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PubMed1995

Novel cytotoxic DNA sequence and minor groove targeted photosensitizers: conjugates of pyrene and netropsin analogues.

The design, syntheses, photochemical and biological properties of conjugates of pyrene with pyrrole- (1) and imidazole-containing (2) analogues of netropsin are reported. The results of an ethidium displacement assay and circular dichroism (CD) titration studies show both compounds bind with a higher affinity to poly(dA-dT) than to poly(dG-dC). In addition they bind as strongly to T4 coliphage DNA as to calf thymus DNA suggesting the binding occurs in the minor groove. The quenching rate constants of the singlet excited states of agents 1 and 2 by molecular oxygen were found to be 8.5 x 10(9) M-1S-1 and 7.7 x 10(9) M-1S-1, suggesting the involvement of singlet oxygen. Both compounds showed some cytotoxicity to human chronic myeloid leukemia K562 cells in the dark. Upon irradiation the activities were significantly enhanced resulting in photoinduced dose modifications of 8 and 14 for 1 and 2, respectively under the conditions employed. Both agents were markedly more phototoxic than 1-pyrenebutyric acid 8. To address the mechanism of action of compounds 1 and 2 their photoactivated abilities to produce DNA strand breaks were measured. Both agents caused increased single strand breakage with increasing UV exposure. The concentrations (EC50) of 1 and 2 needed to cause 50% single-strand cleavage of pBR322 DNA upon UV-A activation were found to be 40 microM and 45 microM, respectively. In contrast, no DNA strand breaks were observed in the dark with either conjugate or with 8 following irradiation. DNA strand breaks were measured in drug treated K562 cells using alkaline elution.(ABSTRACT TRUNCATED AT 250 WORDS)

Antineoplastic Agents↗
PubMed1992

Overproduction and purification of Bacillus subtilis DNA polymerase III.

The objectives of this work were to engineer the cloned polC gene encoding Bacillus subtilis DNA polymerase III for controlled overexpression in Escherichia coli and to devise a facile purification scheme permitting the large-scale production of pure recombinant polymerase. The translational signals of polC were restructured by expression cassette PCR (MacFerrin et al., 1990, Proc. Natl. Acad. Sci. USA 87, 1937-1941), and the modified gene was inserted into the expression plasmid, pKC30 (Rosenberg et al., 1983, in "Methods in Enzymology," Vol. 101, pp. 123-138, Academic Press, San Diego), under the strict control of the coliphage lambda pL promoter and its repressor, cI. When the system was derepressed at 32 degrees C, soluble DNA polymerase III accumulated at levels approximating 2% of total cellular protein. The recombinant protein was purified to greater than 99% purity by utilizing a tandem combination of Cibacron blue agarose, phenyl-Sepharose, and MonoQ FPLC chromatography. The properties of the purified recombinant protein were indistinguishable from those of native B. subtilis DNA polymerase III.

Bacillus subtilis↗
PubMed1988

Chimeric single-stranded DNA phage-plasmid cloning vectors.

A variety of ssDNA phagemid cloning vectors have been constructed that combine the advantages of the filamentous coliphages with a number of plasmid and other bacteriophage-encoded functions. The practical and biological advantages of a chimeric phage-plasmid vector are considerable, and the trend toward converting existing plasmids into ssDNA phagemids and consolidating any number of useful features into one or a few vectors will undoubtedly accelerate. A new helper phage specifically designed for the isolation of large amounts of single-stranded plasmid DNA simplifies the use of these cloning vehicles. Additional refinements in phagemid-helper phage systems should extend the potential of these vectors even further.

Bacteriophages↗
PubMed2006

Biophysical investigation of recombinant K5 lyase: structural implications of substrate binding and processing.

