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PubMed2003

Disinfection efficiency of peracetic acid, UV and ozone after enhanced primary treatment of municipal wastewater.

The City of Montreal Wastewater Treatment Plant uses enhanced physicochemical processes (ferric and/or alum coagulation) for suspended solids and phosphorus removal. The objective of this study was to assess the ability of peracetic acid (PAA), UV, or ozone to inactivate the indicator organisms fecal coliforms, Enterococci, MS-2 coliphage, or Clostridium perfringens in the effluent from this plant. PAA doses to reach the target fecal coliform level of 9000 CFU/100mL exceeded 6 mg/L; similar results were obtained for enterococci, and no inactivation of Clostridium perfringens was observed. However a 1-log reduction of MS-2 occurred at PAA doses of 1.5 mg/L and higher. It was expected that this effluent would have a high ozone demand, and would require relatively high UV fluences, because of relatively high effluent COD, iron and suspended solids concentrations, and low UV transmittance. This was confirmed herein. For UV, the inactivation curve for fecal coliforms showed the typical two-stage shape, with the target of 1000 CFU/100 mL (to account for photoreactivation) occurring in the asymptote zone at fluences >20 mJ/cm(2). In contrast, inactivation curves for MS-2 and Clostridium perfringens were linear. Clostridium perfringens was the most resistant organism. For ozone, inactivation was already observed before any residuals could be measured. The transferred ozone doses to reach target fecal coliform levels ( approximately 2-log reduction) were 30-50 mg/L. MS-2 was less resistant, but Clostridium perfringens was more resistant than fecal coliforms. The different behaviour of the four indicator organisms studied, depending on the disinfectant, suggests that a single indicator organism might not be appropriate. The required dose of any of the disinfectants is unlikely to be economically viable, and upstream changes to the plant will be needed.

Clostridium perfringens↗
PubMed2001

Optimisation and standardisation of a method for detecting and enumerating bacteriophages infecting Bacteroides fragilis.

A method for the detection and enumeration of bacteriophages infecting Bacteroides fragilis has been standardised. The recommended host strain is RYC2056 (ATCC 700786) because of the relatively high counts (10(4)-10(5) PFU/100ml) that it recovers in sewage from very different geographical areas. The addition of 0.25% bile to the culture and assay media and the manipulation of the host strain under strict anaerobic conditions resulted in a significant increase (more than 100%) in the number of phages detected. No other changes in the media and culture conditions resulted in changes in the phage counts detected. However, these increases do not justify changing the culture conditions and media described, taking into consideration that bile renders the media cloudy making it difficult to follow the host growth and that most laboratories do not have the facilities to work under strict anaerobic conditions. Nalidixic acid (100 microg/ml) and kanamycin (100 microg/ml) in the assay medium significantly reduce the background flora from polluted samples without affecting the phage counts. Freezing cultures just before the end of the log-phage growth at (-70+/-10) degrees C with BSA-sucrose as cryoprotector, storing of 1-2 ml in glass vials at (-70+/-10) degrees C and using them directly to inoculate fresh broth allows the obtention of cultures ready for phage enumeration in about 2.5 h. All these developments have been incorporated into a procedure that makes the method for detecting phages infecting B. fragilis as workable as the standardised methods available for the detection of coliphages.

Anaerobiosis↗
PubMed2003

Comparative analysis of viral pathogens and potential indicators in shellfish.

