

Elbakidze, T; Kokashvili, T; Janelidze, N; Porchkhidze, K; Koberidze, T; Tediashvili, M

2015-03-01

Vibrio cholerae, a widely spread bacterium in various marine, fresh, and brackish water environments, can cause a devastating diarrheal disease - cholera and also mild forms of gastroenteritis. Bacterial viruses are natural controllers of bacterial population density in water systems. The goal of this study was to isolate and characterize V. cholerae-specific bacteriophages occurring in the Georgian coastal zone of the Black Sea and inland water reservoirs in the eastern part of Georgia. During 2006-2009, 71 phages lytic to V. cholerae were collected from these aquatic environments. The phage isolation rate was varying from 8% to 15%, depending on the sampling season and site, and the abundance of host bacteria. The majority of phages specific to V. cholerae were collected from freshwater sources. The phage isolation showed seasonal character covering warm period -from April to September. Based on basic characteristics of primary phage isolates (lytic spectrum, virion morphology and DNA restriction profiles) 23 V. cholerae -specific phages were selected for series of consecutive screenings. Comparatively wide spectrum of lytic activity was revealed in case of 14 phages specific to V. cholerae O1, and one phage - VchBS3, active against non-O1 V. cholerae. Three phages active against V. cholerae non-O1 and six V. cholerae O1 -specific phages have been studied in detail for a number of biological features (stability in different solutions, temperature-, pH- and UV- sensitivity, influence of high ionic strength etc.), considered to be additional important characteristics for selection of phages with therapeutic potential.

Characterization of Novel Virulent Broad-Host-Range Phages of Xylella fastidiosa and Xanthomonas

PubMed Central

Ahern, Stephen J.; Das, Mayukh; Bhowmick, Tushar Suvra; Young, Ry

2014-01-01

The xylem-limited bacterium Xylella fastidiosa is the causal agent of several plant diseases, most notably Pierce's disease of grape and citrus variegated chlorosis. We report the isolation and characterization of the first virulent phages for X. fastidiosa, siphophages Sano and Salvo and podophages Prado and Paz, with a host range that includes Xanthomonas spp. Phages propagated on homologous hosts had observed adsorption rate constants of ∼4 × 10−12 ml cell−1 min−1 for X. fastidiosa strain Temecula 1 and ∼5 × 10−10 to 7 × 10−10 ml cell−1 min−1 for Xanthomonas strain EC-12. Sano and Salvo exhibit >80% nucleotide identity to each other in aligned regions and are syntenic to phage BcepNazgul. We propose that phage BcepNazgul is the founding member of a novel phage type, to which Sano and Salvo belong. The lysis genes of the Nazgul-like phage type include a gene that encodes an outer membrane lipoprotein endolysin and also spanin gene families that provide insight into the evolution of the lysis pathway for phages of Gram-negative hosts. Prado and Paz, although exhibiting no significant DNA homology to each other, are new members of the phiKMV-like phage type, based on the position of the single-subunit RNA polymerase gene. The four phages are type IV pilus dependent for infection of both X. fastidiosa and Xanthomonas. The phages may be useful as agents for an effective and environmentally responsible strategy for the control of diseases caused by X. fastidiosa. PMID:24214944

Purification of phage display-modified bacteriophage T4 by affinity chromatography

PubMed Central

2011-01-01

Background Affinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system. Results Elution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml. Conclusions Affinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be considered as a new phage purification method, appropriate for further investigations and development. PMID:21627821

An anti-tumor protein produced by Trichinella spiralis and identified by screening a T7 phage display library, induces apoptosis in human hepatoma H7402 cells

USDA-ARS?s Scientific Manuscript database

Trichinella spiralis infection confers effective resistance to tumor cell expansion. In this study, a T7 phage cDNA display library was constructed to express genes encoded by T. spiralis. Organic phase multi-cell screening was used to sort through candidate proteins in a transfected human chronic m...

Phage Display Breast Carcinoma cDNA Libraries: Isolation of Clones Which Specifically Bind to Membrane Glycoproteins, Mucins, and Endothelial Cell Surface

DTIC Science & Technology

1999-07-01

nutrients and waste and UV 2237 fibrosarcoma sublines (17). Expression of elimination, cell surfaces are also important for the galectin-1, another member of...10B capsid protein. Therefore, this GAG CGG AAA ATG GCA GAC AAT TTT TCG CTC CAT ... vector was chosen to assess the feasibility of phage met Ala Asp

Improved Performance of Loop-Mediated Isothermal Amplification Assays via Swarm Priming.

PubMed

Martineau, Rhett L; Murray, Sarah A; Ci, Shufang; Gao, Weimin; Chao, Shih-Hui; Meldrum, Deirdre R

2017-01-03

This work describes an enhancement to the loop-mediated isothermal amplification (LAMP) reaction which results in improved performance. Enhancement is achieved by adding a new set of primers to conventional LAMP reactions. These primers are termed "swarm primers" based on their relatively high concentration and their ability to create new amplicons despite the theoretical lack of single-stranded annealing sites. The primers target a region upstream of the FIP/BIP primer recognition sequences on opposite strands, substantially overlapping F1/B1 sites. Thus, despite the addition of a new primer set to an already complex assay, no significant increase in assay complexity is incurred. Swarm priming is presented for three DNA templates: Lambda phage, Synechocystis sp. PCC 6803 rbcL gene, and human HFE. The results of adding swarm primers to conventional LAMP reactions include increased amplification speed, increased indicator contrast, and increased reaction products. For at least one template, minor improvements in assay repeatability are also shown. In addition, swarm priming is shown to be effective at increasing the reaction speed for RNA amplification via RT-LAMP. Collectively, these results suggest that the addition of swarm primers will likely benefit most if not all existing LAMP assays based on state-of-the-art, six-primer reactions.

