Gamblers: an Antibiotic-induced Evolvable Cell Subpopulation Differentiated by Reactive-oxygen-induced General Stress Response

SUMMARYAntibiotics can induce mutations that cause antibiotic resistance. Yet, despite their importance, mechanisms of antibiotic-promoted mutagenesis remain elusive. We report that the fluoroquinolone antibiotic ciprofloxacin (cipro) induces mutations that cause drug resistance by triggering differentiation of a mutant-generating cell subpopulation, using reactive oxygen species (ROS) to signal the sigma-S (σS) general-stress response. Cipro-generated DNA breaks activate the SOS DNA-damage response and error-prone DNA polymerases in all cells. However, mutagenesis is restricted to a cell subpopulation in which electron transfer and SOS induce ROS, which activate the σSresponse, allowing mutagenesis during DNA-break repair. When sorted, this small σS-response-“on” subpopulation produces most antibiotic cross-resistant mutants. An FDA-approved drug prevents σSinduction specifically inhibiting antibiotic-promoted mutagenesis. Furthermore, SOS-inhibited cell division, causing multi-chromosome cells, is required for mutagenesis. The data support a model in which within-cell chromosome cooperation together with development of a “gambler” cell subpopulation promote resistance evolution without risking most cells.

Production of Amphidiploids of the Hybrids between Solanum macrocarpon and Eggplant

To restore fertility of the F1 between Solanum macrocarpon and eggplant, amphidiploids were produced through doubling of chromosomes by colchicine treatment. Shoot tips and axillary buds of F1 plants were kept for 2 and 4 days in liquid Murashige and Skoog (MS) media with 0.05% colchicine. As a result of colchicine treatment, two amphidiploids were identified by observing root tip cell chromosome number, stomatal guard cell size, and pollen characteristics. The amphidiploids contained 48 chromosomes, twice the normal diploid number of 24. Stomata size and pollen diameter were significantly larger in amphidiploids than F1. Flower diameter and length and width of anther, petal, and sepal were significantly larger in the amphidiploids than in F1. Pollen stainability was 40% in amphidiploids but only 0.86% in the diploid F1. The amphidiploids, after selfing and backcrossing with S. macrocarpon, set fruits with healthy seeds. Therefore, production of amphidiploids by colchicine treatment restored the pollen and seed fertility of F1 between S. macrocarpon and eggplant. The amphidiploids produced in this study would be very useful in future breeding programs of eggplant.

Cellular Cofactors of Lentiviral Integrase: From Target Validation to Drug Discovery

To accomplish their life cycle, lentiviruses make use of host proteins, the so-called cellular cofactors. Interactions between host cell and viral proteins during early stages of lentiviral infection provide attractive new antiviral targets. The insertion of lentiviral cDNA in a host cell chromosome is a step of no return in the replication cycle, after which the host cell becomes a permanent carrier of the viral genome and a producer of lentiviral progeny. Integration is carried out by integrase (IN), an enzyme playing also an important role during nuclear import. Plenty of cellular cofactors of HIV-1 IN have been proposed. To date, the lens epithelium-derived growth factor (LEDGF/p75) is the best studied cofactor of HIV-1 IN. Moreover, small molecules that block the LEDGF/p75-IN interaction have recently been developed for the treatment of HIV infection. The nuclear import factor transportin-SR2 (TRN-SR2) has been proposed as another interactor of HIV IN-mediating nuclear import of the virus. Using both proteins as examples, we will describe approaches to be taken to identify and validate novel cofactors as new antiviral targets. Finally, we will highlight recent advances in the design and the development of small-molecule inhibitors binding to the LEDGF/p75-binding pocket in IN (LEDGINs).

The Linear Plasmid Prophage Vp58.5 of Vibrio parahaemolyticus Is Closely Related to the Integrating Phage VHML and Constitutes a New Incompatibility Group of Telomere Phages

ABSTRACT Vibrio parahaemolyticus O3:K6 pandemic strains recovered in Chile frequently possess a 42-kb plasmid which is the prophage of a myovirus. We studied the prototype phage VP58.5 and show that it does not integrate into the host cell chromosome but replicates as a linear plasmid (Vp58.5) with covalently closed ends (telomeres). The Vp58.5 replicon coexists with other plasmid prophages (N15, PY54, and ΦKO2) in the same cell and thus belongs to a new incompatibility group of telomere phages. We determined the complete nucleotide sequence (42,612 nucleotides) of the VP58.5 phage DNA and compared it with that of the plasmid prophage. The two molecules share the same nucleotide sequence but are 35% circularly permuted to each other. In contrast to the hairpin ends of the plasmid, VP58.5 phage DNA contains 5′-protruding ends. The VP58.5 sequence is 92% identical to the sequence of phage VHML, which was reported to integrate into the host chromosome. However, the gene order and termini of the phage DNAs are different. The VHML genome exhibits the same gene order as does the Vp58.5 plasmid. VHML phage DNA has been reported to contain terminal inverted repeats. This repetitive sequence is similar to the telomere resolution site (telRL) of VP58.5 which, after processing by the phage protelomerase, forms the hairpin ends of the Vp58.5 prophage. It is discussed why these closely related phages may be so different in terms of their genome ends and their lifestyle.