Shellfish can be responsible of outbreaks of infectious diseases and current health measures do not guarantee the absence of viral pathogens in this product. Here we examine the presence of pathogenic viruses and potential indicators in shellfish in a comparative analysis.Sixty shellfish samples collected in three areas with different levels of faecal contamination were analysed for Escherichia coli, total coliforms, Clostridium perfringens, somatic coliphages, F-specific phages of RNA (F-RNA), bacteriophages infecting Bacteroides fragilis RYC2056, human adenovirus, enterovirus and hepatitis A virus (HAV). Viruses were eluted in a glycine buffer at pH 10. The overall percentage of viral pathogens detected was 47% for human adenoviruses, 19% for enteroviruses and 24% for HAV. Since all the samples positive for enterovirus and HAV were also positives for human adenovirus, the latter may be considered useful as a molecular index of viral contamination in shellfish. No significant differences in the bioaccumulation of bacteria and bacteriophages for oysters or mussels were observed. It was found that the probability of detection of any of the pathogenic virus decreases as the temperature of shellfish growing waters increases. However, the probability of detecting viruses increases when phages of B. fragilis are found. Although more data are needed in order to fulfil the need of viral indicators for controlling the presence of human viruses in shellfish, the obtained results indicate that phages infecting B. fragilis RYC2056 could be a suitable group of bacteriophages to be used as an indicator of the presence of viruses in shellfish.

Adenoviruses, Human↗
PubMed1979

High resolution SEM of bacterial virus T7.

High resolution "low-loss" scanning electron microscopy is a relatively new technique which permits an investigator to examine structures that were formerly visualized exclusively by transmission electron microscopy [1]. This paper presents some images of intact bacterial virus T7, viewed at the ultrastructural level. Due to the high resolution capibility of this technique, and the demanding physical prerequisites for visualization of the specimen, current specimen preparation techniques were modified in order to permit 1--2 nm resolution in surface mode. Using this method of microscopy, it is possible to view clearly this small bacteriophage (the smallest of the T-coliphages), adsorbed to its host bacterium, in a scanning mode at magnification (and resolution) comparable to TEM without resorting to the use of replicas, or reconstruction of a two-dimensional image.

Capsid↗
PubMed2002

Specificity in DNA recognition by phage integrases.

The lambda-related (lambdoid) coliphages are related to one another by frequent natural recombination and maintain a high level of functional polymorphism for several activities of the phages. Arguments are presented that the polymorphism of the integration module results from selection (presumably frequency-dependent) for new (not improved) specificities of site recognition. Analysis of phages lambda and HK022 by Weisberg and collaborators previously showed that changes in five noncontiguous amino acids could switch site recognition specificity. Phage 21 and defective element e14, which integrate at the same site, differ in recognition specificity for both core and arm sites. In vitro assays of e14 and 21 insertion and excision confirm this conclusion. Inhibition by ds arm site oligonucleotides defines the sequence specificity more precisely.

Bacteriophage lambda↗
PubMed1998

A procedure for cloning genomic DNA fragments with increasing thermoresistance.

Genomic DNAs have been cleaved by restriction or sonication, and the resulting double-stranded fragments have been exposed to increasing temperatures. This treatment may induce the helix-coil transition either in a single or in several steps, depending on the size and composition of the duplexes. Eventually, a critical temperature is reached at which each duplex melts completely and the two constitutive single strands separate. A transition interval can thus be defined for each duplex by the temperature at which the earliest strand separation takes place and that at which the most resistant double-stranded core collapses. If solutions containing a mixture of DNA duplexes are exposed to temperatures within their transition intervals, three kinds of molecules should originate: (1) duplexes that have not yet initiated the melting phase; (2) duplexes that have undergone only partial melting; and (3) single strands that derive from fully melted duplexes. If the heated solutions are quickly cooled to 0 degreesC, only the molecules from the first two classes can be ligated to a compatibly ended vector and cloned: class (1) are intact duplexes, and class (2) are molecules that snap immediately back to fully duplex structures: both are double-stranded. Conversely, the single strands of class (3) may not reanneal and thus be neither ligated nor cloned. We have tested the procedure on restricted coliphage lambda DNA, in view of its compartmentalized organization and known sequence. Then, we have applied it to human genomic DNA fragmented by sonication. After cloning of the available duplexes in a bacterial plasmid, libraries of molecules endowed with a progressively higher thermoresistance can be prepared for thermodynamic and genomic studies.

Bacteriophage lambda↗
PubMed2003

Prophage insertion sites.