Genome Integration and Excision by a New Streptomyces Bacteriophage, ϕJoe

PubMed Central

Haley, Joshua A.; Stark, W. Marshall

2016-01-01

ABSTRACT Bacteriophages are the source of many valuable tools for molecular biology and genetic manipulation. In Streptomyces, most DNA cloning vectors are based on serine integrase site-specific DNA recombination systems derived from phage. Because of their efficiency and simplicity, serine integrases are also used for diverse synthetic biology applications. Here, we present the genome of a new Streptomyces phage, ϕJoe, and investigate the conditions for integration and excision of the ϕJoe genome. ϕJoe belongs to the largest Streptomyces phage cluster (R4-like) and encodes a serine integrase. The attB site from Streptomyces venezuelae was used efficiently by an integrating plasmid, pCMF92, constructed using the ϕJoe int-attP locus. The attB site for ϕJoe integrase was occupied in several Streptomyces genomes, including that of S. coelicolor, by a mobile element that varies in gene content and size between host species. Serine integrases require a phage-encoded recombination directionality factor (RDF) to activate the excision reaction. The ϕJoe RDF was identified, and its function was confirmed in vivo. Both the integrase and RDF were active in in vitro recombination assays. The ϕJoe site-specific recombination system is likely to be an important addition to the synthetic biology and genome engineering toolbox. IMPORTANCE Streptomyces spp. are prolific producers of secondary metabolites, including many clinically useful antibiotics. Bacteriophage-derived integrases are important tools for genetic engineering, as they enable integration of heterologous DNA into the Streptomyces chromosome with ease and high efficiency. Recently, researchers have been applying phage integrases for a variety of applications in synthetic biology, including rapid assembly of novel combinations of genes, biosensors, and biocomputing. An important requirement for optimal experimental design and predictability when using integrases, however, is the need for multiple enzymes with different specificities for their integration sites. In order to provide a broad platform of integrases, we identified and validated the integrase from a newly isolated Streptomyces phage, ϕJoe. ϕJoe integrase is active in vitro and in vivo. The specific recognition site for integration is present in a wide range of different actinobacteria, including Streptomyces venezuelae, an emerging model bacterium in Streptomyces research. PMID:28003200

Genomic Characterization of a Novel Phage Found in Black Abalone (Haliotis cracherodii) Infected with Withering Syndrome

NASA Astrophysics Data System (ADS)

Closek, C. J.; Langevin, S.; Burge, C. A.; Crosson, L.; White, S.; Friedman, C. S.

2016-02-01

Withering syndrome (WS), caused by the bacterium Candidatus Xenohaliotis californiensis, a Rickettsia-like organism (RLO), infects many species of abalone. Black abalone (Haliotis cracherodii), one of two endangered species of abalone, has experienced high population losses along the California coast due to WS. Recently, we observed reduced pathogenicity and mortality events in RLO-infected abalone when a novel bacteriophage (phage) was also present. To better understand phage-bacterium dynamics and develop more informative diagnostic tools, we sequenced the genome of the novel phage associated with the RLO responsible for WS. Metagenomic sequencing libraries were prepared with extracted genomic DNA from two experimentally infected H. cracherodii and phage sequences were enriched using hydroxyapatite chromatography normalization. Normalized libraries were individually barcoded and sequenced with Illumina MiSeq. Raw sequence reads were processed using VIrominer and de novo assembly produced one single phage-like contig (35.7Kb) from the experimentally infected abalone. This highly divergent genome had closest homology with a virus associated with abalone shriveling syndrome (SS). Of the 34 predicted ORFs, overlapping homology with the SS virus ranged from 20-72%, demonstrating the phage sequenced is genetically distinct from any known phage. The phage-like sequences represented a significant portion of the total reads sequenced ( 2 million of the 12 million paired-end reads; 17%) and we obtained 94,000X coverage across the novel phage genome. Beyond characterization of this novel phage, which appears to reduce pathogenicity of the RLO, the genome enabled us to develop quantitative PCR and in situ hybridization assays as diagnostic tools. These tools allow us to detect and quantify this phage in the endangered H. cracherodii.

The host outer membrane proteins OmpA and OmpC are associated with the Shigella phage Sf6 virion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao Haiyan, E-mail: zhaohy@ku.ed; Sequeira, Reuben D., E-mail: sequen@ku.ed; Galeva, Nadezhda A., E-mail: galeva@ku.ed

2011-01-20

Assembly of dsDNA bacteriophage is a precisely programmed process. Potential roles of host cell components in phage assembly haven't been well understood. It was previously reported that two unidentified proteins were present in bacteriophage Sf6 virion (Casjens et al, 2004, J.Mol.Biol. 339, 379-394, Fig. 2A). Using tandem mass spectrometry, we have identified the two proteins as outer membrane proteins (OMPs) OmpA and OmpC from its host Shigella flexneri. The transmission electron cryo-microscopy structure of Sf6 shows significant density at specific sites at the phage capsid inner surface. This density fit well with the characteristic beta-barrel domains of OMPs, thus maymore » be due to the two host proteins. Locations of this density suggest a role in Sf6 morphogenesis reminiscent of phage-encoded cementing proteins. These data indicate a new, OMP-related phage:host linkage, adding to previous knowledge that some lambdoid bacteriophage genomes contain OmpC-like genes that express phage-encoded porins in the lysogenic state.« less

Characterization of a new ViI-like Erwinia amylovora bacteriophage phiEa2809.

