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Promoter Sequences Necessary for High-Level Expression of the Plasmid-Associated ampC ß-Lactamase Gene blaMIR-1. Mark D. Reisbig, 2004.Little is known about mechanisms involved in high-level expression of plasmid-associated ampC genes . The sequence for blaMIR-1 has been elucidated, and the gene is not inducible . Although the sequence for the promoter (prA) that drives expression of Enterobacter cloacae chromosomal ampC is present upstream of blaMIR-1, high-level expression from blaMIR-1 is directed from a hybrid promoter (prB) located further upstream of prA . The purpose of this study was to determine the influence of each promoter on blaMIR-1 expression and ß-lactam resistance . RNA expression by deletion clones with both promoters was measured and compared to that by clones in which –35 and/or –10 elements of prA and/or prB were altered . Primer extension revealed two start sites for blaMIR-1 transcription . Expression of blaMIR-1 in clones with both promoters was 171-fold higher than that in clones carrying only prA . In addition, blaMIR-1 expression from prA increased 11-fold in the presence of the prB –10 element compared to expression driven from prA alone . Ceftazidime and cefotaxime MICs increased 42- and 64-fold, respectively, for the clone expressing blaMIR-1 from both promoters compared to expression from prA alone . The upstream promoter prB of blaMIR-1 is solely responsible for high-level expression required for cefotaxime and ceftazidime resistance . These data suggest that resistance to extended-spectrum cephalosporins mediated by noninducible plasmid-associated ampC genes requires the formation of novel promoter elements that are capable of increasing ampC expression . Clinical-Use-Associated Decrease in Susceptibility of Vancomycin-Resistant Enterococcus faecium to Linezolid: a Comparison with Quinupristin-Dalfopristin. Issam I. Raad, 2004.The susceptibility of 135 vancomycin-resistant Enterococcus faecium bacteremic isolates to linezolid and quinupristin-dalfopristin was determined . All were susceptible to linezolid, while 88% were susceptible to quinupristin-dalfopristin prior to the clinical use of the drugs at our hospital . More than 6 months after their clinical use, a decrease in susceptibility was noted for only linezolid at 83% . This was related in part to a single G2576U gene mutation in domain V of the 23S rRNA gene . The Escherichia coli gabDTPC Operon: Specific  -Aminobutyrate Catabolism and Nonspecific Induction.Barbara L. Schneider, 2002.Nitrogen limitation induces the nitrogen-regulated (Ntr) response, which includes proteins that assimilate ammonia and scavenge nitrogen . Nitrogen limitation also induces catabolic pathways that degrade four metabolically related compounds: putrescine, arginine, ornithine, and -aminobutyrate (GABA) . We analyzed the structure, function, and regulation of the gab operon, whose products degrade GABA, a proposed intermediate in putrescine catabolism . We showed that the gabDTPC gene cluster constitutes an operon based partially on coregulation of GabT and GabD activities and the polarity of an insertion in gabT on gabC . A
 gabDT mutant grew normally on all of the nitrogen sources tested except GABA . The unexpected growth with putrescine resulted from specific induction of gab-independent enzymes . Nac was required for gab transcription in vivo and in vitro . Ntr induction did not require GABA, but various nitrogen sources did not induce enzyme activity equally . A gabC (formerly ygaE) mutant grew faster with GABA and had elevated levels of gab operon products, which suggests that GabC is a repressor . GabC is proposed to reduce nitrogen source-specific modulation of expression . Unlike a wild-type strain, a gabC mutant utilized GABA as a carbon source and such growth required
 S . Previous studies showing
S-dependent gab expression in stationary phase involved gabC mutants, which suggests that such expression does not occur in wild-type strains . The seemingly narrow catabolic function of the gab operon is contrasted with the nonspecific (nitrogen source-independent) induction . We propose that the gab operon and the Ntr response itself contribute to putrescine and polyamine homeostasis .
Detection and Enumeration of Aromatic Oxygenase Genes by Multiplex and Real-Time PCR. Brett R. Baldwin, 2003.Our abilities to detect and enumerate pollutant-biodegrading microorganisms in the environment are rapidly advancing with the development of molecular genetic techniques . Techniques based on multiplex and real-time PCR amplification of aromatic oxygenase genes were developed to detect and quantify aromatic catabolic pathways, respectively . PCR primer sets were identified for the large subunits of aromatic oxygenases from alignments of known gene sequences and tested with genetically well-characterized strains . In all, primer sets which allowed amplification of naphthalene dioxygenase, biphenyl dioxygenase, toluene dioxygenase, xylene monooxygenase, phenol monooxygenase, and ring-hydroxylating toluene monooxygenase genes were identified . For each primer set, the length of the observed amplification product matched the length predicted from published sequences, and specificity was confirmed by hybridization . Primer sets were grouped according to the annealing temperature for multiplex PCR permitting simultaneous detection of various genotypes responsible for aromatic hydrocarbon biodegradation . Real-time PCR using SYBR green I was employed with the individual primer sets to determine the gene copy number . Optimum polymerization temperatures for real-time PCR were determined on the basis of the observed melting temperatures of the desired products . When a polymerization temperature of 4 to 5°C below the melting temperature was used, background fluorescence signals were greatly reduced, allowing detection limits of 2 x 102 copies per reaction mixture . Improved in situ microbial characterization will provide more accurate assessment of pollutant biodegradation, enhance studies of the ecology of contaminated sites, and facilitate assessment of the impact of remediation technologies on indigenous microbial populations .
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