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Comparison of Gene Expression Profiles of Candida albicans Azole-Resistant Clinical Isolates and Laboratory Strains Exposed to Drugs Inducing Multidrug Transporters. Mahir Karababa, 2004.Azole resistance in Candida albicans can be due to upregulation of multidrug transporters belonging to ABC (ATP-binding cassette) transporters (CDR1 and CDR2) or major facilitators (CaMDR1) . Upregulation of these genes can also be achieved by exposure to fluphenazine, resulting in specific upregulation of CDR1 and CDR2 and by exposure to benomyl, resulting in specific CaMDR1 upregulation . In this study, these two different states of gene upregulation were used to determine coregulated genes that often share similar functions or similar regulatory regions . The transcript profiles of a laboratory strain exposed to these drugs were therefore determined and compared with those of two matched pairs of azole-susceptible and -resistant strains expressing CDR1 and CDR2 (CDR strains) or CaMDR1 (MDR isolates) . The results obtained revealed that, among 42 commonly regulated genes (8.6% of all regulated genes) between fluphenazine-exposed cells and CDR isolates, the most upregulated were CDR1 and CDR2 as expected, but also IFU5, RTA3 (which encodes putative membrane proteins), HSP12 (which encodes heat shock protein), and IPF4065 (which is potentially involved in stress response) . Interestingly, all but HSP12 and IPF4065 contain a putative cis-acting drug responsive element in their promoters . Among the 57 genes (11.5% of all regulated genes) commonly regulated between benomyl-exposed cells and MDR isolates, the most upregulated were CaMDR1 as expected but also genes with oxido-reductive functions such as IFD genes, IPF5987, GRP2 (all belonging to the aldo-keto reductase family), IPF7817 [NAD(P)H oxido-reductase], and IPF17186 . Taken together, these results show that in vitro drug-induced gene expression only partially mimics expression profiles observed in azole-resistant clinical strains . Upregulated genes in both drug-exposed conditions and clinical strains are drug resistance genes but also genes that could be activated under cell damage conditions . Lipid Accumulation, Lipid Body Formation, and Acyl Coenzyme A Oxidases of the Yeast Yarrowia lipolytica. Kate, 2004.Yarrowia lipolytica contains five acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX5 genes, that catalyze the limiting step of peroxisomal ß-oxidation . In this study, we analyzed morphological changes of Y . lipolytica growing in an oleic acid medium and the effect of POX deletions on lipid accumulation . Protrusions involved in the uptake of lipid droplets (LDs) from the medium were seen in electron micrographs of the surfaces of wild-type cells grown on oleic acid . The number of protrusions and surface-bound LDs increased during growth, but the sizes of the LDs decreased . The sizes of intracellular lipid bodies (LBs) and their composition depended on the POX genotype . Only a few, small, intracellular LBs were observed in the mutant expressing only Aox4p (  pox2
pox3
pox5), but strains expressing either Aox3p or both Aox3p and Aox4p had the same number of LBs as did the wild type . In contrast, strains expressing either Aox2p or both Aox2p and Aox4p formed fewer, but larger, LBs than did the wild type . The size of the LBs increased proportionately with the amount of triacylglycerols in the LBs of the mutants . In summary, Aox2p expression regulates the size of cellular triacylglycerol pools and the size and number of LBs in which these fatty acids accumulate .
