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PubMed1976

Cleavage maps of the filamentous bacteriophages M13, fd, fl, and ZJ/2.

The replicative form DNAs of bacteriophage M13, fd, f1, and ZJ/2 were found to be sensitive to cleavage by the restriction endonucleases endoR-HapII, endoR-HaeII, endoR-HaeIII, endoR-HindII, endoR-AluI, endoR-Hha, and endoR-Hinf. With respect to M13 DNA the number of cleavage sites varied from 21 for endoR-Hinf, 18 for endoR-AluI, 15 for endoR-Hha, 13 for endoR-HapII, 10 for endoR-HaeIII, 3 for endoR-HaeII, to only a single site for endoR-HindII. In contrast to M13, fd and f1, the ZJ/2 DNA molecule was not cleaved by the endoR-HindII endonuclease. No cleavage site on either phage DNA was detected for the endonucleases endoR-Hsu, endoR-EcoRI and endoR-Sma. When compared with M13 DNA, several differences were noted in the number and size of cleavage products obtained with DNA of phage fd, f1, and ZJ/2. From the results of these analyses, using the M13 enzyme cleavage maps as a reference, the endoR-HapII, endoR-HaeII, endoR-HaeIII, endoR-HindII and endoR-AluI maps of phage fd, f1, and ZJ/2 could be constructed. As is expected for very closely related phages, the enzyme cleavage patterns exhibit a high degree of homology. Phage f1 and ZJ/2 are most related since an identical pattern was obtained with seven different restriction endonucleases. Evidence is provided also that f1 is more similar to M13 than to fd. Furthermore, characteristic differences exist within the endoR-Hinf enzyme cleavage pattern of all the four phages tested. Digestion of phage DNA with this enzyme, therefore, provides a new and sensitive method of distinguishing these closely related filamentous coliphages .

Chromosome Mapping↗
PubMed1982

Nucleotide sequence of bacteriophage f1 DNA.

The nucleotide sequence of the DNA of the filamentous coliphage f1 has been determined. In agreement with earlier conclusions, the genome was found to comprise 6,407 nucleotides, 1 less than that of the related phage fd. Phage f1 DNA differs from that of phage M13 by 52 nucleotide changes, which lead to 5 amino acid substitutions in the corresponding proteins of the two phages, and from phage fd DNA by 186 nucleotide changes (including the single-nucleotide deletion), which lead to 12 amino acid differences between the proteins of phages f1 and fd. More than one-half of the nucleotide changes in each case are found in the sequence of 1,786 nucleotides comprising gene IV and the major intergenic region between gene IV and gene II. The sequence of this intergenic region (nucleotides 5501 to 6005) of phage f1 differs from the sequence reported by others through the inclusion of additional single nucleotides in eight positions and of a run of 13 nucleotides between positions 5885 and 5897, a point of uncertainty in the earlier published sequence. The differences between the sequence of bacteriophage f1 DNA now presented and a complete sequence for the DNA previously published by others are discussed, and the f1 DNA sequence is compared with those of bacteriophages M13 and fd.

Amino Acid Sequence↗
PubMed1987

Interference with viral infection by defective RNA replicase.

RNA-dependent RNA and DNA polymerases have a conserved segment, Tyr-X-Asp-Asp (G. Karmer and P. Argos, Nucleic Acids Res. 12:7269-7282, 1984). To investigate the function of this segment, we changed the Gly residue at position 357 in the conserved sequence Tyr-356-Gly-357-Asp-358-Asp-359 of the replicase of RNA coliphage Q beta to Ala, Ser, Pro, Met, or Val and examined the replicase activity in vivo. Cells carrying the variant plasmids lost the replicase activity and severely inhibited the proliferation of phage Q beta (group III) and related phage SP (group IV) by suppressing phage RNA synthesis. In contrast, substitution of the Gly residue at 390 showed only a slight inhibitory effect, although replicase activity was also lost. These results suggest that the cells harboring an altered replicase at the conserved segment can interfere specifically with the wild-type phage and different but related phage infections.

