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Membrane Sphingolipid-Ergosterol Interactions Are Important Determinants of Multidrug Resistance in Candida albicans. Kasturi Mukhopadhyay, 2004.In this study, we examined the importance of membrane ergosterol and sphingolipids in the drug susceptibilities of Candida albicans . We used three independent methods to test the drug susceptibilities of erg mutant cells, which were defective in ergosterol biosynthesis . While spot and filter disk assays revealed that erg2 and erg16 mutant cells of C . albicans became hypersensitive to almost all of the drugs tested (i.e., 4-nitroquinoline oxide, terbinafine, o-phenanthroline, itraconazole, and ketoconazole), determination of the MIC at which 80% of the cells were inhibited revealed more than fourfold increase in susceptibility to ketoconazole and terbinafine . Treatment of wild-type C . albicans cells with fumonisin B1 resulted in 45% inhibition of sphingolipid biosynthesis and caused cells to become hypersensitive to the above drugs . Although erg mutants displayed enhanced membrane fluidity and passive diffusion, these changes alone were not sufficient to elicit the observed hypersusceptibility phenotype of erg mutants . For example, the induction in vitro of a 12% change in the membrane fluidity of C . albicans cells by a membrane fluidizer, benzyl alcohol, did not affect the drug susceptibilities of Candida cells . Additionally, the surface localization of green fluorescent protein-tagged Cdr1p, a major drug efflux pump protein of C . albicans, revealed that any disruption in ergosterol and sphingolipid interactions also interfered with its proper surface localization and functioning . A 50% reduction in the efflux of the Cdr1p substrate, rhodamine 6G, in erg mutant cells or in cells with a reduced sphingolipid content suggested a strong correlation between these membrane lipid components and this major efflux pump protein . Taken together, the results of our study demonstrate for the first time that there is an interaction between membrane ergosterol and sphingolipids, that a reduction in the content of either of these two components results in a disruption of this interaction, and that this disruption has deleterious effects on the drug susceptibilities of C . albicans cells . The Membrane-Bound  -Glucuronidase from Pseudomonas cellulosa Hydrolyzes 4-O-Methyl-D-Glucuronoxylooligosaccharides but Not 4-O-Methyl-D-Glucuronoxylan.Tibor Nagy, 2002.The microbial degradation of xylan is a key biological process . Hardwood 4-O-methyl-D-glucuronoxylans are extensively decorated with 4-O-methyl-D-glucuronic acid, which is cleaved from the polysaccharides by -glucuronidases . In this report we describe the primary structures of the
-glucuronidase from Cellvibrio mixtus (C . mixtus GlcA67A) and the
-glucuronidase from Pseudomonas cellulosa (P . cellulosa GlcA67A) and characterize P . cellulosa GlcA67A . The primary structures of C . mixtus GlcA67A and P . cellulosa GlcA67A, which are 76% identical, exhibit similarities with
-glucuronidases in glycoside hydrolase family 67 . The membrane-associated pseudomonad
-glucuronidase released 4-O-methyl-D-glucuronic acid from 4-O-methyl-D-glucuronoxylooligosaccharides but not from 4-O-methyl-D-glucuronoxylan . We propose that the role of the glucuronidase, in combination with cell-associated xylanases, is to hydrolyze decorated xylooligosaccharides, generated by extracellular hemicellulases, to xylose and 4-O-methyl-D-glucuronic acid, enabling the pseudomonad to preferentially utilize the sugars derived from these polymers .
Chromosomal Expression of the Haemophilus influenzae Hap Autotransporter Allows Fine-Tuned Regulation of Adhesive Potential via Inhibition of Intermolecular Autoproteolysis. Doran L. Fink, 2003.The Haemophilus influenzae Hap autotransporter is a nonpilus adhesin that promotes adherence to respiratory epithelial cells and selected extracellular matrix proteins and facilitates bacterial aggregation and microcolony formation . Hap consists of a 45-kDa outer membrane translocator domain called Hapß and a 110-kDa extracellular passenger domain called HapS . All adhesive activity resides within HapS, which also contains protease activity and directs its own secretion from the bacterial cell surface via intermolecular autoproteolysis . In the present study, we sought to determine the relationship between the magnitude of Hap expression, the efficiency of Hap autoproteolysis, and the level of Hap-mediated adherence and aggregation . We found that a minimum threshold of Hap precursor was required for autoproteolysis and that this threshold approximated expression of Hap from a chromosomal allele, as occurs in H . influenzae clinical isolates . Chromosomal expression of wild-type Hap was sufficient to promote significant adherence to epithelial cells and extracellular matrix proteins, and adherence was enhanced substantially by inhibition of autoproteolysis . In contrast, chromosomal expression of Hap was sufficient to promote bacterial aggregation only when autoproteolysis was inhibited, indicating that the threshold for Hap-mediated aggregation is above the threshold for autoproteolysis . These results highlight the critical role of autoproteolysis and an intermolecular mechanism of cleavage in controlling the diverse adhesive activities of Hap . Integrative Transformation System for the Metabolic Engineering of the Sphingoid Base-Producing Yeast Pichia ciferrii. Jung-Hoon Bae, 2003.We have developed an integrative transformation system for metabolic engineering of the tetraacetyl phytosphingosine (TAPS)-secreting yeast Pichia ciferrii . The system uses (i) a mutagenized ribosomal protein L41 gene of P . ciferrii as a dominant selection marker that confer resistance to the antibiotic cycloheximide and (ii) a ribosomal DNA (rDNA) fragment of P . ciferrii as a target for multicopy gene integration into the chromosome . A locus within the nontranscribed region located between 5S and 26S rDNAs was selected as the integration site . A maximum frequency of integrative transformation of approximately 1,350 transformants/µg of DNA was observed . To improve the de novo synthesis of sphingolipid, the LCB2 gene, encoding a subunit of serine palmitoyltransferase, which catalyzes the first committed step of sphingolipid synthesis, was cloned from P . ciferrii and overexpressed under the control of the P . ciferrii glyceraldehyde-3-phosphate dehydrogenase promoter . After transformation of an LCB2 gene expression cassette, several transformants that contained approximately five to seven copies of transforming DNA in the chromosome and exhibited about 50-fold increase in LCB2 mRNA relative to the wild type were identified . These transformants were observed to produce approximately two times more TAPS than the wild type .
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