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Involvement of a Plasmid-Encoded Type IV Secretion System in the Plant Tissue Watersoaking Phenotype of Burkholderia cenocepacia. Amanda S. Engledow, 2004.Burkholderia cenocepacia strain K56-2, a representative of the Burkholderia cepacia complex, is part of the epidemic and clinically problematic ET12 lineage . The strain produced plant tissue watersoaking [ptw] on onion tissue, which is a plant disease-associated trait. Using plasposon mutagenesis, mutants in the ptw phenotype were generated . The translated sequence of a disrupted gene [ptwD4] from a ptw-negative mutant showed homology to VirD4-like proteins. Analysis of the region proximal to the transfer gene homolog identified a gene cluster located on the 92-kb resident plasmidthat showed homology to type IV secretion systems . The roleof ptwD4, ptwC, ptwB4, and ptwB10 in the expression of ptw activity was determined by conducting site-directed mutagenesis . Theptw phenotype was not expressed by K56-2 derivatives with adisruption in ptwD4, ptwB4, or ptwB10 but was observed in a derivative with a disruption in ptwC . Complementation of ptw-negative K56-2 derivatives in trans resulted in complete restoration of the ptw phenotype . In addition, analysis of culture supernatants revealed that the putative ptw effector[s] was a secreted, heat-stable protein[s] that caused plasmolysis of plant protoplasts . A second chromosomally encoded type IV secretion system with complete homology to the VirB-VirD system was identified in K56-2 . Site-directed mutagenesis of key secretory genes in the VirB-VirD system did not affect expression of the ptw phenotype . Our findings indicate that in strain K56-2, the plasmid-encoded Ptw type IV secretion system is responsible for the secretion of a plant cytotoxic protein[s]. The Pseudomonas fluorescens AlgG Protein, but Not Its Mannuronan C-5-Epimerase Activity, Is Needed for Alginate Polymer Formation. Martin Gimmestad, 2003.Bacterial alginates are produced as 1-4-linked ß-D-mannuronan, followed by epimerization of some of the mannuronic acid residues to  -L-guluronic
acid . Here we report the isolation of four different
epimerization-defective point mutants of the periplasmic Pseudomonas
fluorescens mannuronan C-5-epimerase AlgG . All mutations affected
amino acids conserved among AlgG-epimerases and were clustered
in a part of the enzyme also sharing some sequence similarity to a
group of secreted epimerases previously reported in Azotobacter
vinelandii . An algG-deletion mutant was constructed and found
to produce predominantly a dimer containing a 4-deoxy-L-erythro-hex-4-enepyranosyluronate
residue at the nonreducing end and a mannuronic acid residue at
the reducing end . The production of this dimer is the result of the
activity of an alginate lyase, AlgL, whose in vivo activity is much
more limited in the presence of AlgG . A strain expressing both an
epimerase-defective (point mutation) and a wild-type epimerase was
constructed and shown to produce two types of alginate molecules: one
class being pure mannuronan and the other having the wild-type
content of guluronic acid residues . This formation of two distinct
classes of polymers in a genetically pure cell line can be explained
by assuming that AlgG is part of a periplasmic protein complex .
Posaconazole Is a Potent Inhibitor of Sterol 14  -Demethylation in Yeasts and Molds.Hanan K. Munayyer, 2004.Posaconazole (POS; SCH 56592) is a novel triazole that is active against a wide variety of fungi, including fluconazole-resistant Candida albicans isolates and fungi that are inherently less susceptible to approved azoles, such as Candida glabrata . In this study, we compared the effects of POS, itraconazole (ITZ), fluconazole (FLZ), and voriconazole (VOR) on sterol biosynthesis in strains of C . albicans (both azole-sensitive and azole-resistant strains), C . glabrata, Aspergillus fumigatus, and Aspergillus flavus . Following exposure to azoles, nonsaponifiable sterols were extracted and resolved by liquid chromatography and sterol identity was confirmed by mass spectroscopy . Ergosterol was the major sterol in all but one of the strains; C . glabrata strain C110 synthesized an unusual sterol in place of ergosterol . Exposure to POS led to a decrease in the total sterol content of all the strains tested . The decrease was accompanied by the accumulation of 14 -methylated sterols, supporting the contention that POS inhibits the cytochrome P450 14-demethylase enzyme . The degree of sterol inhibition was dependent on both dose and the susceptibility of the strain tested . POS retained activity against C . albicans isolates with mutated forms of the 14-demethylase that rendered these strains resistant to FLZ, ITZ, and VOR . In addition, POS was a more potent inhibitor of sterol synthesis in A . fumigatus and A . flavus than either ITZ or VOR .
Effect of Granulocyte Colony-Stimulating Factor Combination Therapy on Efficacy of Posaconazole (SCH56592) in an Inhalation Model of Murine Pulmonary Aspergillosis. Andriani C. Patera, 2004.Using an inhalation model of pulmonary aspergillosis, we observed modest differences in the survival rates of mice treated with granulocyte colony-stimulating factor (G-CSF) and posaconazole (POS) and those treated with POS alone . This finding is in contrast to a previous report that suggested that G-CSF had a significant antagonistic effect on the antifungal activity of POS .
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