Coliphages↗
PubMed1971

Isolation and characterization of an Escherichia coli bacteriophage requiring cell wall galactose.

A new coliphage, designated U3, has been selected for the ability to discriminate the presence of galactose in the cell wall of Escherichia coli. U3 attacks E. coli K-12 cells that are able to incorporate galactose into their cell walls, but mutants blocked in the synthesis of uridine diphosphogalactose, the precursor of cell wall galactose, are completely resistant to the phage. U3 is a small, tail-less, approximately spherical phage resembling phiX174 in its physical properties. Its diameter by electron microscopy is 21 to 22 nm, and its particle weight is approximately 4 x 10(6) daltons. Like phiX174, U3 appears to have a single-stranded deoxyribonucleic acid genome and has at least four cistrons.

Adsorption↗
PubMed1987

Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae.

The procaryotic cre-lox site-specific recombination system of coliphage P1 was shown to function in an efficient manner in a eucaryote, the yeast Saccharomyces cerevisiae. The cre gene, which codes for a site-specific recombinase, was placed under control of the yeast GALI promoter. lox sites flanking the LEU2 gene were integrated into two different chromosomes in both orientations. Excisive recombination at the lox sites (as measured by loss of the LEU2 gene) was promoted efficiently and accurately by the Cre protein and was dependent upon induction by galactose. These results demonstrate that a procaryotic recombinase can enter a eucaryotic nucleus and, moreover, that the ability of the Cre recombinase to perform precise recombination events on the chromosomes of S. cerevisiae is unimpaired by chromatin structure.

Coliphages↗
PubMed1993

Mechanisms of genome propagation and helper exploitation by satellite phage P4.

Temperate coliphage P2 and satellite phage P4 have icosahedral capsids and contractile tails with side tail fibers. Because P4 requires all the capsid, tail, and lysis genes (late genes) of P2, the genomes of these phages are in constant communication during P4 development. The P4 genome (11,624 bp) and the P2 genome (33.8 kb) share homologous cos sites of 55 bp which are essential for generating 19-bp cohesive ends but are otherwise dissimilar. P4 turns on the expression of helper phage late genes by two mechanisms: derepression of P2 prophage and transactivation of P2 late-gene promoters. P4 also exploits the morphopoietic pathway of P2 by controlling the capsid size to fit its smaller genome. The P4 sid gene product is responsible for capsid size determination, and the P2 capsid gene product, gpN, is used to build both sizes. The P2 capsid contains 420 capsid protein subunits, and P4 contains 240 subunits. The size reduction appears to involve a major change of the whole hexamer complex. The P4 particles are less stable to heat inactivation, unless their capsids are coated with a P4-encoded decoration protein (the psu gene product). P4 uses a small RNA molecule as its immunity factor. Expression of P4 replication functions is prevented by premature transcription termination effected by this small RNA molecule, which contains a sequence that is complementary to a sequence in the transcript that it terminates.

Amino Acid Sequence↗
PubMed1975

The immune response of phiX 174 in man. III. Evidence for an association between hyposplenism and immunodeficiency in patients with coeliac disease.

The ability of patients with coeliac disease to produce primary and secondary antibody in response to a new antigen has been tested. Eight patients with coeliac disease were injected intravenously with the coliphage phiX 174. Antibody levels were measured throughout the primary response. On day 28 a second injection was given and the secondary response was also studied in detail. The production of antibody in the primary response is lower than in a group of normal subjects. The secondary response is severely reduced and the difference between the coeliac group and controls is highly significant. The secondary response in the coeliac patients contains a much higher proportion of IgM antibody than normal. These abnormalities correlate approximately with clinical status and with hyposplenism. It is concluded that in patients with coeliac disease the ability to produce antibody is impaired, with a defect in switching from IgM to IgG antibody.

