Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A field-scale experiment with a complete randomized block design was performed to study the degradation of buried oil on a shoreline over a period of almost 1 year. The following four treatments were examined in three replicate blocks: two levels of fertilizer treatment of oil-treated plots, one receiving a weekly application of liquid fertilizer and the other treated with a slow-release fertilizer; and two controls, one not treated with oil and the other treated with oil but not with fertilizer. Oil degradation was monitored by measuring carbon dioxide evolution and by chemical analysis of the oil. Buried oil was degraded to a significantly greater extent in fertilized plots, but no differences in oil chemistry were observed between the two different fertilizer treatments, although carbon dioxide production was significantly higher in the oil-treated plots that were treated with slow-release fertilizer during the first 14 days of the experiment. Bacterial communities present in the beach sediments were profiled by denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA gene fragments and 16S rRNA amplified by reverse transcriptase PCR. Similarities between the DGGE profiles were calculated, and similarity matrices were subjected to statistical analysis. These analyses showed that although significant hydrocarbon degradation occurred both in plots treated with oil alone and in the plots treated with oil and liquid fertilizer, the bacterial community structure in these plots was, in general, not significantly different from that in the control plots that were not treated with oil and did not change over time. In contrast, the bacterial community structure in the plots treated with oil and slow-release fertilizer changed rapidly, and there were significant differences over time, as well as between blocks and even within plots. The differences were probably related to the higher concentrations of nutrients measured in interstitial water from the plots treated with slow-release fertilizer. Bacteria with 16S rRNA sequences closely related (>99.7% identity) to Alcanivorax borkumensis and Pseudomonas stutzeri sequences dominated during the initial phase of oil degradation in the plots treated with slow-release fertilizer. Field data were compared to the results of previous laboratory microcosm experiments, which revealed significant differences.
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PMID:Bacterial community dynamics and hydrocarbon degradation during a field-scale evaluation of bioremediation on a mudflat beach contaminated with buried oil. 1512 9

Regulation of medium-chain-length polyhydroxyalkanoate biosynthesis in Pseudomonas aeruginosa and Pseudomonas putida was studied conducting PHA accumulation experiments and transcriptional analysis of PHA biosynthesis genes with wild type strains and rpoN-negative mutants. In P. putida PHA accumulation was RpoN-independent, whereas in P. aeruginosa PHA accumulation was RpoN-dependent. Transcriptional analysis applying reverse transcriptase-polymerase chain reaction showed strong induction of phaG, encoding the transacylase, under nitrogen starvation in P. putida KT2440 and the respective rpoN-negative mutant, indicating an RpoN-independent regulation of phaG. No transcription of phaG and no PHA accumulation was detected in the rpoN-negative mutant of P. aeruginosa neither from gluconate nor from octanoate as carbon source. Alginate-overproducing mutant P. aeruginosa FRD1 showed strongly decreased PHA accumulation from gluconate but no difference in phaC1 (encoding the PHA synthase) transcription, indicating that alginate biosynthesis competes with PHA biosynthesis regarding acetyl-CoA as precursor for both biopolymers. Transcription of phaF and phaI-F was nitrogen independent.
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PMID:Regulation of polyhydroxyalkanoate biosynthesis in Pseudomonas putida and Pseudomonas aeruginosa. 1526 31

