Isolation of additional bacteriophages with genomes of segmented double-stranded RNA.
(65/4361)
Eight different bacteriophages were isolated from leaves of Pisum sativum, Phaseolus vulgaris, Lycopersicon esculentum, Daucus carota sativum, Raphanus sativum, and Ocimum basilicum. All contain three segments of double-stranded RNA and have genomic-segment sizes that are similar but not identical to those of previously described bacteriophage phi6. All appear to have lipid-containing membranes. The base sequences of some of the viruses are very similar but not identical to those of phi6. Three of the viruses have little or no base sequence identity to phi6. Two of the viruses, phi8 and phi12, contain proteins with a size distribution very different from that of phi6 and do not package genomic segments of phi6. Whereas phi6 attaches to host cells by means of a pilus, several of the new isolates attach directly to the outer membrane. Although the normal hosts of these viruses seem to be pseudomonads, those viruses that attach directly to the outer membrane can establish carrier states in Escherichia coli or Salmonella typhimurium. One of the isolates, phi8, can form plaques on heptoseless strains of S. typhimurium. (+info)
The recognition of a noncanonical RNA base pair by a zinc finger protein.
(66/4361)
BACKGROUND: The zinc finger (ZF) is the most abundant nucleic-acid-interacting protein motif. Although the interaction of ZFs with DNA is reasonably well understood, little is known about the RNA-binding mechanism. We investigated RNA binding to ZFs using the Zif268-DNA complex as a model system. Zif268 contains three DNA-binding ZFs; each independently binds a 3 base pair (bp) subsite within a 9 bp recognition sequence. RESULTS: We constructed a library of phage-displayed ZFs by randomizing the alpha helix of the Zif268 central finger. Successful selection of an RNA binder required a noncanonical base pair in the middle of the RNA triplet. Binding of the Zif268 variant to an RNA duplex containing a G.A mismatch (rG.A) is specific for RNA and is dependent on the conformation of the mismatched middle base pair. Modeling and NMR analyses revealed that the rG.A pair adopts a head-to-head configuration that counterbalances the effect of S-puckered riboses in the backbone. We propose that the structure of the rG.A duplex is similar to the DNA in the original Zif268-DNA complex. CONCLUSIONS: It is possible to change the specificity of a ZF from DNA to RNA. The ZF motif can use similar mechanisms in binding both types of nucleic acids. Our strategy allowed us to rationalize the interactions that are possible between a ZF and its RNA substrate. This same strategy can be used to assess the binding specificity of ZFs or other protein motifs for noncanconical RNA base pairs, and should permit the design of proteins that bind specific RNA structures. (+info)
Identification of mimotope peptides which bind to the mycotoxin deoxynivalenol-specific monoclonal antibody.
(67/4361)
Monoclonal antibody 6F5 (mAb 6F5), which recognizes the mycotoxin deoxynivalenol (DON) (vomitoxin), was used to select for peptides that mimic the mycotoxin by employing a library of filamentous phages that have random 7-mer peptides on their surfaces. Two phage clones selected from the random peptide phage-displayed library coded for the amino acid sequences SWGPFPF and SWGPLPF. These clones were designated DONPEP.2 and DONPEP.12, respectively. The results of a competitive enzyme-linked immunosorbent assay (ELISA) suggested that the two phage displayed peptides bound to mAb 6F5 specifically at the DON binding site. The amino acid sequence of DONPEP.2 plus a structurally flexible linker at the C terminus (SWGPFPFGGGSC) was synthesized and tested to determine its ability to bind to mAb 6F5. This synthetic peptide (designated peptide C430) and DON competed with each other for mAb 6F5 binding. When translationally fused with bacterial alkaline phosphatase, DONPEP.2 bound specifically to mAb 6F5, while the fusion protein retained alkaline phosphatase activity. The potential of using DONPEP.2 as an immunochemical reagent in a DON immunoassay was evaluated with a DON-spiked wheat extract. When peptide C430 was conjugated to bovine serum albumin, it elicited antibody specific to peptide C430 but not to DON in both mice and rabbits. In an in vitro translation system containing rabbit reticulocyte lysate, synthetic peptide C430 did not inhibit protein synthesis but did show antagonism toward DON-induced protein synthesis inhibition. These data suggest that the peptides selected in this study bind to mAb 6F5 and that peptide C430 binds to ribosomes at the same sites as DON. (+info)
Sunlight inactivation of fecal bacteriophages and bacteria in sewage-polluted seawater.