K5 lyase of coliphage K5A degrades the K5 polysaccharide of encapsulated E. coli strains expressing the K5 antigen thereby contributing to virus binding and infection. We have investigated the affinities of the recombinant enzyme for different GAG ligands by isothermal fluorescence titrations and correlated them with substrate processing and protein structural changes. Chondroitin sulfate (CS) and heparan sulfate (HS) bound to K5 lyase with a Kd of 0.5 microM whereas heparin exhibited a Kd=1.1 microM. The natural substrate K5 polysaccharide displayed a similar apparent affinity as CS and HS but was the only ligand of the enzyme which induced a large structural rearrangement of the protein as detected by far-UV CD spectroscopy. Since significant enzymatic degradation was only found for the K5 polysaccharide peaking at 44 degrees C, but binding was also detected for heparin, we propose that the K5 lyase is able to discriminate between specific (acetylated/non-sulfated) and unspecific (acetylated/sulfated) ligands by its heparin binding motif in the C-terminus. This is proposed to be the origin for the enzyme's residual HS degrading activity.

Chondroitin Sulfates↗
PubMed2006

Pure human urine is a good fertiliser for cucumbers.

Human urine obtained from separating toilets was tested as a fertiliser for cultivation of outdoor cucumber (Cucumis sativus L.) in a Nordic climate. The urine used contained high amounts of nitrogen with some phosphorus and potassium, but numbers of enteric microorganisms were low even though urine had not been preserved before sampling. The cucumber yield after urine fertilisation was similar or slightly better than the yield obtained from control rows fertilised with commercial mineral fertiliser. None of the cucumbers contained any enteric microorganisms (coliforms, enterococci, coliphages and clostridia). In the taste assessment, 11 out of 20 persons could recognise which cucumber of three cucumbers was different but they did not prefer one over the other cucumber samples, since all of them were assessed as equally good.

Cucumis sativus↗
PubMed2004

Dimerization of bacteriophage P2 integrase is not required for binding to its DNA target but for its biological activity.

Coliphage P2 integrates into the host chromosome upon lysogenization via site-specific recombination mediated by the phage integrase (Int). P2 integrase belongs to the tyrosine family of recombinases. In this work, it is shown that P2 integrase forms dimers but not oligomers in the absence of its DNA target. Furthermore, the C-terminal end of the protein and amino acid (aa) E197 have been found to be involved in dimerization. Amino acid E197 is located in a conserved region of the tyrosine recombinases that has not previously been implicated in dimerization. The dimerization deficient mutants were unaffected in binding to its phage attachment site (attP) substrate, but had a reduced ability to complement an int-defective prophage.

Amino Acid Sequence↗
PubMed2005

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

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.

Amino Acid Sequence↗
PubMed2026

Insights into Genome Ejection by a Therapeutic phiKMV-like Bacteriophage.

Ar-KM is a phiKMV-like therapeutic bacteriophage used in clinical candidate phage therapy cocktails to treat lung infections caused by P. aeruginosa. Here, we present an integrative structural atlas of Ar-KM proteins using cryo-EM, proteomics, and bioinformatics. From a single purified Ar-KM preparation, we identified three distinct populations: mature DNA-filled virions, open-nozzle particles with ejection proteins extending from the tail, and closed-nozzle empty particles. Near-atomic-resolution reconstructions of all three states enabled us to build atomic models for eleven structural proteins. The mature virion revealed the pre-ejection conformation of three ejection proteins, gp41, gp42, and gp43, homologous to coliphage T7's gp14, gp15, and gp16, respectively. Unlike T7, peptidoglycan hydrolase activity associated with the ejectosome resides in the gp15-like periplasmic tunnel protein gp42, whereas in T7 the lysozyme-like domain is located at the N-terminus of gp16, underscoring the structural plasticity and evolutionary mosaicity of ejection proteins. We further identified a short α-helical factor, gp34, present in eight copies at the mismatched interface between the portal barrel and gp41. Gp34 forms a cage within the nozzle, acting as a molecular wedge that stabilizes the open conformation and permits gp41 to assemble into a hexameric channel during ejection. Evolutionarily, gp34 appears to be an ortholog of the essential gene gp7.3 in phage T7 and is conserved across sequenced phiKMV-like phages. We propose that this protein functions as an ejection protein assembly factor, stabilizing the open nozzle during infection and allowing the coordinated exit of ejection proteins and their assembly into a DNA-ejectosome.