Adult↗
PubMed1981

Genetic studies of H group plasmids by bacteriophage P1 transduction.

Bacteriophage P1 transduction was used to study the incompatibility group H1 plasmid pRG1251, molecular weight 120 x 10(6), and the incompatibility group H2 plasmid pSD114, molecular weight 166 x 10(6). The order of resistance (R) determinants on pSD114 was deduced from transduction and segregation experiments to be chloramphenicol-tetracycline-kanamycin-streptomycin. Resistance to tellurium and to coliphages, which are properties also encoded by many H2 plasmids, were not transduced with the other markers. On pRG1251, the ampicillin and tetracycline resistance markers appear to be located together, as do the chloramphenicol, streptomycin, and sulfamethoxazole resistance markers. Frequently, blocks of R determinants were transposed to the P1 genome or to the Escherichia coli chromosome. P1 DNA was isolated which carried the chloramphenicol, streptomycin, and sulfamethoxazole markers from pRG1251 and had a molecular weight of 64 x 10(6). Other P1 prophages carried R determinants from pSD114 and had molecular weights of 86 x 10(6). A plasmid of molecular weight 124 x 10(6) was also isolated which contained incompatibility determinants from P1 (incompatibility group Y) and from the H2 group plasmid. The mechanism of formation of these unusual plasmid species is discussed.

Anti-Bacterial Agents↗
PubMed1982

Incidence of plasmid DNA in Salmonella strains isolated from clinical sources in Ontario, Canada, during 1979 and 1980.

Gel electrophoresis of DNA from 70 clinical strains of Salmonella revealed a heterogenous plasmid population. Plasmid DNA, ranging in molecular weight from 1.4 X 10(6) to 145 X 10(6), was demonstrated in 26 of 32 antibiotic-resistant strains. Several resistant strains carried up to six plasmids; however, of these, five strains which were multiply resistant contained a single plasmid of molecular weight 54 X 10(6) to 145 X 10(6). Only one incompatibility group H2 (IncH2) plasmid (pDT28) was detected in a strain of S. heidelberg; thus, this represents a reduction in the prevalence of these plasmids in Ontario Salmonella strains since 1974. The pDT28 plasmid resembled other IncH2 plasmids by its high molecular weight (145 X 10(6) ) and by virtue of its temperature-sensitive mode of transfer, resistance to tellurium, and inhibition of coliphage development. Of the 38 antibiotic-susceptible Salmonella strains, approximately half contained plasmids, ranging in molecular weight from 1.4 X 10(6) to 60 X 10(6). The plasmid-containing antibiotic-susceptible strains carried either a group of two to four small plasmids, with molecular weights less than 4.5 X 10(6), or a single large plasmid of molecular weight 23 X 10(6) or 60 X 10(6).

Anti-Bacterial Agents↗
PubMed1987

Effects of temperature upon the cell-free translation system from Coxiella burnetii.

The rate and extent of coliphage Q beta RNA translation by cell-free extracts prepared from Coxiella burnetii were studied. When translations were conducted at temperatures elevated above 37 degrees C, both polypeptide elongation and frequency of initiation were by comparison increased. The ratios of products synthesized from the polycistronic phage mRNA also changed upon increases in translation temperature, especially at 45 degrees C. Although the organism is a moderate acidophile, initiation of protein synthesis in extracts did not occur below pH 6.2, and was superior when the pH was 6.8-7.2. The results are discussed in context with the known physiological characteristics of this obligate intracellular bacterium.

Animals↗
PubMed1989

Isolation of marine bacteria with antiviral properties.

