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Characterization of NorR Protein, a Multifunctional Regulator of norA Expression in Staphylococcus aureus. Que Chi Truong-Bolduc, 2003.We characterized a Staphylococcus aureus norA gene expression regulator, NorR, initially identified from its binding to the norA promoter . The norR gene was 444 bp in length, located  7
kb upstream from the norA gene, and encoded a predicted 17.6-kDa
protein . Overexpression of norR in wild-type S . aureus
strain ISP794 led to a fourfold decrease in sensitivity to quinolones
and ethidium bromide and an increase in the level of norA
transcripts, suggesting that NorR acts as a positive regulator of
norA expression . Overexpression of norR in sarA and
agr mutants did not alter quinolone sensitivity or levels of
norA transcription, indicating that the presence of these two
global regulatory systems is necessary for NorR to affect the
expression of norA . Insertion and disruption of norR in
ISP794 increased resistance to quinolones by 4- to 16-fold but had no
effect on norA transcription, suggesting that NorR acts as a
repressor for another unidentified efflux pump or pumps . These
mutants also exhibited an exaggerated clumping phenotype in liquid
media, which was complemented fully by a plasmid-encoded norR
gene . Collectively, these results indicate that NorR is a
multifunctional regulator, affecting cell surface properties as well
as the expression of NorA and likely other multidrug resistance
efflux pumps .
Effects of Drug Resistance Mutations L100I and V106A on the Binding of Pyrrolobenzoxazepinone Nonnucleoside Inhibitors to the Human Immunodeficiency Virus Type 1 Reverse Transcriptase Catalytic Complex. Giada A. Locatelli, 2004.We have previously described a novel class of nonnucleoside reverse transcriptase (RT) inhibitors, the pyrrolobenzoxazepinone (PBO) and the pyridopyrrolooxazepinone (PPO) derivatives, which were effective inhibitors of human immunodeficiency virus type 1 (HIV-1) RT, either wild type or carrying known drug resistance mutations (G . Campiani et al., J . Med . Chem . 42:4462-4470, 1999) . The lead compound of the PPO class, (R)-(–)-PPO464, was shown to selectively target the ternary complex formed by the viral RT with its substrates nucleic acid and nucleotide (G . Maga et al., J . Biol . Chem . 276:44653-44662, 2001) . In order to better understand the structural basis for this selectivity, we exploited some PBO analogs characterized by various substituents at C-3 and by different inhibition potencies and drug resistance profiles, and we studied their interaction with HIV-1 RT wild type or carrying the drug resistance mutations L100I and V106A . Our kinetic and thermodynamic analyses showed that the formation of the complex between the enzyme and the nucleotide increased the inhibition potency of the compound PBO354 and shifted the free energy (energy of activation,  G#) for inhibitor binding toward more negative values . The V106A mutation conferred resistance to PBO 354 by increasing its dissociation rate from the enzyme, whereas the L100I mutation mainly decreased the association rate . This latter mutation also caused a severe reduction in the catalytic efficiency of the RT . These results provide a correlation between the efficiency of nucleotide utilization by RT and its resistance to PBO inhibition .
The gnyRDBHAL Cluster Is Involved in Acyclic Isoprenoid Degradation in Pseudomonas aeruginosa. A. L. Díaz-Pérez, 2004.Pseudomonas aeruginosa PAO1 mutants affected in the ability to degrade acyclic isoprenoids were isolated with transposon mutagenesis . The gny cluster (for geranoyl), which encodes the enzymes involved in the lower pathway of acyclic isoprenoid degradation, was identified . The gny cluster is constituted by five probable structural genes, gnyDBHAL, and a possible regulatory gene, gnyR . Mutations in the gnyD, gnyB, gnyA, or gnyL gene caused inability to assimilate acyclic isoprenoids of the citronellol family of compounds . Transcriptional analysis showed that expression of the gnyB gene was induced by citronellol and repressed by glucose, whereas expression of the gnyR gene had the opposite behavior . Western blot analysis of citronellol-grown cultures showed induction of biotinylated proteins of 70 and 73 kDa, which probably correspond to 3-methylcrotonoyl-coenzyme A (CoA) carboxylase and geranoyl-CoA carboxylase (GCCase) alpha subunits, respectively . The 73-kDa biotinylated protein, identified as the  -GCCase subunit, is encoded by gnyA . Intermediary metabolites of the isoprenoid pathway, citronellic and geranic acids, were shown to accumulate in gnyB and gnyA mutants . Our data suggest that the protein products encoded in the gny cluster are the ß and
subunits of geranoyl-CoA carboxylase (GnyB and GnyA), the citronelloyl-CoA dehydrogenase (GnyD), the
 -carboxygeranoyl-CoA hydratase (GnyH), and the 3-hydroxy--carboxygeranoyl-CoA lyase (GnyL) . We conclude that the gnyRDBHAL cluster is involved in isoprenoid catabolism .
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