Pseudomonas phages↗
PubMed2004

The use of degenerate-primed random amplification of polymorphic DNA (DP-RAPD) for strain-typing and inferring the genetic similarity among closely related viruses.

Often it is necessary to distinguish among strains of closely related viruses, as well as infer the genetic relatedness within large groups of viruses. Current methods for strain-typing viruses are time-consuming, require significant quantities of extracted DNA, and/or may require a priori genetic information. In this study we modified random amplification of polymorphic DNA (RAPD) by using a degenerate primer to produce unique and reproducible banding patterns from viral genomes. In the degenerate-primed RAPD analysis (DP-RAPD), a selection of algal virus and bacteriophage strains were profiled that encompassed an array of genome sizes and virus families. Closely related viruses (e.g. strains infecting Micromonas pusilla) generated similar, yet unique DP-RAPD patterns that could be readily distinguished from viruses within the same family (Phycodnaviridae) infecting a Chlorella-like alga. As well, marine vibriophage from the families Myoviridae, Siphoviridae, and Podoviridae showed high diversity and were distinct from coliphage and cyanophage. Contamination of host DNA, even at levels above those that would normally be encountered, did not interfere with the viral patterns. These findings describe a rapid, PCR-based tool for strain-typing viral isolates that allows inferences to be made on genetic relatedness within groups of closely related viruses.

Caudovirales↗
PubMed2005

Enumeration of bacteriophages in water by different laboratories of the European Union in two interlaboratory comparison studies.

As part of a European project on bacteriophages in bathing waters two interlaboratory comparison studies were carried out (May 1997 and March 1998). During these studies phage reference materials as well as naturally polluted standard samples were analysed in 16 European laboratories. Three groups of bacteriophages were tested using standardised methods: somatic coliphages, F-specific RNA-phages and phages of Bacteroides fragilis. Many of the participating laboratories applied one or more of the phage methods for the first time, after a one-week training session in a central laboratory. Nevertheless, the values of repeatability (r=1.35-1.38 calculated on log(10)-scale) and reproducibility (R=1.52-2.04 calculated on log(10)-scale) when analysing phage reference materials were close to the theoretical optimum for a Poisson distribution. When analysing the naturally polluted samples more variation in results within and between laboratories was found (r=1.63-2.34; R=3.10-5.72), in comparison with the results obtained with the pure phage reference materials.

Bacteriophages↗
PubMed2004

UV disinfection of indigenous aerobic spores: implications for UV reactor validation in unfiltered waters.

Conventional validation testing of UV reactors use cultured microorganisms spiked into test water flowing through a reactor. These tests are limited by the microbe titer it is possible to grow, thus limiting the size of the reactor it is possible to validate. The goal of this study was to examine the UV inactivation of indigenous aerobic spores naturally occurring in raw/unfiltered water supplies and to assess their use as an alternative indicator for validation testing of UV reactor performance, specifically for unfiltered water supplies planning large UV reactors. These spores were found in all raw waters tested in concentrations ranging between 20 and 12,000 CFU/100 mL and were very resistant to UV irradiation compared to a range of different microbes in the literature (i.e. adenovirus, MS-2 coliphage, and Cryptosporidium parvum). The inactivation of indigenous natural aerobic spores followed first-order kinetics with an inactivation coefficient ranging between 0.013 and 0.022 cm2/mJ with a high correlation coefficient. It was determined that naturally occurring aerobic spores, well characterized with respect to UV 253.7 nm inactivation, can be a useful tool when validating plant performance, and might also be used as a regular monitor of UV fluence and performance in a water treatment plant.

Bacteria, Aerobic↗
PubMed2004

The use of microbial tracers to monitor seasonal variations in effluent retention in a constructed wetland.