PubMed

Lagonenko, Alexander L; Sadovskaya, Olga; Valentovich, Leonid N; Evtushenkov, Anatoly N

2015-04-01

Erwinia amylovora is a Gram-negative plant pathogenic bacteria causing fire blight disease in many Rosaceae species. A novel E. amylovora bacteriophage, phiEa2809, was isolated from symptomless apple leaf sample collected in Belarus. This phage was also able to infect Pantoea agglomerans strains. The genome of phiEa2809 is a double-stranded linear DNA 162,160 bp in length, including 145 ORFs and one tRNA gene. The phiEa2809 genomic sequence is similar to the genomes of the Serratia plymutica phage MAM1, Shigella phage AG-3, Dickeya phage vB DsoM LIMEstone1 and Salmonella phage ViI and lacks similarity to described E. amylovora phage genomes. Based on virion morphology (an icosahedral head, long contractile tail) and genome structure, phiEa2809 was classified as a member of Myoviridae, ViI-like bacteriophages group. PhiEa2809 is the firstly characterized ViI-like bacteriophage able to lyse E. amylovora. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Modular architecture of the T4 phage superfamily: A conserved core genome and a plastic periphery

DOE Office of Scientific and Technical Information (OSTI.GOV)

Comeau, Andre M.; Bertrand, Claire; Letarov, Andrei

2007-06-05

Among the most numerous objects in the biosphere, phages show enormous diversity in morphology and genetic content. We have sequenced 7 T4-like phages and compared their genome architecture. All seven phages share a core genome with T4 that is interrupted by several hyperplastic regions (HPRs) where most of their divergence occurs. The core primarily includes homologues of essential T4 genes, such as the virion structure and DNA replication genes. In contrast, the HPRs contain mostly novel genes of unknown function and origin. A few of the HPR genes that can be assigned putative functions, such as a series of novelmore » Internal Proteins, are implicated in phage adaptation to the host. Thus, the T4-like genome appears to be partitioned into discrete segments that fulfil different functions and behave differently in evolution. Such partitioning may be critical for these large and complex phages to maintain their flexibility, while simultaneously allowing them to conserve their highly successful virion design and mode of replication.« less

The action of Escherichia coli CRISPR–Cas system on lytic bacteriophages with different lifestyles and development strategies

PubMed Central

Strotskaya, Alexandra; Savitskaya, Ekaterina; Metlitskaya, Anastasia; Morozova, Natalia; Datsenko, Kirill A.; Semenova, Ekaterina

2017-01-01

Abstract CRISPR–Cas systems provide prokaryotes with adaptive defense against bacteriophage infections. Given an enormous variety of strategies used by phages to overcome their hosts, one can expect that the efficiency of protective action of CRISPR–Cas systems against different viruses should vary. Here, we created a collection of Escherichia coli strains with type I-E CRISPR–Cas system targeting various positions in the genomes of bacteriophages λ, T5, T7, T4 and R1-37 and investigated the ability of these strains to resist the infection and acquire additional CRISPR spacers from the infecting phage. We find that the efficiency of CRISPR–Cas targeting by the host is determined by phage life style, the positions of the targeted protospacer within the genome, and the state of phage DNA. The results also suggest that during infection by lytic phages that are susceptible to CRISPR interference, CRISPR–Cas does not act as a true immunity system that saves the infected cell but rather enforces an abortive infection pathway leading to infected cell death with no phage progeny release. PMID:28130424

Survival of prototype strains of somatic coliphage families in environmental waters and when exposed to UV low-pressure monochromatic radiation or heat.

PubMed

Lee, Hee Suk; Sobsey, Mark D

2011-06-01

The potential use of specific somatic coliphage taxonomic groups as viral indicators based on their persistence and prevalence in water was investigated. Representative type strains of the 4 major somatic coliphage taxonomic groups were seeded into reagent water and an ambient surface water source of drinking water and the survival of the added phages was measured over 90 days at temperatures of 23-25 and 4 °C. Microviridae (type strain PhiX174), Siphoviridae (type strain Lambda), and Myoviridae (type strain T4) viruses were the most persistent in water at the temperatures tested. The Microviridae (type strain PhiX174) and the Siphoviridae (type strain Lambda) were the most resistant viruses to UV radiation and the Myoviridae (type strain T4) and the Microviridae (type strain PhiX174) were the most resistant viruses to heat. Based on their greater persistence in water over time and their relative resistance to heat and/or UV radiation, the Myoviridae (type strain T4), the Microviridae (type strain PhiX174), and the Siphoviridae (type strain Lambda) were the preferred candidate somatic coliphages as fecal indicator viruses in water, with the Microviridae (type strain PhiX174) the most resistant to these conditions overall. Copyright © 2011 Elsevier Ltd. All rights reserved.