Insertion of viral DNA into host chromosomes is an ancient process essential for propagation in the proviral form. Many present-day bacteriophages insert at specific sites on the host chromosome. Insertion by two coliphage families (lambdoid and P4-like) is compared. For both families, insertion sites frequently lie within tRNA genes. The lambdoid phages insert at anticodon loops, whereas the p4-like phages insert in the TpsiC loops downstream from them. The association of both groups with tRNA genes suggests that the primordial insertion site of both groups may have been within a tRNA gene. The integrase proteins used in phage insertion may have originated at that stage, with subsequent diversification of specificity.

Attachment Sites, Microbiological↗
PubMed1999

Four dimers of lambda repressor bound to two suitably spaced pairs of lambda operators form octamers and DNA loops over large distances.

Transcription factors that are bound specifically to DNA often interact with each other over thousands of base pairs [1] [2]. Large DNA loops resulting from such interactions have been observed in Escherichia coli with the transcription factors deoR [3] and NtrC [4], but such interactions are not, as yet, well understood. We propose that unique protein complexes, that are not present in solution, may form specifically on DNA. Their uniqueness would make it possible for them to interact tightly and specifically with each other. We used the repressor and operators of coliphage lambda to construct a model system in which to test our proposition. lambda repressor is a dimer at physiological concentrations, but forms tetramers and octamers at a hundredfold higher concentration. We predict that two lambda repressor dimers form a tetramer in vitro when bound to two lambda operators spaced 24 bp apart and that two such tetramers interact to form an octamer. We examined, in vitro, relaxed circular plasmid DNA in which such operator pairs were separated by 2,850 bp and 2,470 bp. Of these molecules, 29% formed loops as seen by electron microscopy (EM). The loop increased the tightness of binding of lambda repressor to lambda operator. Consequently, repression of the lambda PR promoter in vivo was increased fourfold by the presence of a second pair of lambda operators, separated by a distance of 3,600 bp.

DNA, Viral↗
PubMed1997

Structure-activity relationship of a series of C-terminus modified aminoalkyl, diaminoalkyl- and anilino-containing analogues of the benzoic acid mustard distamycin derivative tallimustine: synthesis, DNA binding and cytotoxicity studies.

As part of our investigations into the design of more cytotoxic analogues of the experimental anticancer drug tallimustine, 1, C-terminus modified aminoalkyl-, 2a-c, diaminoalkyl-, 3, and anilino-containing, 4, derivatives have been synthesized. Compounds 2a-c differ by 2, 3, or 4 methylene units in the C-terminus, respectively. Results from an ethidium displacement study on poly(dA-dT), poly(dG-dC), calf thymus DNA and T4 coliphage DNA showed that compounds 2-4 interact in the minor groove of the polynucleotides with a preference for poly(dA-dT) over poly(dG-dC). Compound 4 bound more weakly to the DNAs than 2a-c and 3. Using a CD dilution assay compounds 2a-c and 3 were demonstrated to bind irreversibly to calf thymus DNA. The sequence selectivity by which compounds 2-4 alkylate DNA was demonstrated using a Taq polymerase stop assay. All the compounds alkylated preferentially at the 3'-purine residue in a 5'-TTTTGPu-3' sequence (Pu = A or G). This observed sequence specificity is similar to that of tallimustine and a related compound 5. At an equimolar concentration the aminoalkyl compounds 2a-c (2b > 2a > 2c), and diaminoalkyl compound 3 were more efficient at alkylating these sequences than the anilino compound 4. Following a one hour exposure of human chronic myeloid leukemia K562 cells, compounds 2b and 3 have lower IC50 values (1.64 microM and 3.03 microM, respectively) than tallimustine (5 microM) and similar values to a related compound 5 (2.2 microM). The order of cytotoxicity for all the compounds is 2b > 5 > 3 > 2a > 1 > 2c = 4. These results indicate that the cytotoxicities of these compounds are related to their relative ability to alkylate the consensus DNA binding sequence.

Alkylation↗
PubMed1998

Structural analysis of the Spiroplasma virus, SpV4: implications for evolutionary variation to obtain host diversity among the Microviridae.