Molecular Characterization of the PceA Reductive Dehalogenase of Desulfitobacterium sp . Strain Y51. Akiko Suyama, 2002.The tetrachloroethene (PCE) reductive dehalogenase (encoded by the pceA gene and designated PceA dehalogenase) of Desulfitobacterium sp . strain Y51 was purified and characterized . The expression of the enzyme was highly induced in the presence of PCE and trichloroethene (TCE) . The purified enzyme catalyzed the reductive dehalogenation of PCE via TCE to cis-1,2-dichloroethene at a specific activity of 113.6 nmol · min-1 · mg of protein-1 . The apparent Km values for PCE and TCE were 105.7 and 535.3 µM, respectively . Chlorinated ethenes other than PCE and TCE were not dehalogenated . However, the enzyme exhibited dehalogenation activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane, and 1,1,2,2-tetrachloroethane . The pceA gene of Desulfitobacterium sp . strain Y51 was identified in a 2.8-kb DNA fragment and used to express the protein in Escherichia coli for the preparation of antibodies . Immunoblot analyses located PceA in the periplasm of the cell . Extent of Genetic Lesions of the Arginine and Pyrimidine Biosynthetic Pathways in Lactobacillus plantarum, L . paraplantarum, L . pentosus, and L . casei: Prevalence of CO2-Dependent Auxotrophs and Characterization of Deficient arg Genes in L . plantarum. Françoise Bringel, 2003.Lactic acid bacteria require rich media since, due to mutations in their biosynthetic genes, they are unable to synthesize numerous amino acids and nucleobases . Arginine biosynthesis and pyrimidine biosynthesis have a common intermediate, carbamoyl phosphate (CP), whose synthesis requires CO2 . We investigated the extent of genetic lesions in both the arginine biosynthesis and pyrimidine biosynthesis pathways in a collection of lactobacilli, including 150 strains of Lactobacillus plantarum, 32 strains of L . pentosus, 15 strains of L . paraplantarum, and 10 strains of L . casei . The distribution of prototroph and auxotroph phenotypes varied between species . All L . casei strains, no L . paraplantarum strains, two L . pentosus strains, and seven L . plantarum strains required arginine for growth . Arginine auxotrophs were more frequently found in L . plantarum isolated from milk products than in L . plantarum isolated from fermented plant products or humans; association with dairy products might favor arginine auxotrophy . In L . plantarum the argCJBDF genes were functional in most strains, and when they were inactive, only one gene was mutated in more than one-half of the arginine auxotrophs . Random mutation may have generated these auxotrophs since different arg genes were inactivated (there were single point mutations in three auxotrophs and nonrevertible genetic lesions in four auxotrophs) . These data support the hypothesis that lactic acid bacteria evolve by progressively loosing unnecessary genes upon adaptation to specific habitats, with genome evolution towards cumulative DNA degeneration . Although auxotrophy for only uracil was found in one L . pentosus strain, a high CO2 requirement (HCR) for arginine and pyrimidine was common; it was found in 74 of 207 Lactobacillus strains tested . These HCR auxotrophs may have had their CP cellular pool-related genes altered or deregulated . Extremely Acidophilic Protists from Acid Mine Drainage Host Rickettsiales-Lineage Endosymbionts That Have Intervening Sequences in Their 16S rRNA Genes. Brett J. Baker, 2003.During a molecular phylogenetic survey of extremely acidic (pH < 1), metal-rich acid mine drainage habitats in the Richmond Mine at Iron Mountain, Calif., we detected 16S rRNA gene sequences of a novel bacterial group belonging to the order Rickettsiales in the Alphaproteobacteria . The closest known relatives of this group (92% 16S rRNA gene sequence identity) are endosymbionts of the protist Acanthamoeba . Oligonucleotide 16S rRNA probes were designed and used to observe members of this group within acidophilic protists . To improve visualization of eukaryotic populations in the acid mine drainage samples, broad-specificity probes for eukaryotes were redesigned and combined to highlight this component of the acid mine drainage community . Approximately 4% of protists in the acid mine drainage samples contained endosymbionts . Measurements of internal pH of the protists showed that their cytosol is close to neutral, indicating that the endosymbionts may be neutrophilic . The endosymbionts had a conserved 273-nucleotide intervening sequence (IVS) in variable region V1 of their 16S rRNA genes . The IVS does not match any sequence in current databases, but the predicted secondary structure forms well-defined stem loops . IVSs are uncommon in rRNA genes and appear to be confined to bacteria living in close association with eukaryotes . Based on the phylogenetic novelty of the endosymbiont sequences and initial culture-independent characterization, we propose the name "Candidatus Captivus acidiprotistae." To our knowledge, this is the first report of an endosymbiotic relationship in an extremely acidic habitat .
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