Triplication of a four-gene set during evolution of the goat beta-globin locus produced three genes now expressed differentially during development.

PubMed Central

Townes, T M; Fitzgerald, M C; Lingrel, J B

1984-01-01

Distinct hemoglobins are synthesized in goats at different stages of development, similar to humans. Embryonic hemoglobins (zeta 2 epsilon 2 and alpha 2 epsilon 2) are synthesized initially and are followed sequentially by fetal (alpha 2 beta F2), preadult (alpha 2 beta C2), and adult (alpha 2 beta A2) hemoglobins. To help understand the basis of these switches, the genes of the beta-globin locus have been cloned and their linkage arrangement has been determined by the isolation of lambda phage carrying overlapping inserts of genomic goat DNA. The locus extends over 120 kilobase pairs and consists of 12 genes arranged in the following order: epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F. Comparison of the nucleotide sequence of the 12 genes shows that the locus is organized into three homologous four-gene sets that presumably evolved by the triplication of an ancestral set of four genes (epsilon-epsilon-psi beta-beta). Interestingly, the three genes (beta C, beta A, and beta F) located at the ends of the four-gene sets are expressed at different stages of development. Therefore, the goat beta F-, beta C-, and beta A-globin genes appear to have evolved by a mechanism that includes the triplication of 40-50 kilobase pairs of DNA and the recruitment of newly formed genes for expression in fetal, preadult, and adult life. PMID:6593719

Viruses Infecting a Freshwater Filamentous Cyanobacterium (Nostoc sp.) Encode a Functional CRISPR Array and a Proteobacterial DNA Polymerase B.

PubMed

Chénard, Caroline; Wirth, Jennifer F; Suttle, Curtis A

2016-06-14

Here we present the first genomic characterization of viruses infecting Nostoc, a genus of ecologically important cyanobacteria that are widespread in freshwater. Cyanophages A-1 and N-1 were isolated in the 1970s and infect Nostoc sp. strain PCC 7210 but remained genomically uncharacterized. Their 68,304- and 64,960-bp genomes are strikingly different from those of other sequenced cyanophages. Many putative genes that code for proteins with known functions are similar to those found in filamentous cyanobacteria, showing a long evolutionary history in their host. Cyanophage N-1 encodes a CRISPR array that is transcribed during infection and is similar to the DR5 family of CRISPRs commonly found in cyanobacteria. The presence of a host-related CRISPR array in a cyanophage suggests that the phage can transfer the CRISPR among related cyanobacteria and thereby provide resistance to infection with competing phages. Both viruses also encode a distinct DNA polymerase B that is closely related to those found in plasmids of Cyanothece sp. strain PCC 7424, Nostoc sp. strain PCC 7120, and Anabaena variabilis ATCC 29413. These polymerases form a distinct evolutionary group that is more closely related to DNA polymerases of proteobacteria than to those of other viruses. This suggests that the polymerase was acquired from a proteobacterium by an ancestral virus and transferred to the cyanobacterial plasmid. Many other open reading frames are similar to a prophage-like element in the genome of Nostoc sp. strain PCC 7524. The Nostoc cyanophages reveal a history of gene transfers between filamentous cyanobacteria and their viruses that have helped to forge the evolutionary trajectory of this previously unrecognized group of phages. Filamentous cyanobacteria belonging to the genus Nostoc are widespread and ecologically important in freshwater, yet little is known about the genomic content of their viruses. Here we report the first genomic analysis of cyanophages infecting filamentous freshwater cyanobacteria, revealing that their gene content is unlike that of other cyanophages. In addition to sharing many gene homologues with freshwater cyanobacteria, cyanophage N-1 encodes a CRISPR array and expresses it upon infection. Also, both viruses contain a DNA polymerase B-encoding gene with high similarity to genes found in proteobacterial plasmids of filamentous cyanobacteria. The observation that phages can acquire CRISPRs from their hosts suggests that phages can also move them among hosts, thereby conferring resistance to competing phages. The presence in these cyanophages of CRISPR and DNA polymerase B sequences, as well as a suite of other host-related genes, illustrates the long and complex evolutionary history of these viruses and their hosts. Copyright © 2016 Chénard et al.

Tubulin homolog TubZ in a phage-encoded partition system

PubMed Central

Oliva, María A.; Martin-Galiano, Antonio J.; Sakaguchi, Yoshihiko; Andreu, José M.

2012-01-01

Partition systems are responsible for the process whereby large and essential plasmids are accurately positioned to daughter cells during bacterial division. They are typically made of three components: a centromere-like DNA zone, an adaptor protein, and an assembling protein that is either a Walker-box ATPase (type I) or an actin-like ATPase (type II). A recently described type III segregation system has a tubulin/FtsZ-like protein, called TubZ, for plasmid movement. Here, we present the 2.3 Å structure and dynamic assembly of a TubZ tubulin homolog from a bacteriophage and unravel the Clostridium botulinum phage c-st type III partition system. Using biochemical and biophysical approaches, we prove that a gene upstream from tubZ encodes the partner TubR and localize the centromeric region (tubS), both of which are essential for anchoring phage DNA to the motile TubZ filaments. Finally, we describe a conserved fourth component, TubY, which modulates the TubZ-R-S complex interaction. PMID:22538818

Recombination and Transfection Mapping of Cistron 5 of Bacteriophage Sp82g

PubMed Central

Green, D. MacDonald; Urban, Margeret I.

