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Role of the Multidrug Efflux System MexXY in the Emergence of Moderate Resistance to Aminoglycosides among Pseudomonas aeruginosa Isolates from Patients with Cystic Fibrosis. Christelle Vogne, 2004.This study investigates the role of active efflux system MexXY in the emergence of aminoglycoside (AG) resistance among cystic fibrosis (CF) isolates of Pseudomonas aeruginosa . Three genotypically related susceptible and resistant (S/R) bacterial pairs and three other AG-resistant CF strains were compared to four non-CF strains moderately resistant to AGs . As demonstrated by immunoblot experiments, pump MexY was strongly overproduced in all of the resistant bacteria . This MexXY upregulation was associated with a 2- to 16-fold increase in the MICs of AGs in the S/R pairs and lower intracellular accumulation of dihydrostreptomycin . Alterations in mexZ, the repressor gene of operon mexXY, were found in all of the AG-resistant CF isolates and in one non-CF strain . Complementation of these bacteria with a plasmid-borne mexZ gene dramatically reduced the MICs of AGs, thus highlighting the role played by MexXY in the development of moderate resistance in CF patients . In contrast, complementation of the three non-CF strains showing wild-type mexZ genes left residual levels of resistance to AGs . These data indicate that a locus different from mexZ may be involved in overproduction of MexXY and that other nonenzymatic mechanisms contribute to AG resistance in P . aeruginosa . Bacillus subtilis Mutant LicT Antiterminators Exhibiting Enzyme I- and HPr-Independent Antitermination Affect Catabolite Repression of the bglPH Operon. Cordula Lindner, 2002.The Bacillus subtilis antiterminator LicT regulates the expression of bglPH and bglS, which encode the enzymes for the metabolism of aryl-ß-glucosides and the ß-glucanase BglS . The N-terminal domain of LicT (first 55 amino acids) prevents the formation of  -independent terminators on the respective transcripts by binding to target sites overlapping these terminators . Proteins of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulate the antitermination activity of LicT by phosphorylating histidines in its two PTS regulation domains (PRDs) . Phosphorylation at His-100 in PRD-1 requires the PTS proteins enzyme I and HPr and the phosphorylated permease BglP and inactivates LicT . During transport and phosphorylation of aryl-ß-glucosides, BglP is dephosphorylated, which renders LicT active and thus leads to bglPH and bglS induction . In contrast, phosphorylation at His-207 and/or His-269 in PRD-2, which requires only enzyme I and HPr, is absolutely necessary for LicT activity and bglPH and bglS expression . We isolated spontaneous licT mutants expressing bglPH even when enzyme I and HPr were absent (as indicated by the designation "Pia" [PTS-independent antitermination]) . Introduced in a ptsHI+ strain, two classes of licT(Pia) mutations could be distinguished . Mutants synthesizing LicT(Pia) antiterminators altered in PRD-2 still required induction by aryl-ß-glucosides, whereas mutations affecting PRD-1 caused constitutive bglPH expression . One of the two carbon catabolite repression (CCR) mechanisms operative for bglPH requires the
-independent terminator and is probably prevented when LicT is activated by P His-HPr-dependent phosphorylation in PRD-2 (where the prefix "P" stands for "phospho") . During CCR, the small amount of PHis-HPr present in cells growing on repressing PTS sugars probably leads to insufficient phosphorylation at PRD-2 of LicT and therefore to reduced bglPH expression . In agreement with this concept, mutants synthesizing a PHis-HPr-independent LicT(Pia) had lost LicT-modulated CCR .
Use of a Green Fluorescent Protein-Based Reporter Fusion for Detection of Nitric Oxide Produced by Denitrifiers. Shixue Yin, 2003.To determine if green fluorescent protein could be used as a reporter for detecting nitric oxide production, gfp was fused to nnrS from Rhodobacter sphaeroides 2.4.3 . nnrS was chosen because its expression requires nitric oxide . The presence of the fusion in R . sphaeroides 2.4.3 resulted in a significant increase in fluorescent intensity of the cells, but only when nitrite reductase was active . Cells lacking nitrite reductase activity and consequently the ability to generate nitric oxide were only weakly fluorescent when grown under denitrification-inducing conditions . One of the R . sphaeroides strains unable to generate nitric oxide endogenously was used as a reporter to detect exogenously produced nitric oxide . Incubation of this strain with sodium nitroprusside, a nitric oxide generator, significantly increased its fluorescence intensity . Mixing of known denitrifiers with the reporter strain also led to significant increases in fluorescence intensity, although the level varied depending on the denitrifier used . The reporter was tested on unknown isolates capable of growing anaerobically in the presence of nitrate, and one of these was able to induce expression of the fusion . Analysis of the 16S rRNA gene sequence of this isolate placed it within the Thauera aromatica subgroup, which is known to contain denitrifiers . These experiments demonstrate that this green fluorescent protein-based assay provides a useful method for assessing the ability of bacteria to produce nitric oxide .
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