BACKGROUND: Spiroplasma virus, SpV4, is a small, non-enveloped virus that infects the helical mollicute Spiroplasma melliferum. SpV4 exhibits several similarities to the Chlamydia phage, Chp1, and the Coliphages alpha 3, phi K, G4 and phi X174. All of these viruses are members of the Microviridae. These viruses have isometric capsids with T = 1 icosahedral symmetry, cause lytic infections and are the only icosahedral phages that contain single-stranded circular DNA genomes. The aim of this comparative study on these phages was to understand the role of their capsid proteins during host receptor recognition. RESULTS: The three-dimensional structure of SpV4 was determined to 27 A resolution from images of frozen-hydrated particles. Cryo-electron microscopy (cryo-EM) revealed 20, approximately 54 A long, 'mushroom-like' protrusions on the surface of the capsid. Each protrusion comprises a trimeric structure that extends radially along the threefold icosahedral axes of the capsid. A 71 amino acid portion of VP1 (the SpV4 capsid protein) was shown, by structural alignment with the atomic structure of the F capsid protein of phi X174, to represent an insertion sequence between the E and F strands of the eight-stranded antiparallel beta-barrel. Secondary structure prediction of this insertion sequence provided the basis for a probable structural motif, consisting of a six-stranded antiparallel beta sheet connected by small turns. Three such motifs form the rigid stable trimeric structures (mushroom-like protrusions) at the threefold axes, with hydrophobic depressions at their distal surface. CONCLUSIONS: Sequence alignment and structural analysis indicate that distinct genera of the Microviridae might have evolved from a common primordial ancestor, with capsid surface variations, such as the SpV4 protrusions, resulting from gene fusion events that have enabled diverse host ranges. The hydrophobic nature of the cavity at the distal surface of the SpV4 protrusions suggests that this region may function as the receptor-recognition site during host infection.

Amino Acid Sequence↗
PubMed2003

Isolation of Qbeta polymerase complexes containing mutant species of elongation factor Tu.

The RNA genome of coliphage Qbeta is replicated by a complex of four proteins, one of them being the translation elongation factor Tu. The role of EF-Tu in this RNA polymerase complex is still unclear, but the obligate presence of translationally functional EF-Tu in the cell hampers the use of conventional mutational analysis. Therefore, we designed a system based on affinity chromatography and could separate two types of complexes by placing an affinity tag on mutated EF-Tu species. Thus, we were able to show a direct link between the vital tRNA binding property of EF-Tu and polymerase activity.

Allolevivirus↗
PubMed2002

Entropic recoil separation of long DNA molecules.

A novel technique that can rapidly separate long-strand polymers according to length is presented. The separation mechanism is mediated by a confinement-induced entropic force at the abrupt interface between regions of vastly different configuration entropy. To demonstrate this technique, DNA molecules were partially inserted into a dense array of nanopillars (an entropically unfavorable region) using a pulsed electric field and allowed to relax to their natural state by removal of the field. Molecules of dissimilar lengths (T2 and T7 coliphage DNA) were inserted into this region in such a way that shorter molecules were fully inserted in this region, while longer molecules remained partially across the interface. The longer T2 molecules were observed to recoil entirely out of the pillar array, leaving the shorter T7 molecules inserted, and effecting separation of the two species in a single step. To show how this method can be used for separation of unknown samples, the inserting electric field was pulsed for progressively longer times, allowing passage of progressively longer molecules and producing the equivalent of a conventional electropherogram. The effects limiting resolution in this device are discussed, and the expected separating power of a multistage device is reported. The extracted resolution and running separation time compare favorably with current conventional separation techniques.

Algorithms↗
PubMed2002

Nine-analyte detection using an array-based biosensor.

A fluorescence-based multianalyte immunosensor has been developed for simultaneous analysis of multiple samples. While the standard 6 x 6 format of the array sensor has been used to analyze six samples for six different analytes, this same format has the potential to allow a single sample to be tested for 36 different agents. The method described herein demonstrates proof of principle that the number of analytes detectable using a single array can be increased simply by using complementary mixtures of capture and tracer antibodies. Mixtures were optimized to allow detection of closely related analytes without significant cross-reactivity. Following this facile modification of patterning and assay procedures, the following nine targets could be detected in a single 3 x 3 array: Staphylococcal enterotoxin B, ricin, cholera toxin, Bacillus anthracis Sterne, Bacillus globigii, Francisella tularensis LVS, Yersiniapestis F1 antigen, MS2 coliphage, and Salmonella typhimurium. This work maximizes the efficiency and utility of the described array technology, increasing only reagent usage and cost; production and fabrication costs are not affected.