1972-01-01

Recombination between transfecting SP82G DNA molecules has been studied in Bacillus subtilis. Recombinant progeny issuing from transfected cells show many of the features that characterize progeny production in multiplicity reactivated bacteriophage, such as: majority recombinant clones, non-reciprocity of recombinant clones and the frequent absence of input alleles. While transfection substantially lowers the linkage observed between markers in normal phage crosses, linkage is observed at small map distances in transfection either by plating transfected bacteria or the progeny phage. Maps constructed from transfection crosses are identical to those of normal phage crosses, except in magnitude.—Examination of the concentration response of two marker biparental crosses, and three marker triparental crosses using transfecting DNA leads to the conclusion that at all concentrations, transfective centers are saturated with respect to the number of molecules that can be taken up. Thus, the frequency of recombinant infective centers, or recombinant progeny is independent of concentration effects. PMID:17248556

Stability engineering of anti-EGFR scFv antibodies by rational design of a lambda-to-kappa swap of the VL framework using a structure-guided approach.

PubMed

Lehmann, Andreas; Wixted, Josephine H F; Shapovalov, Maxim V; Roder, Heinrich; Dunbrack, Roland L; Robinson, Matthew K

2015-01-01

Phage-display technology facilitates rapid selection of antigen-specific single-chain variable fragment (scFv) antibodies from large recombinant libraries. ScFv antibodies, composed of a VH and VL domain, are readily engineered into multimeric formats for the development of diagnostics and targeted therapies. However, the recombinant nature of the selection strategy can result in VH and VL domains with sub-optimal biophysical properties, such as reduced thermodynamic stability and enhanced aggregation propensity, which lead to poor production and limited application. We found that the C10 anti-epidermal growth factor receptor (EGFR) scFv, and its affinity mutant, P2224, exhibit weak production from E. coli. Interestingly, these scFv contain a fusion of lambda3 and lambda1 V-region (LV3 and LV1) genes, most likely the result of a PCR aberration during library construction. To enhance the biophysical properties of these scFvs, we utilized a structure-based approach to replace and redesign the pre-existing framework of the VL domain to one that best pairs with the existing VH. We describe a method to exchange lambda sequences with a more stable kappa3 framework (KV3) within the VL domain that incorporates the original lambda DE-loop. The resulting scFvs, C10KV3_LV1DE and P2224KV3_LV1DE, are more thermodynamically stable and easier to produce from bacterial culture. Additionally, C10KV3_LV1DE and P2224KV3_LV1DE retain binding affinity to EGFR, suggesting that such a dramatic framework swap does not significantly affect scFv binding. We provide here a novel strategy for redesigning the light chain of problematic scFvs to enhance their stability and therapeutic applicability.

Three novel Pseudomonas phages isolated from composting provide insights into the evolution and diversity of tailed phages.

PubMed

Amgarten, Deyvid; Martins, Layla Farage; Lombardi, Karen Cristina; Antunes, Luciana Principal; de Souza, Ana Paula Silva; Nicastro, Gianlucca Gonçalves; Kitajima, Elliott Watanabe; Quaggio, Ronaldo Bento; Upton, Chris; Setubal, João Carlos; da Silva, Aline Maria

2017-05-04

Among viruses, bacteriophages are a group of special interest due to their capacity of infecting bacteria that are important for biotechnology and human health. Composting is a microbial-driven process in which complex organic matter is converted into humus-like substances. In thermophilic composting, the degradation activity is carried out primarily by bacteria and little is known about the presence and role of bacteriophages in this process. Using Pseudomonas aeruginosa as host, we isolated three new phages from a composting operation at the Sao Paulo Zoo Park (Brazil). One of the isolated phages is similar to Pseudomonas phage Ab18 and belongs to the Siphoviridae YuA-like viral genus. The other two isolated phages are similar to each other and present genomes sharing low similarity with phage genomes in public databases; we therefore hypothesize that they belong to a new genus in the Podoviridae family. Detailed genomic descriptions and comparisons of the three phages are presented, as well as two new clusters of phage genomes in the Viral Orthologous Clusters database of large DNA viruses. We found sequences encoding homing endonucleases that disrupt a putative ribonucleotide reductase gene and an RNA polymerase subunit 2 gene in two of the phages. These findings provide insights about the evolution of two-subunits RNA polymerases and the possible role of homing endonucleases in this process. Infection tests on 30 different strains of bacteria reveal a narrow host range for the three phages, restricted to P. aeruginosa PA14 and three other P. aeruginosa clinical isolates. Biofilm dissolution assays suggest that these phages could be promising antimicrobial agents against P. aeruginosa PA14 infections. Analyses on composting metagenomic and metatranscriptomic data indicate association between abundance variations in both phage and host populations in the environment. The results about the newly discovered and described phages contribute to the understanding of tailed bacteriophage diversity, evolution, and role in the complex composting environment.

