The lytic bacteriophage T4 uses multiple mechanisms to initiate the replication of its DNA. Initiation occurs predominantly at replication origins at early times of infection, but there is a switch to genetic recombinationdependent initiation at late times of infection. The T4 insertion-substitution system was used to create a deletion in the T4 dda gene, which encodes a 5'-3' DNA (11). In vitro, the gene 41 DNA helicase processively unwinds DNA in the 5'-to-3' direction (i.e., moving along the lagging strand at the replication fork) (25, 31) and greatly stimulates the rate of DNA strand displacement DNA synthesis at a replication fork. It also interacts with the T4 gene 61 protein (the DNA primase that makes the RNA primers for Okazaki fragment synthesis) to form the T4 primosome (25,29).The molecular events that lead to the loading of the gene 41 protein at replication origins during early times of infection are not known, but the phenotype of T4 dda helicase mutants suggests that the Dda protein plays a role. Little (24) found that infections with phage carrying deletions that remove a significant fraction of the DNA between genes 39 and 56 (which delete the dda gene) show a substantial delay in DNA synthesis at early times of infection, but-because nearly normal amounts of DNA are eventually produced-phage burst size is reduced only slightly. We have previously shown that the dda gene is essential only when the phage also carries a mutation in T4 gene 59 (10, 13; this gene is discussed below). Unfortunately, the dda mutant and dda 59 double mutant phage strains used in those studies either carried extensive deletions that also removed genes flanking the dda gene or contained mutations in additional genes. As a result, it was uncertain whether the phenotype of these phage was due only to a dda deficiency.Although the physiological role of the Dda helicase is not clear, a great deal has been learned about its biochemical properties from in vitro studies. The Dda protein was originally isolated as a DNA-dependent ATPase by Ebisuzaki and coworkers (2, 7). Like the gene 41 helicase, the Dda protein unwinds DNA in the 5'-to-3' direction, stimulates the rate of DNA strand displacement DNA synthesis at an in vitro * Corresponding author. Fax: (415) 476-0806. replication fork, and removes DNA-binding proteins that block replication fork movement (la, 16). Since no increase in the rate of replication fork movement is observed when the Dda protein is added to in vitro reactions that have been stimulated by the gene 41 protein, the two DNA helicases do not appear to act synergistically at the fork (16). The Dda protein differs from the gene 41 protein by (i) acting distributively (continuously dissociating and reassociating with the DNA molecule being unwound) rather than processively and (ii) not forming a primosome with the gene 61 protein (16).Although no UV sensitivity or genetic recombination deficiencies have been detected during growth of dda mutant phage, involvement of the Dda protein in DNA recombinat...
Uptake of tetracyclines into Escherichia coli was assessed with a strain carrying a tetA-lacZ translational fusion, in which expression of the enzyme is controlled by the pSC101 tetR repressor gene, by examining beta-galactosidase induction. The ability of tetracycline analogues to induce beta-galactosidase synthesis was correlated with their hydrophobicity, such that hydrophobic analogues were poor enzyme inducers. Treatment of E. coli with polymyxin B nonapeptide (PMBN) rendered cells more permeable to minocycline, but not to tetracycline.
A sensitive microbiological detection system for tetracyclines, utilizing an Escherichia coli strain containing a cloned tetA-lacZ gene fusion, is described. Expression of beta-galactosidase by the fusion plasmid pUB3610 remained subject to regulatory control by the TetR repressor protein, with the presence of tetracyclines in the growth medium leading to a 12-fold induction of beta-galactosidase synthesis. Because synthesis of beta-galactosidase was influenced to a small extent by the carbon source and the addition of cyclic AMP to the medium, cells were grown in the presence of cyclic AMP to enhance the sensitivity of the assay. All commonly marketed tetracyclines and some derivatives at concentrations as low as 0.1 ng/ml could be detected in the growth medium. A plate assay utilizing the fusion plasmid that detects 1 ng of tetracycline has also been developed.
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