The effects of imipenem and meropenem on the transcriptional expression of resistance-related genes oprD, oprM and oprN in Pseudomonas aeruginosa were studied by quantitative real-time PCR. Four strains were examined: the type strain PT5 (PAO1), its derivatives M7 and PT149, and a clinical isolate, PaKT3. The derivative M7 is a nalB mutant, overexpressing the MexAB-OprM pump, and the derivative PT149 is a nfxC-type mutant, overexpressing the MexEF-OprN pump while it is down-regulated for the OprD protein. After 18 h incubation in broth, the cultures were divided into three portions. Two were supplemented with antibiotics and the other was left antibiotic-free as the control. After a further 45 min incubation, total RNA was isolated from the strains by guanidine denaturation and acid-phenol/chloroform extraction. DNA-free total RNAs were immediately reverse-transcribed by MMuLV reverse transcriptase. Concentrations of mRNAs obtained by quantitative PCR were expressed relative to uninduced portions of the strains. The results showed that oprD was relatively stable against carbapenem antibiotics. oprM was induced significantly by imipenem in only one strain and oprN was induced by imipenem in most of the strains. The responses at the mRNA level found here were unexpected and suggested a chaotic, unpredictable regulatory mechanism.
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PMID:Effect of carbapenems on the transcriptional expression of the oprD, oprM and oprN genes in Pseudomonas aeruginosa. 1531

Fluoroquinolones and carbapenems are important drug classes used for the treatment of Pseudomonas aeruginosa infections. However, overexpression of the P. aeruginosa efflux pump, MexEF-OprN, can provide dual resistance to both fluoroquinolones and carbapenems. Recently, a hospital in Texas encountered several P. aeruginosa isolates resistant to both of these drug classes. The purpose of this study was to determine whether the mechanism responsible for this multidrug resistance involved the overexpression of MexEF-OprN. To test this hypothesis, 7 clinical isolates from the Texas hospital were analyzed for clonality, antimicrobial susceptibility, expression of the porin, oprD, and four multidrug-resistant efflux pumps (mexAB-oprM, mexCD-oprJ, mexEF-oprN, and mexXY), quinolone resistance-determining region mutations, and beta-lactamase production. Two groups of isolates possessed similar pulse field gel electrophoresis patterns indicating their genetic relatedness. In addition to fluoroquinolone and carbapenem resistance, each isolate also displayed varying degrees of susceptibility to additional beta-lactams tested and resistance to gentamicin. Interestingly, none of the 7 clinical isolates overexpressed mexEF-oprN as determined by semiquantitative reverse transcriptase polymerase chain reaction, but overexpression of mexXY was observed in 6 of the 7 isolates. All 7 isolates showed a decrease of OprD in the outer membrane and a reduction in transcriptional expression of oprD compared to wild-type strain, PAO1. These results demonstrate that multidrug resistance to the fluoroquinolones and carbapenems in these clinical isolates was not a result of the overexpression of the mexEF-oprN pump. Instead, resistance to these two agents seemed to arise through independent mutational events.
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PMID:Multidrug resistance associated with mexXY expression in clinical isolates of Pseudomonas aeruginosa from a Texas hospital. 1538 Feb 77

The expression of iron regulated genes in bacteria is typically controlled by the ferric uptake regulator (Fur) protein, a global transcriptional repressor that regulates functions as diverse as iron acquisition, oxidative stress, and virulence. We have identified a fur homologue in Dichelobacter nodosus, the causative agent of ovine footrot, and shown that it complements an Escherichia coli fur mutant. Homology modeling of the D. nodosus Fur protein with the recently solved crystal structure of Fur from Pseudomonas aeruginosa indicated extensive structural conservation. As Southern hybridization analysis of different clinical isolates of D. nodosus indicated that the fur gene was present in all of these strains, the fur gene was insertionally inactivated to determine its functional role. Analysis of these mutants by various techniques did not indicate any significant differences in the expression of known virulence genes or in iron-dependent growth. However, we determined several Fur regulatory targets by two-dimensional gel electrophoresis coupled with mass spectrometry. Analysis of proteins from cytoplasmic, membrane, and extracellular fractions revealed numerous differentially expressed proteins. The transcriptional basis of these differences was analyzed by using quantitative reverse transcriptase PCR. Proteins with increased expression in the fur mutant were homologues of the periplasmic iron binding protein YfeA and a cobalt chelatase, CbiK. Down-regulated proteins included a putative manganese superoxide dismutase and ornithine decarboxylase. Based on these data, it is suggested that in D. nodosus the Fur protein functions as a regulator of iron and oxidative metabolism.
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PMID:Identification of a Dichelobacter nodosus ferric uptake regulator and determination of its regulatory targets. 1560 21