(68/4361)
Sunlight inactivation rates of somatic coliphages, F-specific RNA bacteriophages (F-RNA phages), and fecal coliforms were compared in seven summer and three winter survival experiments. Experiments were conducted outdoors, using 300-liter 2% (vol/vol) sewage-seawater mixtures held in open-top chambers. Dark inactivation rates (k(D)s), measured from exponential survival curves in enclosed (control) chambers, were higher in summer (temperature range: 14 to 20 degrees C) than in winter (temperature range: 8 to 10 degrees C). Winter k(D)s were highest for fecal coliforms and lowest for F-RNA phages but were the same or similar for all three indicators in summer. Sunlight inactivation rates (k(S)), as a function of cumulative global solar radiation (insolation), were all higher than the k(D)s with a consistent k(S) ranking (from greatest to least) as follows: fecal coliforms, F-RNA phages, and somatic coliphages. Phage inactivation was exponential, but bacterial curves typically exhibited a shoulder. Phages from raw sewage exhibited k(S)s similar to those from waste stabilization pond effluent, but raw sewage fecal coliforms were inactivated faster than pond effluent fecal coliforms. In an experiment which included F-DNA phages and Bacteroides fragilis phages, the k(S) ranking (from greatest to least) was as follows: fecal coliforms, F-RNA phages, B. fragilis phages, F-DNA phages, and somatic coliphages. In a 2-day experiment which included enterococci, the initial concentration ranking (from greatest to least: fecal coliforms, enterococci, F-RNA phages, and somatic coliphages) was reversed during sunlight exposure, with only the phages remaining detectable by the end of day 2. Inactivation rates under different optical filters decreased with the increase in spectral cutoff wavelength (50% light transmission) and indicated that F-RNA phages and fecal coliforms are more susceptible than somatic coliphages to longer solar wavelengths, which predominate in seawater. The consistently superior survival of somatic coliphages in our experiments suggests that they warrant further consideration as fecal, and possibly viral, indicators in marine waters. (+info)
The Shigella flexneri bacteriophage Sf6 tailspike protein (TSP)/endorhamnosidase is related to the bacteriophage P22 TSP and has a motif common to exo- and endoglycanases, and C-5 epimerases.
(69/4361)
The temperate bacteriophage Sf6 infects Shigella flexneri strains of serotype X or Y, converting them into serotypes 3a or 3b, respectively. The tailspike protein (TSP) of Sf6 possesses endo-1,3-alpha-L-rhamnosidase (endorhamnosidase) activity which results in cleavage of the lipopolysaccharide O-antigen receptor during the adsorption of the phage to the cell surface. When used in Southern hybridization, a P22 gene 9 (encoding P22 TSP) DNA probe hybridized with restriction fragment Pstl-7 of Sf6. DNA sequencing and analysis of Pstl-7 and the adjacent Pstl-8 fragment revealed an open reading frame (ORF1) of 1872 bp (624 amino acids) bearing amino acid sequence homology to the bacteriophage P22 TSP N-terminal head-binding domain. High conservation of key residues was suggestive of similar secondary and tertiary N-terminal protein structure and a similar function of the Sf6 TSP in this region. In addition, an amino acid sequence motif (DFGX3DGX6AX3A) was identified between residues 164 and 184 which was also found to exist in various prokaryotic and eukaryotic exo-/endoglycanases, C-5 epimerases and bacteriophage proteins. Expression of ORF1 from a T7 promoter produced a 67 kDa protein (detected by L-[35S]methionine labelling and SDS-PAGE). Assay of heat-treated cytoplasmic extracts containing the ORF1-encoded protein by incubation with whole Sh. flexneri Y cells demonstrated that O-antigen hydrolysis activity was present; ORF1 therefore encodes Sf6 TSP. Sf6 TSP exhibited specific and preferential activity for long-chain Sh. flexneri serotype X or Y O-antigen, cleavage of which resulted in the release of oligosaccharide fragments, consistent with octasaccharides in size, as detected by fluorophore-assisted carbohydrate electrophoresis (FACE). (+info)
Selection for improved subtiligases by phage display.
(70/4361)
Engineering enzyme activity has been challenging because of uncertainties in structure-function relationships and difficulties in screening a large number of mutant enzymes. A product capture strategy using phage display is presented here for the selection of improved enzymes from a large library of variants (>10(9) independently derived mutants). Subtiligase, a double mutant of subtilisin BPN' that catalyzes the ligation of peptides, was displayed on phage. Twenty-five active site residues were randomly mutated in groups of four or five to yield six different libraries that were independently sorted. Variants that ligated a biotin peptide onto their own extended N termini were selectively captured. Mutant subtiligases were identified that had increased ligase activity. The selection also yielded unexpected subtiligase mutants having residues known to improve the stability and oxidative resistance of wild-type subtilisin. These studies are exemplary for the use of phage to improve enzyme function when it is closely linked to a selectable catalytic event. (+info)
Flagellar determinants of bacterial sensitivity to chi-phage.
(71/4361)
Bacteriophage chi is known to infect motile strains of enteric bacteria by adsorbing randomly along the length of a flagellar filament and then injecting its DNA into the bacterial cell at the filament base. Here, we provide evidence for a "nut and bolt" model for translocation of phage along the filament: the tail fiber of chi fits the grooves formed by helical rows of flagellin monomers, and active flagellar rotation forces the phage to follow the grooves as a nut follows the threads of a bolt. (+info)
Isolation of peptides that mimic epitopes on a malarial antigen from random peptide libraries displayed on phage.
(72/4361)
The ring-infected erythrocyte surface antigen (RESA) is a dense-granule protein of Plasmodium falciparum which binds to the cytoskeletal structure of the erythrocyte after parasite invasion. It is currently under trial as a vaccine candidate. In an effort to characterize further the antibody responses to this antigen, we have panned two independent libraries of random peptides expressed on the surface of filamentous phage with a monoclonal antibody (MAb 18/2) against RESA. One library consisted of a potentially constrained 17-mer peptide fused with the gpVIII phage coat protein, and the other displayed an unconstrained 15-mer as a fusion with the minor phage coat protein gpIII. Several rounds of biopanning resulted in enrichment from both libraries clones that interacted specifically with MAb 18/2 in protein-blotting and enzyme-linked immunosorbent assay experiments. Nucleotide sequencing of the random oligonucleotide insert revealed a common predominant motif: (S/T)AVDD. Several other clones had related but degenerate motifs. Thus, a monoclonal antibody against a malarial antigen can select common mimotopes from different random peptide libraries. We envisage many uses for this technology in malaria research. (+info)