Temperate Phages Acquire DNA from Defective Prophages by Relaxed Homologous Recombination: The Role of Rad52-Like Recombinases

PubMed Central

De Paepe, Marianne; Hutinet, Geoffrey; Son, Olivier; Amarir-Bouhram, Jihane; Schbath, Sophie; Petit, Marie-Agnès

2014-01-01

Bacteriophages (or phages) dominate the biosphere both numerically and in terms of genetic diversity. In particular, genomic comparisons suggest a remarkable level of horizontal gene transfer among temperate phages, favoring a high evolution rate. Molecular mechanisms of this pervasive mosaicism are mostly unknown. One hypothesis is that phage encoded recombinases are key players in these horizontal transfers, thanks to their high efficiency and low fidelity. Here, we associate two complementary in vivo assays and a bioinformatics analysis to address the role of phage encoded recombinases in genomic mosaicism. The first assay allowed determining the genetic determinants of mosaic formation between lambdoid phages and Escherichia coli prophage remnants. In the second assay, recombination was monitored between sequences on phage λ, and allowed to compare the performance of three different Rad52-like recombinases on the same substrate. We also addressed the importance of homologous recombination in phage evolution by a genomic comparison of 84 E. coli virulent and temperate phages or prophages. We demonstrate that mosaics are mainly generated by homology-driven mechanisms that tolerate high substrate divergence. We show that phage encoded Rad52-like recombinases act independently of RecA, and that they are relatively more efficient when the exchanged fragments are divergent. We also show that accessory phage genes orf and rap contribute to mosaicism. A bioinformatics analysis strengthens our experimental results by showing that homologous recombination left traces in temperate phage genomes at the borders of recently exchanged fragments. We found no evidence of exchanges between virulent and temperate phages of E. coli. Altogether, our results demonstrate that Rad52-like recombinases promote gene shuffling among temperate phages, accelerating their evolution. This mechanism may prove to be more general, as other mobile genetic elements such as ICE encode Rad52-like functions, and play an important role in bacterial evolution itself. PMID:24603854

Molecular mechanisms of CRISPR-mediated microbial immunity.

PubMed

Gasiunas, Giedrius; Sinkunas, Tomas; Siksnys, Virginijus

2014-02-01

Bacteriophages (phages) infect bacteria in order to replicate and burst out of the host, killing the cell, when reproduction is completed. Thus, from a bacterial perspective, phages pose a persistent lethal threat to bacterial populations. Not surprisingly, bacteria evolved multiple defense barriers to interfere with nearly every step of phage life cycles. Phages respond to this selection pressure by counter-evolving their genomes to evade bacterial resistance. The antagonistic interaction between bacteria and rapidly diversifying viruses promotes the evolution and dissemination of bacteriophage-resistance mechanisms in bacteria. Recently, an adaptive microbial immune system, named clustered regularly interspaced short palindromic repeats (CRISPR) and which provides acquired immunity against viruses and plasmids, has been identified. Unlike the restriction–modification anti-phage barrier that subjects to cleavage any foreign DNA lacking a protective methyl-tag in the target site, the CRISPR–Cas systems are invader-specific, adaptive, and heritable. In this review, we focus on the molecular mechanisms of interference/immunity provided by different CRISPR–Cas systems.

Cypermethrin and lambda-cyhalothrin induced in vivo alterations in nucleic acids and protein contents in a freshwater catfish, Clarias batrachus (Linnaeus; Family-Clariidae).

PubMed

Kumar, Amit; Sharma, Bechan; Pandey, Ravi S

2009-08-01

The fresh water fish, Clarias batrachus, were exposed to sub-acute concentrations of cypermethrin and lambda-cyhalothrin for 96 h to assess their impact on the levels of nucleic acids and protein in different organs of fish. DNA content was found almost unchanged with a single exception of liver, which showed significant increment in the levels of DNA in response to the separate treatments of both compounds. Both RNA and protein contents declined in brain, liver, and muscle while sharp increase was observed in gills. However, in kidney, RNA contents depicted significant enhancement only at higher concentrations, with initial decline at lower concentrations. The trends of alterations in RNA/DNA and protein/DNA ratios were quite similar to the corresponding results explained above for RNA and protein. The results clearly indicated that both of these pyrethroids exerted their effects at transcriptional and translational levels while DNA synthesis was found to be unaffected by these compounds with an exception of liver.

Highly Sensitive Bacteriophage-Based Detection of Brucella abortus in Mixed Culture and Spiked Blood

PubMed Central

Sergueev, Kirill V.; Filippov, Andrey A.; Nikolich, Mikeljon P.

2017-01-01

For decades, bacteriophages (phages) have been used for Brucella species identification in the diagnosis and epidemiology of brucellosis. Traditional Brucella phage typing is a multi-day procedure including the isolation of a pure culture, a step that can take up to three weeks. In this study, we focused on the use of brucellaphages for sensitive detection of the pathogen in clinical and other complex samples, and developed an indirect method of Brucella detection using real-time quantitative PCR monitoring of brucellaphage DNA amplification via replication on live Brucella cells. This assay allowed the detection of single bacteria (down to 1 colony-forming unit per milliliter) within 72 h without DNA extraction and purification steps. The technique was equally efficient with Brucella abortus pure culture and with mixed cultures of B. abortus and α-proteobacterial near neighbors that can be misidentified as Brucella spp., Ochrobactrum anthropi and Afipia felis. The addition of a simple short sample preparation step enabled the indirect phage-based detection of B. abortus in spiked blood, with the same high sensitivity. This indirect phage-based detection assay enables the rapid and sensitive detection of live B. abortus in mixed cultures and in blood samples, and can potentially be applied for detection in other clinical samples and other complex sample types. PMID:28604602

5' modification of duplex DNA with a ruthenium electron donor-acceptor pair using solid-phase DNA synthesis

NASA Technical Reports Server (NTRS)

Frank, Natia L.; Meade, Thomas J.

