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Saccharomyces cerevisiae Multidrug Transporter Qdr2p (Yil121wp): Localization and Function as a Quinidine Resistance Determinant. Rita C. Vargas, 2004.This work reports the functional analysis of Saccharomyces cerevisiae open reading frame YIL121w, encoding a member of a family of drug:H+ antiporters with 12 predicted membrane-spanning segments (DHA12 family) . Like its close homologue Qdr1p, Yil121wp was localized at the plasma membrane, and its increased expression also led to increased tolerance to the antiarrhythmia and antimalarial drug quinidine . The quinidine resistance phenotype was confirmed for different yeast strains and growth media, including a prototrophic strain, and YIL121w was named the QDR2 gene . Both QDR1 and QDR2 were also implicated in yeast resistance to the herbicide barban (4-chloro-2-butynyl [3-chlorophenyl] carbamate), and the genes are functionally interchangeable with respect to both resistance phenotypes . The average intracellular pH of a yeast population challenged with quinidine added to the acidic growth medium was significantly below the intracellular pH of the unstressed population, suggesting plasma membrane permeabilization by quinidine with consequent increase of the H+ influx rate . For the same extracellular quinidine concentration, internal acidification was more intense for the  qdr2 deletant compared with the parental strain . Although QDR2 transcription was not enhanced in response to quinidine, the results confirmed that Qdr2p is involved in the active export of quinidine out of the cell, thus conferring resistance to the drug .
Morphological, Chemical, and Genetic Diversity of Tropical Marine Cyanobacteria Lyngbya spp . and Symploca spp . (Oscillatoriales). Robert W. Thacker, 2004.Although diverse natural products have been isolated from the benthic, filamentous cyanobacterium Lyngbya majuscula, it is unclear whether this chemical variation can be used to establish taxonomic relationships among disparate collections . We compared morphological characteristics, secondary-metabolite compositions, and partial 16S ribosomal DNA (rDNA) sequences among several collections of L . majuscula Gomont, Lyngbya spp., and Symploca spp . from Guam and the Republic of Palau . The morphological characteristics examined were cell length, cell width, and the presence or absence of a calyptra . Secondary metabolites were analyzed by two-dimensional thin-layer chromatography . Each collection possessed a distinct cellular morphology that readily distinguished Lyngbya spp . from Symploca spp . Each collection yielded a unique chemotype, but common chemical characteristics were shared among four collections of L . majuscula . A phylogeny based on secondary-metabolite composition supported the reciprocal monophyly of Lyngbya and Symploca but yielded a basal polytomy for Lyngbya . Pairwise sequence divergence among species ranged from 10 to 14% across 605 bp of 16S rDNA, while collections of L . majuscula showed 0 to 1.3% divergence . Although the phylogeny of 16S rDNA sequences strongly supported the reciprocal monophyly of Lyngbya and Symploca as well as the monophyly of Lyngbya bouillonii and L . majuscula, genetic divergence was not correlated with chemical and morphological differences . These data suggest that 16S rDNA sequence analyses do not predict chemical variability among Lyngbya species . Other mechanisms, including higher rates of evolution for biosynthetic genes, horizontal gene transfer, and interactions between different genotypes and environmental conditions, may play important roles in generating qualitative and quantitative chemical variation within and among Lyngbya species . Coordination of Ubiquinol Oxidase and Cytochrome cbb3 Oxidase Expression by Multiple Regulators in Rhodobacter capsulatus. Danielle L. Swem, 2002.Rhodobacter capsulatus utilizes two terminal oxidases for aerobic respiration, cytochrome cbb3 and ubiquinol oxidase . To determine the transcription factors involved in terminal oxidase expression, ccoN-lacZ and cydA-lacZ protein fusions were assayed in a variety of regulatory mutants . The results of this and previous studies indicate that cytochrome cbb3 expression is controlled by regB-regA, fnrL, and hvrA and that ubiquinol oxidase expression is controlled by regB-regA, fnrL, hvrA, crtJ, and aerR . Origin Binding Activity of the Mycobacterial Plasmid pAL5000 Replication Protein RepB Is Stimulated through Interactions with Host Factors and Coupled Expression of repA. Abhijit Basu, 2002.The minimal replication region of the mycobacterial plasmid pAL5000 encompasses the replication origin (ori) and two tandemly organized replication genes, repA and repB, the functions of which are not clearly known . It was observed that when the repA and repB genes were expressed in Escherichia coli, a strong ori binding activity was generated in the host cells . Inactivation of repB led to a complete loss of activity, whereas inactivation of repA