Effluent retention in a constructed wetland was determined using both microbial and chemical tracers. Seasonal variation in effluent retention was the main focus of the study. The biotracers used in the study were the coliphage MS2, a bacteriophage of Enterobacter cloacae and antibiotic resistant endospores of Bacillus globigii. Two separate tracer runs were conducted, Winter high flow (January 2002) and Summer low flow (June 2002). The three biotracers were evaluated simultaneously on both occasions, with the commonly used chemical tracer, rhodamine WT, a bright red fluorescent dye, being evaluated during the final experiment. The Winter tracer run was conducted during a typical Winter storm, with a mean effluent discharge of 4.1 ls(-1). Tracer recovery was 98% MS2, 91% Ent. cloacae phage and 2% endospore. Effluent retention was estimated at between 2 and 4 h at 90% phage tracer recovery. The Summer tracer run was conducted at a typical site operating discharge rate of 0.8 ls(-1). Tracer recovery was 23% MS2, 36% Ent. cloacae phage, 8% rhodamine and 14% for the endospores. Effluent retention was estimated at between 11 and 18 h at 90% of phage tracer recovery. Initial results are encouraging and indicate bacteriophage to have further potential as tracing agents in wetlands.

Bacillus↗
PubMed2005

Feasibility of the silver-UV process for drinking water disinfection.

A synergistic effect between cationic silver and UV radiation (silver-UV disinfection) has been observed that can appreciably enhance inactivation of viruses. The purpose of this work was to assess the feasibility of this technique for drinking water disinfection and evaluate the effects of selected impurities, found in fresh water, and common parameters on inactivation of the coliphage MS-2 with the silver-UV process. Turbidity (kaolin), calcium hardness, carbonate alkalinity, and pH did not significantly degrade inactivation. Inactivation was reduced in the presence of chloride, at concentrations greater than 30 mg/L, and in water samples with UV-254 absorbance values greater than ca. 0.1 cm(-1). Inactivation of MS-2 with silver-UV disinfection was also reduced at high phosphate concentrations (above ca. 5 mM). Silver-UV inactivation of MS-2 increased with increases in temperature between 10 and 20 degrees C. Silver-UV inactivation of MS-2 was increased by greater than 1-log over UV alone, in two untreated fresh water sources, which indicates that silver-UV may be a viable treatment technology. An assessment of operation and management costs suggests that an increase in inactivation of MS-2 with silver-UV disinfection could be economically beneficial.

Chlorides↗
PubMed2003

Determinants that target the integrase of phage HK022 into the mammalian nucleus.

The integrase (Int) protein of coliphage HK022 catalyzes the site-specific integration and excision of the phage into and from its Escherichia coli host chromosome. Int expressed from a plasmid in COS1 monkey cells is localized in the nucleus, as is a fusion protein between Int and the green fluorescent protein (GFP). Mutation analysis of the GFP-Int fusion has revealed in Int two regions of positively charged amino acid residues that cooperate in the nuclear localization. One region harbors residues Arg90 and Arg93. The other, which spans residues 307-340 belongs to the catalytic domain of Int, is rich in basic residues and is strongly conserved within the Int protein family. Being localized in the nucleus renders Int of HK022 as a potential recombinase for site-specific gene manipulations in mammals.

Amino Acid Sequence↗
PubMed1984

Recombinational hotspot activity of Chi-like sequences.

Chi sites, consisting of the nucleotide octamer 5' G-C-T-G-G-T-G-G 3', stimulate coliphage lambda recombination mediated by the Escherichia coli RecBC recombination pathway. In a sensitive genetic assay using phage lambda crosses, three of four Chi-like sequences tested, namely 5' A-C-T-G-G-T-G-G 3', 5' G-T-T-G-G-T-G-G 3' and 5' G-C-T-A-G-T-G-G 3', had about 6%, 11% and 38% of full Chi activity, respectively. We conclude that certain Chi-like sequences manifest a spectrum of recombinational hotspot activities and may account for RecBC-mediated generalized recombination of lambda lacking Chi sites.

