Transduction of Myxococcus virescens by coliphage P1CM: generation of plasmids containing both phage and Myxococcus genes.
(17/3698)
Chloramphenicol-resistant Myxococcus virescens were obtained by infecting myxococci with Escherichia coli specialized transducing phage P1CM. The drug-resistant myxococci were phenotypically unstable. They contained more than one type of plasmid; these plasmids were not found in the parent strain. Chloramphenicol-resistant E. coli were obtained by transformation with either a fraction of myxococcal DNA containing the plasmids or with P1CM prophage DNA. These transformants contained plasmids. Escherichia coli transformed by DNA from the myxococci contained both P1CM and myxococcal genes. Individual transformant clones differed in the genetic make-up of their plasmids. Among the myxococcal genes expressed in these plasmid-harbouring E. coli strains were a capacity for self-transmissibility and a pattern of phage sensitivity characteristic of R factor incompatibility group W. Escherichia coli transformed with P1CM prophage contained incomplete P1CM genomes; none of the chloramphenicol-resistant transformants produced P1CM phage particles. The significance of these findings for an understanding of mechanisms for the generation of R factors is discussed. (+info)
Role of TolR N-terminal, central, and C-terminal domains in dimerization and interaction with TolA and tolQ.
(18/3698)
The Tol-PAL system of Escherichia coli is a multiprotein system involved in maintaining the cell envelope integrity and is necessary for the import of some colicins and phage DNA into the bacterium. It is organized into two complexes, one near the outer membrane between TolB and PAL and one in the cytoplasmic membrane between TolA, TolQ, and TolR. In the cytoplasmic membrane, all of the Tol proteins have been shown to interact with each other. Cross-linking experiments have shown that the TolA transmembrane domain interacts with TolQ and TolR. Suppressor mutant analyses have localized the TolQ-TolA interaction to the first transmembrane domain of TolQ and have shown that the third transmembrane domain of TolQ interacts with the transmembrane domain of TolR. To get insights on the composition of the cytoplasmic membrane complex and its possible contacts with the outer membrane complex, we focused our attention on TolR. Cross-linking and immunoprecipitation experiments allowed the identification of Tol proteins interacting with TolR. The interactions of TolR with TolA and TolQ were confirmed, TolR was shown to dimerize, and the resulting dimer was shown to interact with TolQ. Deletion mutants of TolR were constructed, and they allowed us to determine the TolR domains involved in each interaction. The TolR transmembrane domain was shown to be involved in the TolA-TolR and TolQ-TolR interactions, while TolR central and C-terminal domains appeared to be involved in TolR dimerization. The role of the TolR C-terminal domain in the TolA-TolR interaction and its association with the membranes was also demonstrated. Furthermore, phenotypic studies clearly showed that the three TolR domains (N terminal, central, and C terminal) and the level of TolR production are important for colicin A import and for the maintenance of cell envelope integrity. (+info)
Sunlight inactivation of fecal bacteriophages and bacteria in sewage-polluted seawater.
(19/3698)
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)
Mutations altering the cellular localization of the phage lambda receptor, an Escherichia coli outer membrane protein.
(20/3698)
Two mutant strains of Escherichia coli have been isolated in which the cellular location of an outer membrane protein, the phage lambda receptor (the lamB gene product), is altered. These mutations were initially selected in a strain containing a lamB-lacZ fusion. In the parent strain the protein coded for by the hybrid gene is located, at least in part, in the outer membrane. In the mutants it is located in the cytoplasm. The mutations responsible for the alteration of cellular location lie very early in the lamB gene, in a region corresponding to the NH2-terminus of the lambda receptor protein. One of these mutations is a small deletion internal to the lamB gene. When this mutation is present in an otherwise wild-type lamB gene, the protein produced is of lower molecular weight than normal receptor. The other mutation behaves as a point mutation; when it is present in an otherwise normal lamB gene, reversion can be demonstrated. The molecular weight of this mutant protein, which is located in the cytoplasm, is larger than that of the wild-type gene product by approximately 2000. It is suggested that these two mutations are in the portion of the lamB gene coding for a signal sequence and thereby block export of the protein. (+info)
Characterization of the oriI and oriII origins of replication in phage-plasmid P4.