We report in this study the isolation of marine bacteria with antiviral properties that have been tentatively classified as Moraxella. These bacteria retained their virucidal capacity after prolonged subcultivation in the laboratory. The virus-inactivating agent could not be separated from the viable marine bacteria, indicating that the active agent(s) either remains associated to the microorganisms or has a very short lifetime, or both. The antiviral capacity of the isolated microorganisms was highly specific for poliovirus. No virucidal effect was observed against other strains of enteroviruses, such as Coxsackie and ECHO virus, rotavirus SA11, or bacteriophages proposed as indicators of the virological quality of water, such as coliphage f2 and bacteriophage B40-8, which infects Bacteroides fragilis.

Bacteria↗
PubMed1991

[Bacteriological quality of groundwaters in cemeteries].

Groundwater samples collected by piezometers from three cemeteries in geologically distinct areas of S. Paulo and Santos, Brazil, were analysed in order to determine their hygienic and sanitary conditions. Fecal coliformes, fecal streptococci, sulfite reducer clostridia and Salmonella were searched for the purpose of evaluating sanitary conditions, and total coliforms, heterotrophic bacteria, proteolitic and lipolitic microorganisms for evaluating hygienic conditions. In some samples, nitrate levels were also determined. It was discovered that these waters do not present adequate sanitary and hygienic conditions and that, in some cases, nitrate levels were extremely high (75.7 mg/l). In most samples, higher levels of fecal streptococci and sufite reducer clostridia than fecal coliforms were detected, which seems to show that the two former indicators would be more appropriate for evaluating the sanitary conditions of this kind of water. Salmonella were detected in only one of 44 samples analysed and coliphages in none. In the statistical analysis, the correlation matrix showed significant correlations among three fecal pollution indicators, as well as among anaerobic and aerobic heterotrophs and lipolitic bacteria. A direct relationship between the deterioration of water quality and the geological and hydrogeological conditions of the environment studied was observed. When cemeteries are constructed these conditions should, therefore, be taken into consideration.

Clostridium↗
PubMed2005

Application of microbial risk assessment to the development of standards for enteric pathogens in water used to irrigate fresh produce.

Microbial contamination of the surfaces of cantaloupe, iceberg lettuce, and bell peppers via contact with irrigation water was investigated to aid in the development of irrigation water quality standards for enteric bacteria and viruses. Furrow and subsurface drip irrigation methods were evaluated with the use of nonpathogenic surrogates, coliphage PRD1, and Escherichia coli ATCC 25922. The concentrations of hepatitis A virus (HAV) and Salmonella in irrigation water necessary to achieve a 1:10,000 annual risk of infection, the acceptable level of risk used for drinking water by the U.S. Environmental Protection Agency, were calculated with a quantitative microbial risk assessment approach. These calculations were based on the transfer of the selected nonpathogenic surrogates to fresh produce via irrigation water, as well as previously determined preharvest inactivation rates of pathogenic microorganisms on the surfaces of fresh produce. The risk of infection was found to be variable depending on type of crop, irrigation method, and days between last irrigation event and harvest. The worst-case scenario, in which produce is harvested and consumed the day after the last irrigation event and maximum exposure is assumed, indicated that concentrations of 2.5 CFU/100 ml of Salmonella and 2.5 x 10(-5) most probable number per 100 ml of HAV in irrigation water would result in an annual risk of 1:10,000 when the crop was consumed. If 14 days elapsed before harvest, allowing for die-off of the pathogens, the concentrations were increased to 5.7 x 10(3) Salmonella per 100 ml and 9.9 x 10(-3) HAV per 100 ml.

Agriculture↗
PubMed1978

Adjuvant effect of a peptidoglycan attached covalently to a synthetic antigen provoking anti-phage antibodies.

The synthetic antigen denoted P2-A--L, comprising the fragment P2 of the coat protein of MS-2 coliphage attached to multichain poly-DL-alanine, served for the immunization of guinea-pigs. Immunization was carried out either in phosphate buffered saline (PBS) or in Freund's incomplete adjuvant (FIA) in the presence or absence of a small molecular weight peptidoglycan prepared from Bacillus megaterium, which was checked for its adjuvant effect. The various antisera were assessed by their capacity to neutralize MS-2 bacteriophage viability. When injected in PBS or FIA, P2-A--L did not elicit any measurable anti-phage activity. Addition of the peptidoglycan by simple mixing did not bring about a significant increase in antibody production. However, when the peptidoglycan was chemically linked to the P2-A--L conjugate, it had a marked adjuvant effect when the material was administered in FIA, almost identical to the extent of the effect of Freund's complete adjuvant.