Antigens↗
PubMed2004

Correlation of mass spectrometry identified bacterial biomarkers from a fielded pyrolysis-gas chromatography-ion mobility spectrometry biodetector with the microbiological gram stain classification scheme.

A pyrolysis-gas chromatography-ion mobility spectrometry (Py-GC-IMS) briefcase system has been shown to detect and classify deliberately released bioaerosols in outdoor field scenarios. The bioaerosols included Gram-positive and Gram-negative bacteria, MS-2 coliphage virus, and ovalbumin protein species. However, the origin and structural identities of the pyrolysate peaks in the GC-IMS data space, their microbiological information content, and taxonomic importance with respect to biodetection have not been determined. The present work interrogates the identities of the peaks by inserting a time-of-flight mass spectrometry system in parallel with the IMS detector through a Tee connection in the GC module. Biological substances producing ion mobility peaks from the pyrolysis of microorganisms were identified by their GC retention time, matching of their electron ionization mass spectra with authentic standards, and the National Institutes for Standards and Technology mass spectral database. Strong signals from 2-pyridinecarboxamide were identified in Bacillus samples including Bacillus anthracis, and its origin was traced to the cell wall peptidoglycan macromolecule. 3-Hydroxymyristic acid is a component of lipopolysaccharides in the cell walls of Gram-negative organisms. The Gram-negative Escherichia coli organism showed significant amounts of 3-hydroxymyristic acid derivatives and degradation products in Py-GC-MS analyses. Some of the fatty acid derivatives were observed in very low abundance in the ion mobility spectra, and the higher boiling lipid species were absent. Evidence is presented that the Py-GC-ambient temperature and pressure-IMS system generates and detects bacterial biochemical information that can serve as components of a biological classification scheme directly correlated to the Gram stain reaction in microorganism taxonomy.

Biomarkers↗
PubMed1992

Phosphorylation of Escherichia coli translation initiation factors by the bacteriophage T7 protein kinase.

The lytic coliphage T7 encodes a serine/threonine-specific protein kinase which supports viral reproduction under suboptimal growth conditions. Expression of the protein kinase in T7-infected Escherichia coli cells results in the phosphorylation of 30S ribosomal subunit protein S1, and initiation factors IF1, IF2, and IF3, as determined by high-resolution two-dimensional gel electrophoresis and specific immunoprecipitation analysis. Phosphorylation occurs either exclusively on threonine (IF1, IF3, S1) or on serine and threonine (IF2). There is no phosphorylation of these proteins in uninfected cells or in cells infected with T7 lacking the protein kinase function. Phosphorylation of the initiation factors coincides with the onset of T7 late protein synthesis, occurring 9-12-min postinfection. T7 late protein synthesis, otherwise inhibited in ColIb plasmid-containing cells, is specifically supported by expression of the protein kinase. These results provide the first evidence for the functional involvement of protein phosphorylation in the control of bacterial translation.

Electrophoresis, Polyacrylamide Gel↗
PubMed2001

Structure and organization of bacteriophage Pf3 probed by Raman and ultraviolet resonance Raman spectroscopy.