had a partial effect, indicating that while repB plays an important role in the process, its activity is stimulated through coexpression of repA . However, this stimulatory effect could be demonstrated only when expression of repA and that of repB were coupled . At a relatively high concentration (1,000 nM), the purified RepB protein was found to form an ori complex with low specificity, which was sensitive to high salt concentrations and challenge by a nonspecific competitor . In contrast, the complex formed by an extract of repA-repB-expressing cells was highly specific and was resistant to both types of challenges . At a 10-fold-lower concentration, RepB did not exhibit ori binding activity, but it could nevertheless form a salt-resistant ori complex in vitro, provided that host factors were present . Antibody supershift experiments indicated that RepB is a key component of the specific complex formed by extracts prepared from E . coli cells expressing the repA and repB genes and also from mycobacterial cells harboring pAL5000-derived vectors . The results indicate that in vivo RepB interacts with host factors and forms an ori complex, but this activity is maximal only when there is coupled expression of repA . Pseudomonas syringae Exchangeable Effector Loci: Sequence Diversity in Representative Pathovars and Virulence Function in P . syringae pv . syringae B728a. Wen-Ling Deng, 2003.Pseudomonas syringae is a plant pathogen whose pathogenicity and host specificity are thought to be determined by Hop/Avr effector proteins injected into plant cells by a type III secretion system . P . syringae pv . syringae B728a, which causes brown spot of bean, is a particularly well-studied strain . The type III secretion system in P . syringae is encoded by hrp (hypersensitive response and pathogenicity) and hrc (hrp conserved) genes, which are clustered in a pathogenicity island with a tripartite structure such that the hrp/hrc genes are flanked by a conserved effector locus and an exchangeable effector locus (EEL) . The EELs of P . syringae pv . syringae B728a, P . syringae strain 61, and P . syringae pv . tomato DC3000 differ in size and effector gene composition; the EEL of P . syringae pv . syringae B728a is the largest and most complex . The three putative effector proteins encoded by the P . syringae pv . syringae B728a EEL—HopPsyC, HopPsyE, and HopPsyV—were demonstrated to be secreted in an Hrp-dependent manner in culture . Heterologous expression of hopPsyC, hopPsyE, and hopPsyV in P . syringae pv . tabaci induced the hypersensitive response in tobacco leaves, demonstrating avirulence activity in a nonhost plant . Deletion of the P . syringae pv . syringae B728a EEL strongly reduced virulence in host bean leaves . EELs from nine additional strains representing nine P . syringae pathovars were isolated and sequenced . Homologs of avrPphE (e.g., hopPsyE) and hopPsyA were particularly common . Comparative analyses of these effector genes and hrpK (which flanks the EEL) suggest that the EEL effector genes were acquired by horizontal transfer after the acquisition of the hrp/hrc gene cluster but before the divergence of modern pathovars and that some EELs underwent transpositions yielding effector exchanges or point mutations producing effector pseudogenes after their acquisition . Coupling of Bacterial Endosymbiont and Host Mitochondrial Genomes in the Hydrothermal Vent Clam Calyptogena magnifica. Luis A. Hurtado, 2003.The hydrothermal vent clam Calyptogena magnifica (Bivalvia: Vesicomyidae) depends for its nutrition on sulfur-oxidizing symbiotic bacteria housed in its gill tissues . This symbiont is transmitted vertically between generations via the clam's eggs; however, it remains uncertain whether occasionally symbionts are horizontally transmitted or acquired from the environment . If symbionts are transmitted strictly vertically through the egg cytoplasm, inheritance of symbiont lineages should behave as if coupled to the host's maternally inherited mitochondrial DNA . This coupling would be obscured, however, with low rates of horizontal or environmental transfers, the equivalent of recombination between host lineages . Population genetic analyses of C . magnifica clams and associated symbionts from eastern Pacific hydrothermal vents clearly supported the hypothesis of strictly maternal cotransmission . Host mitochondrial and symbiont DNA sequences were coupled in a clam population that was polymorphic for both genetic markers . These markers were not similarly coupled with sequence variation at a nuclear gene locus, as expected for a randomly mating sexual population . Phylogenetic analysis of the two cytoplasmic genes also revealed no evidence for recombination . The tight association between vesicomyid clams and their vertically transmitted bacterial endosymbionts is phylogenetically very young (<50 million years) and may serve as a model for the origin and evolution of eukaryotic organelles .
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