Adjuvants, Immunologic↗
PubMedDate not supplied

[Microbiological quality of the Catania coastal sea water].

This study was carried out from 1997 to 1998 along a selected coastal area near Catania to ascertain bacteriological and virological quality of marine waters. 44 seawater samples, collected from 4 stations, were assayed for the presence of total and fecal coliforms, fecal streptococci, coliphages, Salmonellae and enteric viruses. Two stations localized at canal outfalls showed high levels of fecal pollution. The other stations were of good microbiological quality and showed a limited number of samples exceeding the standards laid down as guide values for bathing waters by Italian normative during the bathing period. Salmonellae were isolated in 8 out of 44 sea water samples (18%). Their presence was ascertained mainly in samples of the two polluted stations. Enteroviruses were not isolated. Enteric viruses such as Reoviruses were isolated from all stations, in 12 out of 44 samples (27%). The presence of these viruses was ascertained only during autumnal and winter seasons. The results of this study showed that, notwithstanding some stations showed high levels of bacteriological indicators of fecal pollution and presence of Salmonellae, enteroviruses growing on cell cultures were not isolated. Reoviruses confirmed their high diffusion in marine waters.

Bathing Beaches↗
PubMed2002

Comparison of the quantity and quality of the microbiological content of sludge in countries with low and high content of pathogens.

The parasites present in the residual sludge generate by the diverse treatment processes of residual water vary, depending on the socioeconomic and epidemiological conditions of different communities. And although data exist at the global level regarding the microbiological quality of sludge, these data are practically nonexistent in developing countries. Therefore, this project compares the content of the four principal groups of parasites in residual sludge from the United States and Mexico. The marked differences between the orders of magnitude found in the two countries (coliphages 10(3)-10(6) and 1.3 x 10(5) PFU/gTS, fecal coliforms 10(7)-10(10) and 10(7), Salmonella typhi 10(6)-10(8) and 10(3) MPN/gTS, Giardia lamblia 10(2)-10(4) and 10(2) cysts/gTS and helminth ova from 73-177 and <1 to 10 viable HO/gTS, respectively) will permit proposing real, effective and economical stabilization processes (undoubtedly different from those of industrialized countries), and will thus take advantage of the benefits represented by the reuse and disposal of sludge, in addition to developing regulations in agreement with the conditions of each region.

Animals↗
PubMed2004

Pathogen removal efficiency from UASB + BF effluent using conventional and UV post-treatment systems.

The aim of this study was to verify the efficiency of removal of microorganisms in effluents of a Wastewater Treatment Plant (WWTP) comprising an association of a UASB reactor followed by three submerged aerated biofilters (BAF) and one tertiary filter. The WWTP designed to treat domestic wastewater from a population of 1,000 inhabitants showed high removal efficiency for organic matter and suspended solids. Helminth eggs were also efficiently removed from the tertiary effluent and were found in the sludge from the UASB reactor; however, removal of bacteria in this system was very low. To enhance the efficiency of the system, the effluent from tertiary filters was submitted to UV disinfection in a real scale reactor. Our results showed that UV irradiation was very effective at lowering the concentrations of E. coli, thermotolerant coliforms and coliphages to acceptable levels for agricultural reuse. Salmonella spp. and helminth eggs were seeded into the tertiary effluent before passing through the UV reactor. Salmonella was not found in the final effluent, but helminth eggs were not completely inactivated by UV irradiation and viable eggs were detected after 28 d of incubation.

Agriculture↗