Bacteriophage inactivation at the air-water-solid interface in dynamic batch systems. (1/413)

Bacteriophages have been widely used as surrogates for human enteric viruses in many studies on virus transport and fate. In this investigation, the fates of three bacteriophages, MS2, R17, and phiX174, were studied in a series of dynamic batch experiments. Both MS2 and R17 readily underwent inactivation in batch experiments where solutions of each phage were percolated through tubes packed with varying ratios of glass and Teflon beads. MS2 and R17 inactivation was the result of exposure to destructive forces at the dynamic air-water-solid interface. phiX174, however, did not undergo inactivation in similar studies, suggesting that this phage does not accumulate at air-water interfaces or is not affected by interfacial forces in the same manner. Other batch experiments showed that MS2 and R17 were increasingly inactivated during mixing in polypropylene tubes as the ionic strength of the solution was raised (phiX174 was not affected). By the addition of Tween 80 to suspensions of MS2 and R17, phage inactivation was prevented. Our data suggest that viral inactivation in simple dynamic batch experiments is dependent upon (i) the presence of a dynamic air-water-solid interface (where the solid is a hydrophobic surface), (ii) the ionic strength of the solution, (iii) the concentration of surface active compounds in the solution, and (iv) the type of virus used.  (+info)

Replication fork assembly at recombination intermediates is required for bacterial growth. (2/413)

PriA, a 3' --> 5' DNA helicase, directs assembly of a primosome on some bacteriophage and plasmid DNAs. Primosomes are multienzyme replication machines that contribute both the DNA-unwinding and Okazaki fragment-priming functions at the replication fork. The role of PriA in chromosomal replication is unclear. The phenotypes of priA null mutations suggest that the protein participates in replication restart at recombination intermediates. We show here that PriA promotes replication fork assembly at a D loop, an intermediate formed during initiation of homologous recombination. We also show that DnaC810, encoded by a naturally arising intergenic suppressor allele of the priA2::kan mutation, bypasses the need for PriA during replication fork assembly at D loops in vitro. These findings underscore the essentiality of replication fork restart at recombination intermediates under normal growth conditions in bacteria.  (+info)

Virus passage through track-etch membranes modified by salinity and a nonionic surfactant. (3/413)

Why do viruses sometimes not pass through larger pores in track-etch filters? Increasing the salinity (0.8 to 160 mM Na+) decreased phiX174 and PRD1 passage through track-etch polycarbonate membranes (sodium dodecyl sulfate coated but not polyvinylpyrrolidone coated) and PRD1 passage through polyester membranes. Undiminished passage when 0.1% Tween 80 was added implied that nonionic virus adsorption occurred and indicated that high levels of salinity decreased virus passage by decreasing electrostatic repulsion that prevented adsorption.  (+info)

Different trajectories of parallel evolution during viral adaptation. (4/413)

The molecular basis of adaptation is a major focus of evolutionary biology, yet the dynamic process of adaptation has been explored only piecemeal. Experimental evolution of two bacteriophage lines under strong selection led to over a dozen nucleotide changes genomewide in each replicate. At least 96 percent of the amino acid substitutions appeared to be adaptive, and half the changes in one line also occurred in the other. However, the order of these changes differed between replicates, and parallel substitutions did not reflect the changes with the largest beneficial effects or indicate a common trajectory of adaptation.  (+info)

Evolutionary reversals during viral adaptation to alternating hosts. (5/413)

Experimental adaptation of the bacteriophage phiX174 to a Salmonella host depressed its ability to grow on the traditional Escherichia host, whereas adaptation to Escherichia did not appreciably affect growth on Salmonella. Continued host switching consistently exhibited this pattern. Growth inhibition on Escherichia resulted from two to three substitutions in the major capsid gene. When these phages were forced to grow again on Escherichia, fitness recovery occurred predominantly by reversions at these same sites, rather than by second-site compensatory changes, the more frequently observed mechanism in most microbial systems. The affected residues lie on the virion surface and they alter attachment efficiency, yet they occur in a region distinct from a putative binding region previously identified from X-ray crystallography. These residues not only experienced high rates of evolution in our experiments, but also exhibited high levels of radical amino acid variation among phiX174 and its known relatives, consistent with a history of adaptation involving these sites.  (+info)