The Pseudomonas bacteriophage Pf3 is a long and narrow filament consisting of a covalently closed DNA single strand of 5833 bases sheathed by approximately 2500 copies of a 44-residue subunit. Ultraviolet resonance Raman spectra excited at 257, 244, 238, and 229 nm and off-resonance Raman spectra excited at 514.5 nm are reported for Pf3 in both H2O and D2O solutions. The key Raman bands are assigned to specific protein and DNA groups of the native virion assembly. The results are compared with proposed assembly models and Raman spectra recently reported for the isomorphous (class II) Pseudomonas phage Pf1 and the morphologically distinct (class I) coliphage fd [Wen, Z. Q., Overman, S. A., and Thomas, G. J. , Jr. (1997) Biochemistry 36, 7810-7820; Wen, Z. Q., Armstrong, A., and Thomas, G. J., Jr. (1999) Biochemistry 38, 3148-3156]. Surprisingly, deoxynucleosides of the packaged DNA genome of Pf3 adopt the same conformation (C3'-endo/anti) found for DNA packaged in the class I fd virion rather than that (C2'-endo/anti) associated with DNA in the isomorphous Pf1 virion. However, DNA base stacking in Pf3, as judged by Raman hypochromic effects, differs significantly from that occurring in either Pf1 or fd. Thus, the single-stranded DNA genomes of Pf3, Pf1, and fd are all organized differently within their respective capsids, implying that local subunit-DNA interactions may be important in determining the structure specific to each native assembly. The present study confirms a completely alpha-helical secondary structure for the Pf3 subunit and an unusual indolyl ring environment for the subunit tryptophan residue (Trp-38).

Amides↗
PubMed1987

Lambda cro repressor complex with OR3 DNA: 15N NMR observations.

15N NMR studies of the coliphage lambda cro repressor are presented. The protein has been uniformally labeled with 15N, and individual amino acids have been incorporated. Although the four C-terminal residues (63-66) were not located in the original crystallographic studies of the protein [Anderson, W.F., Ohlendorf, D.H., Takeda, Y., & Matthews, B.W. (1981) Nature (London) 290, 754], it has been proposed that the C-terminus is involved in DNA binding [Ohlendorf, D.H., Anderson, W.F., Fisher, R.G., Takeda, Y., & Matthews, B.W. (1982) Nature (London) 298, 718]. These experiments give direct verification of that proposal. [15N]Amide resonances are assigned for residues 56, 62, 63, and 66 in the C-terminus by enzymatic digestion and by 13C-15N double-labeling experiments. 15N[1H] nuclear Overhauser effects show that the C-terminus is mobile on a nanosecond time scale. Exchange experiments using distortionless enhancement via polarization transfer, which is sensitive to proton exchange on the 1/JNH (10 ms) time scale, indicate that the amide protons in the C-terminus are freely accessible to solvent. It is thus a flexible arm in solution. The binding of both specific operator and nonspecific DNA is shown to reduce both the mobility and the degree of solvent exposure of this arm. Two-dimensional 15N-1H correlation experiments using 15N-labeled cro reveal inconsistencies with previously reported 1H NMR assignments for the lysine amides [Weber, P.L., Wemmer, D.E., & Reid, B.R. (1985) Biochemistry 24, 4553]. This result suggests that those assignments require reexamination, illustrating the utility of 15N labeling for obtaining 1H resonance assignments of biomolecules. Furthermore, isomerization of the peptide bond of Pro-59, which has been previously suggested (Weber et al., 1985) and which would significantly affect the properties of the C-terminal arm, is shown to not occur.

Bacteriophage lambda↗
PubMed1981

Primary structure of the phage P22 repressor and its gene c2.

The amino acid sequence of the Salmonella phage P2 repressor and the DNA sequence of its gene c2 have been determined. Sequential Edman degradations on intact P22 repressor and repressor peptides generated by proteolytic and chemical cleavages have been overlapped to give approximately 97% of the complete protein sequence. Additionally, the nucleotide sequence of the P22 c2 repressor gene has been determined by DNA sequencing techniques. The DNA sequence and partial protein sequence are collinear and together define the complete amino acid sequence of P22 repressor. The repressor is a single-chain 216 amino acid polypeptide. Basic residues in the sequence tend to be clustered, and residues 9-20 are highly basic, containing five arginyl and three lysyl residues. The carboxyl-terminal 133 amino acids of the c2 repressor are homologous to the carboxyl-terminal sequence of the coliphage lambda cI repressor. The amino-terminal sequences of these two repressors show little similarity.

Amino Acid Sequence↗