2003-01-01

Incorporation of metalated nucleosides into DNA through covalent modification is crucial to measurement of thermal electron-transfer rates and the dependence of these rates with structure, distance, and position. Here, we report the first synthesis of an electron donor-acceptor pair of 5' metallonucleosides and their subsequent incorporation into oligonucleotides using solid-phase DNA synthesis techniques. Large-scale syntheses of metal-containing oligonucleotides are achieved using 5' modified phosporamidites containing [Ru(acac)(2)(IMPy)](2+) (acac is acetylacetonato; IMPy is 2'-iminomethylpyridyl-2'-deoxyuridine) (3) and [Ru(bpy)(2)(IMPy)](2+) (bpy is 2,2'-bipyridine; IMPy is 2'-iminomethylpyridyl-2'-deoxyuridine) (4). Duplexes formed with the metal-containing oligonucleotides exhibit thermal stability comparable to the corresponding unmetalated duplexes (T(m) of modified duplex = 49 degrees C vs T(m) of unmodified duplex = 47 degrees C). Electrochemical (3, E(1/2) = -0.04 V vs NHE; 4, E(1/2) = 1.12 V vs NHE), absorption (3, lambda(max) = 568, 369 nm; 4, lambda(max) = 480 nm), and emission (4, lambda(max) = 720 nm, tau = 55 ns, Phi = 1.2 x 10(-)(4)) data for the ruthenium-modified nucleosides and oligonucleotides indicate that incorporation into an oligonucleotide does not perturb the electronic properties of the ruthenium complex or the DNA significantly. In addition, the absence of any change in the emission properties upon metalated duplex formation suggests that the [Ru(bpy)(2)(IMPy)](2+)[Ru(acac)(2)(IMPy)](2+) pair will provide a valuable probe for DNA-mediated electron-transfer studies.

Characterization of a new phage, termed ϕA318, which is specific for Vibrio alginolyticus.

PubMed

Lin, Ying-Rong; Chiu, Chi-Wen; Chang, Feng-Yi; Lin, Chan-Shing

2012-05-01

Vibrio alginolyticus is an opportunistic pathogen of animals and humans; its related strains can also produce tetrodotoxin and hemolysins. A new phage, ϕA318, which lysed its host V. alginolyticus with high efficiency, was characterized. The burst size of ϕA318 in V. alginolyticus was 72 PFU/bacterium at an MOI of 1 at room temperature; the plaque size was as large as 5 mm in diameter. Electron microscopy (EM) of the phage particles revealed a 50- to 55-nm isomorphous icosahedral head with a 12-nm non-contractile tail, similar to the T7-like phages of the family Podoviridae. Phylogenetic analysis based on complete sequences of the DNA-directed RNA polymerase gene revealed that ϕA318 had 28-47% amino acid identity to enterobacteria phages T7 and SP6, and other Vibrio phages, and the phylogenetic distance suggested that ϕA318 could be classified as a new T7-like bacteriophage. Nevertheless, several motifs in the ϕA318 phage RNA polymerase were highly conserved, including DFRGR (T7-421 motif), DG (T7-537 motif), PSEKPQDIYGAVS (T7-563 motif), RSMTKKPVMTL PYGS (T7-627 motif), and HDS (T7-811 motif). Genetic analysis indicated that phage ϕA318 is not a thermostable direct hemolysin producer. The results suggest that the MOI should be higher than 0.1 to prevent the chance of hemolysin production by the bacteria before they are lysed by the phage.

In vitro high throughput phage display selection of ovarian cancer avid phage clones for near-infrared optical imaging.

PubMed

Soendergaard, Mette; Newton-Northup, Jessica R; Deutscher, Susan L

2014-01-01

Ovarian cancer is among the leading causes of cancer deaths in women, and is the most fatal gynecological malignancy. Poor outcomes of the disease are a direct result of inadequate detection and diagnostic methods, which may be overcome by the development of novel efficacious screening modalities. However, the advancement of such technologies is often time-consuming and costly. To overcome this hurdle, our laboratory has established a time and cost effective method of selecting and identifying ovarian carcinoma avid bacteriophage (phage) clones using high throughput phage display technology. These phage clones were selected from a filamentous phage fusion vector (fUSE5) 15-amino acid peptide library against human ovarian carcinoma (SKOV-3) cells, and identified by DNA sequencing. Two phage clones, pM6 and pM9, were shown to exhibit high binding affinity and specificity for SKOV-3 cells using micropanning, cell binding and fluorescent microscopy studies. To validate that the binding was mediated by the phage-displayed peptides, biotinylated peptides (M6 and M9) were synthesized and the specificity for ovarian carcinoma cells was analyzed. These results showed that M6 and M9 bound to SKOV-3 cells in a dose-response manner and exhibited EC50 values of 22.9 ± 2.0 μM and 12.2 ± 2.1μM (mean ± STD), respectively. Based on this, phage clones pM6 and pM9 were labeled with the near-infrared fluorophore AF680, and examined for their pharmacokinetic properties and tumor imaging abilities in vivo. Both phage successfully targeted and imaged SKOV-3 tumors in xenografted nude mice, demonstrating the ability of this method to quickly and cost effectively develop novel ovarian carcinoma avid phage.