Alleles↗
PubMed2001

Assessment and impact of microbial fecal pollution and human enteric pathogens in a coastal community.

The goals of this study were to assess watersheds impacted by high densities of OSDS (onsite sewage disposal systems) for evidence of fecal contamination and evaluate the occurrence of human pathogens in coastal waters off west Florida. Eleven stations (representing six watersheds) were intensively sampled for microbial indicators of fecal pollution (fecal coliform bacteria, enterococci, Clostridium perfringens and coliphage) and the human enteric pathogens, Cryptosporidium, Giardia, and enteroviruses during the summer rainy season (May-September 1996). Levels of all indicators ranged between < 5 and > 4000 CFU/100 ml. Cryptosporidium and Giardia were detected infrequently (6.8% and 2.3% of samples tested positive, respectively). Conversely, infectious enteroviruses were detected at low levels in 5 of the 6 watersheds sampled. Using cluster analysis, sites were grouped into two categories, high and low risks, based on combined levels of indicators. These results suggest that stations of highest pollution risk were located within areas of high OSDS densities. Furthermore, data indicate a subsurface transport of contaminated water to surface waters. The high prevalence of enteroviruses throughout the study area suggests a chronic pollution problem and potential risk to recreational swimmers in and around Sarasota Bay.

Bacteria↗
PubMed2003

The complete sequence of marine bacteriophage VpV262 infecting vibrio parahaemolyticus indicates that an ancestral component of a T7 viral supergroup is widespread in the marine environment.

The 46,012-bp sequence of the marine bacteriophage VpV262 infecting the bacterium Vibrio parahaemolyticus is reported. The VpV262 sequence reveals that it is a distant relative of marine Roseophage SIO1, and an even more distant relative of coliphage T7. VpV262 and SIO1 appear to represent a widespread marine phage group that lacks an RNA polymerase gene and is ancestral to the T7-like phages. We propose that this group together with the T7-like phages be designated as the T7 supergroup. The ancestral head structure gene module for the T7 supergroup was reconstructed by using sensitive biased Psi-blast searches supplemented by statistical support derived from gene order. In the early and replicative segments, these phages have participated in extensive interchange with the viral gene pool. VpV262 carries a different replicative module than SIO1 and the T7-like phages.

Bacteriophage T7↗
PubMed2003

Microbial contamination of two urban sandstone aquifers in the UK.

Development of urban groundwater has historically been constrained by concerns about its quality. Rising urban water tables and overabstraction from rural aquifers in the UK have led to a renewed interest in urban groundwater, particularly the possibility of finding water of acceptable quality at depth. This study assessed the microbial quality of groundwater collected from depth-specific intervals over a 15-month period within the Permo-Triassic Sherwood Sandstone aquifers underlying the cities of Nottingham and Birmingham. Sewage-derived bacteria (thermotolerant coliforms, faecal streptococci and sulphite-reducing clostridia) and viruses (enteroviruses, Norwalk-like viruses, coliphage) were regularly detected to depths of 60 m in the unconfined sandstone and to a depth of 91 m in the confined sandstone. Microbial concentrations varied temporally and spatially but increased frequency of contamination with depth coincided with geological heterogeneities such as fissures and mudstone bands. Significantly, detection of Norwalk-like viruses and Coxsackievirus B4 in groundwater corresponded with seasonal variations in virus discharge to the sewer system. The observation of low levels of sewage-derived microbial contaminants at depth in the Triassic Sandstone aquifer is explained by the movement of infinitesimal proportions of bulk (macroscopic) groundwater flow along preferential pathways (e.g., fissures, bedding planes). The existence of very high microbial populations at source (raw sewage) and their extremely low detection limits at the receptor (multilevel piezometer) enable these statistically extreme (microscopic) flows to be traced. Rapid penetration of microbial contaminants into sandstone aquifers, not previously reported, highlights the vulnerability of sandstone aquifers to microbial contamination.

Cities↗