Azotobacter vinelandii belongs to the y subdivision of eubacteria and has one of the highest respiratory rates. It is considered to be among the probable progenitors of mitochondria. Group II introns were originally identified on organelle genomes. Analysis of the A. vinelandii genome for the presence of group II introns using a deduced group II intron consensus sequence identified two putative introns. The first intron (AVI) which was found to be inserted in the groEL, an essential gene, was already characterized. Our study identified another group II intron (AV2) in A. vinelandii genome. This intron is inserted in a mobile genetic element, similar to most of the group II introns in bacteria, which in this case is a transposase like gene, tnpAl. This putative TnpAl protein is 52% identical to TnpA, the transposase of bacteriophage Lambda, and 85% identical to TnpAl of Pseudomonas stutzeri. Sequence analysis showed that this intron encodes a reverse transcriptase (RT) like motif in domain IV, similar to other group II introns. The RT of this intron open reading frame (ORF) is 53% homologous with that of AVI intron and 66% homologous with that of Pseudomonas putida (Tn5041c) intron. Secondary structure analysis showed that this intron has the typical sub-group IIB1 structure, but the EBS2-IBS2 interaction appears to be missing. Using the RNA generated by in vitro transcription of the intron sequence with its flanking exons, in vitro splicing experiments were performed. It was found that the AV2 intron is functional, despite of lacking the EBS2-IBS2 interaction that plays a role in exon recognition.
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PMID:Analysis of the genome of Azotobacter vinelandii revealed the presence of two genetically distinct group II introns on the chromosome. 1613 25

Pseudomonas aeruginosa poses a serious risk in individuals suffering from cystic fibrosis (CF). Strains colonizing the CF lung are generally motile but frequently convert to a nonmotile phenotype as the disease progresses. In many cases, this is coordinately regulated with the overproduction of the exopolysaccharide alginate. Both the expression of alginate (mucoidy) and the loss of flagellum synthesis may provide the bacterium with a selective advantage in the CF lung. Previously published data showed that the regulation of alginate production and flagellum biosynthesis in the CF isolate FRD1 is inversely controlled by the alternative sigma factor AlgT. In this study, we observed that in CF isolates, the mucoid and the nonmotile phenotypes occur predominantly together. Using microarrays, we compared the transcriptomes of isogenic AlgT(+) and AlgT(-) P. aeruginosa and discovered that AlgT significantly downregulated the majority of flagellar genes. A pronounced inhibitory effect was observed in several genes essential for proper flagellum expression, including fleQ, which encodes an essential flagellar regulator. The microarray data were confirmed by reverse transcriptase PCR analysis and promoter fusion assays in isogenic AlgT(+) and AlgT(-) strains. Transmission electron microscopy, motility assays, and Western blots showed that ectopic expression of FleQ in mucoid, nonmotile CF isolates restored flagellum biosynthesis and motility. Together, these data show that AlgT mediates the negative control of flagellum expression by inhibiting the expression of the flagellar regulator fleQ.
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PMID:The alternative sigma factor AlgT represses Pseudomonas aeruginosa flagellum biosynthesis by inhibiting expression of fleQ. 1629 68