Transfer of immune complexes from erythrocyte CR1 to mouse macrophages. (6/413)

We are developing a potential therapeutic approach for removing pathogens from the circulation of primates in which the pathogen is bound to the complement receptor (CR1) on E using a bispecific mAb complex, a heteropolymer (HP). We have used mAb this approach to demonstrate that cleared prototype pathogens are localized to, phagocytosed in, and destroyed in the liver. Extension of this work to a clinical setting will require a detailed understanding of the mechanism by which the E-bound immune complex substrates are transferred to fixed tissue macrophages in the liver, the transfer reaction. Therefore, we examined an in vitro system to study this process using bacteriophage phiX174 as a model pathogen. E containing phiX174 (bound via an anti-CR1/anti-phiX174 HP) were incubated with P388D1 murine macrophages, and the two cell types were separated by centrifugation through Ficoll. Both E and macrophages were then probed and analyzed by RIA or flow cytometry. The results indicate that all three components of the E-bound IC (phiX174, HP, and CR1) were removed from the E and internalized by the macrophages. We found that transfer requires the Fc portion of IgG, because little transfer of phiX174 occurs when it is bound to E CR1 using a HP containing only Fab fragments. These findings, taken in the context of other studies, suggest a general mechanism for the transfer reaction in which Fc receptors facilitate close juxtaposition of the macrophage to the E-bound IC which then allows a macrophage-associated protease to cleave CR1. The released IC are then internalized and processed by the macrophages.  (+info)

Purification and characterization of DnaC810, a primosomal protein capable of bypassing PriA function. (7/413)

Escherichia coli strains lacking PriA are severely compromised in their ability to repair UV-damaged DNA and to perform homologous recombination. These phenotypes arise because of a lack of PriA-directed replication fork assembly at recombination intermediates such as D-loops. Naturally arising suppressor mutations in dnaC restore strains carrying the priA2::kan null allele to wild-type function. We have cloned one such gene, dnaC810, and overexpressed, purified, and characterized the DnaC810 protein. DnaC810 can support a PriA-independent synthesis of phiX174 complementary strand DNA. This can be attributed to its ability, unlike wild-type DnaC, to catalyze a SSB-insensitive general priming reaction with DnaB and DnaG on any SSB-coated single-stranded DNA. Gel mobility shift analysis revealed that DnaC810 could load DnaB directly to SSB-coated single-stranded DNA as well as to D loop DNA. This explains the ability of DnaC810 to bypass the requirement for PriA, PriB, PriC, and DnaT during replication fork assembly at recombination intermediates.  (+info)

Characterization of the binding of spike H protein of bacteriophage phiX174 with receptor lipopolysaccharides. (8/413)

The spike H protein of bacteriophage phiX174 was prepared as a hexa histidine-tagged fusion (HisH). On enzyme-linked plate assaying, HisH was found to bind specifically to the lipopolysaccharides (LPSs) of phiX174-sensitive strains, Escherichia coli C and Salmonella typhimurium Ra chemotype, having the complete oligosaccharide sequence of the R-core on the LPSs. In sharp contrast, HisH bound weakly to the LPSs of phiX174-insensitive strains, i.e. E. coli F583 (Rd(2)) lacking some terminal saccharides and E. coli O111: B4 (smooth strain) having additional O-repeats on the R-core. The fluorescence spectra of HisH changed dose-dependently in the case of the LPS of E. coli C, the intensity increasing and the emission peak shifting to the shorter wavelength side, which was attributable to the hydrophobic interaction of HisH with the LPS. The binding equilibrium was analyzed by fluorometric titration to determine the dissociation constant K(d), 7.02 +/- 0.37 microM, and the Gibbs free energy change DeltaG(0), -29.1 kJ mol(-1) (at 22 degrees C, pH 7.4). Based on the temperature dependence of (K)d in a van't Hoff plot, the standard enthalpy change DeltaH(0) and the entropy change DeltaS(0) were calculated to be +23.7 kJ mol(-1) and 179 J mol(-1) K(-1) at 22 degrees C, respectively, and this binding was thereby concluded to be an entropy-driven reaction.  (+info)

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