Competition between conjugation and M13 phage infection in Escherichia coli in the absence of selection pressure: a kinetic study.

PubMed

Wan, Zhenmao; Goddard, Noel L

2012-10-01

Inter- and intraspecies horizontal gene transfer enabled by bacterial secretion systems is a powerful mechanism for bacterial genome plasticity. The type IV secretion system of Escherichia coli, encoded by the F plasmid, enables cell-to-cell contact and subsequent DNA transfer known as conjugation. Conjugation is compromised by phage infection that specifically targets the secretion machinery. Hence, the use of phages to regulate the spread of genes, such as acquired antibiotic resistance or as general biosanitation agents, has gained interest. To predict the potential efficacy, the competition kinetics must first be understood. Using quantitative PCR to enumerate genomic loci in a resource-limited batch culture, we quantify the infection kinetics of the nonlytic phage M13 and its impact on conjugation in the absence of selection pressure (isogenic set). Modeling the resulting experimental data reveals the cellular growth rate to be reduced to 60% upon phage infection. We also find a maximum phage infection rate of 3×10(-11) mL phage(-1) min(-1) which is only 1 order of magnitude slower than the maximum conjugation rate (3×10(-10) mL cell(-1) min(-1)), suggesting phages must be in significant abundance to be effective antagonists to horizontal gene transfer. In the regime where the number of susceptible cells (F(+)) and phages are equal upon initial infection, we observe the spread of the conjugative plasmid throughout the cell population despite phage infection, but only at 10% of the uninfected rate. This has interesting evolutionary implications, as even in the absence of selection pressure, cells maintain the ability to conjugate despite phage vulnerability and the associated growth consequences.

Biology and Genomics of an Historic Therapeutic Escherichia coli Bacteriophage Collection.

PubMed

Baig, Abiyad; Colom, Joan; Barrow, Paul; Schouler, Catherine; Moodley, Arshnee; Lavigne, Rob; Atterbury, Robert

2017-01-01

We have performed microbiological and genomic characterization of an historic collection of nine bacteriophages, specifically infecting a K1 E. coli O18:K1:H7 ColV + strain. These phages were isolated from sewage and tested for their efficacy in vivo for the treatment of systemic E. coli infection in a mouse infection model by Smith and Huggins (1982). The aim of the study was to identify common microbiological and genomic characteristics, which co-relate to the performance of these phages in in vivo study. These features will allow an informed selection of phages for use as therapeutic agents. Transmission electron microscopy showed that six of the nine phages were Podoviridae and the remaining three were Siphoviridae . The four best performing phages in vivo belonged to the Podoviridae family. In vitro , these phages exhibited very short latent and rise periods in our study. In agreement with their microbiological profiles, characterization by genome sequencing showed that all six podoviruses belong to the Autographivirinae subfamily. Of these, four were isolates of the same species (99% identity), whereas two had divergent genomes compared to other podoviruses. The Siphoviridae phages, which were moderate to poor performers in vivo , exhibited longer latent and rise periods in vitro . Two of the three siphoviruses were closely related to each other (99% identity), but all can be associated with the Guernseyvirinae subfamily. Genome sequence comparison of both types of phages showed that a gene encoding for DNA-dependent RNA polymerase was only present in phages with faster replication cycle, which may account for their better performance in vivo . These data define a combination of microbiological, genomic and in vivo characteristics which allow a more rational evaluation of the original in vivo data and pave the way for the selection of phages for future phage therapy trails.

Phage Conversion for β-Lactam Antibiotic Resistance of Staphylococcus aureus from Foods.

PubMed

Lee, Young-Duck; Park, Jong-Hyun

2016-02-01

Temperate phages have been suggested to carry virulence factors and other lysogenic conversion genes that play important roles in pathogenicity. In this study, phage TEM123 in wild-type Staphylococcus aureus from food sources was analyzed with respect to its morphology, genome sequence, and antibiotic resistance conversion ability. Phage TEM123 from a mitomycin C-induced lysate of S. aureus was isolated from foods. Morphological analysis under a transmission electron microscope revealed that it belonged to the family Siphoviridae. The genome of phage TEM123 consisted of a double-stranded DNA of 43,786 bp with a G+C content of 34.06%. A bioinformatics analysis of the phage genome identified 43 putative open reading frames (ORFs). ORF1 encoded a protein that was nearly identical to the metallo-β-lactamase enzymes that degrade β-lactam antibiotics. After transduction to S. aureus with phage TEM123, the metallo-β-lactamase gene was confirmed in the transductant by PCR and sequencing analyses. In a β-lactam antibiotic susceptibility test, the transductant was more highly resistant to β-lactam antibiotics than S. aureus S133. Phage TEM123 might play a role in the transfer of β-lactam antibiotic resistance determinants in S. aureus. Therefore, we suggest that the prophage of S. aureus with its exotoxin is a risk factor for food safety in the food chain through lateral gene transfer.