(21/3698)
In the Escherichia coli phage-plasmid P4, two partially overlapping replicons with bipartite ori sites coexist. The essential components of the oriI replicon are the alpha and cnr genes and the ori1 and crr sites; the oriII replicon is composed of the alpha gene, with the internal ori2 site, and the crr region. The P4 alpha protein has primase and helicase activities and specifically binds type I iterons, present in ori1 and crr. Using a complementation test for plasmid replication, we demonstrated that the two replicons depend on both the primase and helicase activities of the alpha protein. Moreover, neither replicon requires the host DnaA, DnaG, and Rep functions. The bipartite origins of the two replicons share the crr site and differ for ori1 and ori2, respectively. By deletion mapping, we defined the minimal ori1 and ori2 regions sufficient for replication. The ori1 site was limited to a 123-bp region, which contains six type I iterons spaced regularly close to the helical periodicity, and a 35-bp AT-rich region. Deletion of one or more type I iterons inactivated oriI. Moreover, insertion of 6 or 10 bp within the ori1 region also abolished replication ability, suggesting that the relative arrangement of the iterons is relevant. The ori2 site was limited to a 36-bp P4 region that does not contain type I iterons. In vitro, the alpha protein did not bind ori2. Thus, the alpha protein appears to act differently at the two origins of replication. (+info)
Use of a linear multicopy vector based on the mini-replicon of temperate coliphage N15 for cloning DNA with abnormal secondary structures.
(22/3698)
A new cloning vector pN15L is described. It is a linear 13.8 kb plasmid based on the coliphage N15 mini-replicon. The vector capacity exceeds 50 kb and the copy number is 250 per Escherichia coli chromosome. We show that some artificial and natural palindromes and approximately 5% of human DNA Bgl II fragments can be cloned effectively in linear vector pN15L, whereas they either sharply reduce the copy number of circular vector pUC19 or cannot be cloned at all. We conclude that pN15L may be usefully employed to clone large imperfect palindromes and some abnormal sequences of human DNA. (+info)
Exonuclease associated with bacteriophage T5-Induced DNA polymerase.
(23/3698)
T-5-induced DNA polymerase has been shown to possess a 3' leads to 5'-exonucleolytic activity. The exonuclease acts on both native and denatured DNA, but the apparent rate of degradation of denatured DNA is about five times faster than that for native DNA. The enzyme appears to act only on 3'-OH ends and produces mainly 5'-dNMP's. Like polymerase activity, exonuclease activity shows a pH optimum around 8.6. Mg2+, dithiothreitol, and N-ethylmaleimide had identical effects on both the activities. Nicked DNA was almost totally protected from exonuclease action under synthetic conditions, i.e., in the presence of 4dNTP's. Denatured DNA was partly degraded in the early phase of incubation with 4dNTP's, presumably due to unhybridized tails at the 3'-OH primer ends. However, the exonuclease activity was operative in both cases under synthetic conditions, as evidenced by template-dependent conversion of [3H]dTTP to [3H]dTMP. (+info)
Selection of ganglioside GM1-binding peptides by using a phage library.
(24/3698)
Ganglioside Gal beta1 --> 3GalNAc beta1 --> 4(NeuAc alpha2 --> 3) Gal beta1 --> 4Glc beta1 -->1'Cer (GM1)-binding peptides were obtained from a phage-displayed pentadecapeptide library by an affinity selection. The selection processes were in situ-monitored by a quartz-crystal microbalance method, on which a ganglioside GM1 monolayer was transferred. After five rounds of biopanning, the DNA sequencing of 18 selected phages showed that only three individual clones were selected. The peptide sequences of the random region were found to be DFRRLPGAFWQLRQP, GWWYKGRARPVSAVA and VWRLLAPPFSNRLLP. Binding constants of these phage clones to the GM1 monolayer were 10(10) M(-1). Three synthetic pentadecapeptides inhibited the binding of cholera toxin B subunit to the GM1 monolayer with an IC50 of 24, 13 and 1.0 microM, respectively. These peptides will be useful for searching functional roles of ganglioside GMI. (+info)