Reverse transcription of RNA molecules inside intact bacterial cells was carried out by using reverse transcriptase with a single oligonucleotide complementary to specific 16S rRNA or mRNA sequences. Fluorescently labeled nucleotides were incorporated into each transcribed cDNA inside cells. This protocol is termed in situ reverse transcription (ISRT). In this study, by using species-specific primers targeting unique regions of the 16S rRNA sequences, ISRT was used successfully to detect and enumerate the two lignin-degrading bacteria Microbulbifer hydrolyticus IRE-31 and Sagittula stellata E-37 in culture mixtures and complex enrichment communities selected for lignin degradation. Image analysis revealed that M. hydrolyticus IRE-31 and S. stellata E-37 accounted for approximately 30 and 2%, respectively, of the total bacterial cells in lignin enrichment communities. Populations estimated by ISRT were comparable to those estimated by in situ hybridization (ISH) techniques and to those estimated by hybridization against extracted community DNA. ISRT was also successfully used to detect Pseudomonas putida F1 expressing the todC1 gene in seawater exposed to toluene vapor. ISRT provided a higher signal intensity than ISH, especially when targeting mRNA. The calculated pixel intensities resulting from ISRT were up to 4.2 times greater than those from ISH. This suggests that multiple incorporation of fluorescently labeled nucleotides into cDNA provides a high sensitivity for phylogenetic identification of bacterial populations as well as detection of cells expressing a specific functional gene within complex bacterial communities.
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PMID:In situ reverse transcription, an approach to characterize genetic diversity and activities of prokaryotes. 1653 53

In this study we examined polyhydroxyalkanoate (PHA) synthases phaC1 and phaC2 gene expression in two strains of Pseudomonas corrugata (Pc) grown in a minimum mineral medium with related (oleic acid and octanoate) or unrelated (glucose) carbon sources. Analysis of transcription was performed by Northern blot and conventional reverse transcriptase (RT) polymerase chain reaction (PCR). In addition, we developed a RT-real-time PCR method to quantitatively evaluate phaC1 (Pc) and phaC2 (Pc) gene expression. Primers and a TaqMan probe were designed for the specific detection of both synthase transcripts as well as of the housekeeping 16S rRNA, and the relative expression of target genes was calculated. We showed that phaC1 (Pc) and phaC2 (Pc) were not cotranscribed and, on the contrary, were independently regulated. In cultures grown with oleic acid as the sole carbon source, only the expression of phaC1 (Pc) was induced (a tenfold increase after 72 h of culture), whereas that of phaC2 (Pc) remained unchanged. In cultures grown with glucose or sodium octanoate, the expression of both phaC1 (Pc) and phaC2 (Pc) was upregulated but at different rates. Cellular PHA content was compared to the gene expression of the PHA synthases and significant correlations were found between PHA production and phaC1 (Pc)/phaC2 (Pc) expression.
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PMID:Regulation of polyhydroxyalkanoate synthases (phaC1 and phaC2) gene expression in Pseudomonas corrugata. 1660 28

Carbapenems are important agents for the therapy of infections due to multidrug-resistant Pseudomonas aeruginosa; the development of carbapenem resistance hampers effective therapeutic options. To assess the mechanisms leading to resistance, 33 clinical isolates with differing degrees of carbapenem susceptibility were analyzed for the expression of the chromosomal beta-lactamase (ampC), the porin that is important for the entry of carbapenems (oprD), and the proteins involved in four efflux systems (mexA, mexC, mexE, and mexX). Real-time reverse transcriptase PCR was performed using primers and fluorescent probes for each of the target genes. The sequencing of regulatory genes (ampR, mexR, nalC, nalD, mexT, and mexZ) was also performed. Diminished expression of oprD was present in all imipenem- and meropenem-resistant isolates but was not required for ertapenem resistance. Increased expression of ampC was not observed in several isolates that were overtly resistant to carbapenems. Increased expression of several efflux systems was observed in many of the carbapenem-resistant isolates. Increased efflux activity correlated with high-level ertapenem resistance and reduced susceptibility to meropenem and aztreonam. Most isolates with increased expression of mexA had mutations affecting nalC and/or nalD. Two isolates with mutations leading to a premature stop codon in mexZ had markedly elevated mexX expressions, although mutations in mexZ were not a prerequisite for overexpression. beta-Lactam resistance in clinical isolates of P. aeruginosa is a result of the interplay between diminished production of oprD, increased activity of ampC, and several efflux systems.
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PMID:Interplay of efflux system, ampC, and oprD expression in carbapenem resistance of Pseudomonas aeruginosa clinical isolates. 1664 29


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