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:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Strains of Pseudomonas aeruginosa causing pulmonary infection in cystic fibrosis patients are often mucoid because of the synthesis of a capsular polysaccharide called alginate. Regulation of alginate biosynthesis includes the algB gene product (AlgB), which belongs to a class of proteins that control gene transcription in response to environmental stimuli. In this study, a homolog of the DNA-binding-and-bending protein integration host factor (IHF) and the positive regulatory gene algT were shown to be involved in algB expression. An algB-cat gene fusion was constructed on a low-copy-number, broad-host-range plasmid. In alginate-producing (Alg+) P. aeruginosa, levels of chloramphenicol acetyltransferase from algB-cat were twofold higher than in spontaneous Alg- or algT::Tn501 mutant strains, indicating that the mucoid status of the cell influences algB transcription. An algB transcription initiation site was identified 286 nucleotides upstream of translation initiation and revealed an Escherichia coli sigma 70-like promoter. Sequences in the algB promoter region were highly similar to the consensus E. coli IHF binding site. In DNA gel band mobility shift assays, a protein present in extracts from IHF+ E. coli strains and IHF purified from E. coli bound specifically to these algB DNA fragments, while extracts prepared from isogenic IHF- E. coli strains failed to alter the mobility of algB DNA fragments containing the consensus IHF binding site. A protein in cell extracts prepared from P. aeruginosa strains also demonstrated binding to algB fragments containing the IHF binding site, and the position of the complex formed with these extracts was identical to that of the complex formed with purified IHF. Moreover, this binding could be inhibited by anti-IHF antibodies. To test the role of the IHF site in algB regulation, site-specific mutations in the algB IHF site, based on changes which severely affect IHF binding in E. coli, were generated. When either purified E. coli IHF or extracts from P. aeruginosa were used in DNA binding studies, the algB mutant DNAs were severely reduced in IHF binding. Mutations affecting IHF binding at the algB promoter were introduced into the algB-cat plasmid, and all resulted in severely impaired transcriptional activity in Alg- and algT mutant strains of P. aeruginosa. However, these mutations resulted in similar or slightly reduced algB-cat transcription in Alg+ and algB::Tn501 mutant strains. Thus, the algT product plays a positive role in the high-level expression of algB in mucoid cells, whereas as protein present in P.aeruginosa extracts which is likely an IHF homolog plays a positive role in maintaining a basal level of algB expression in nonmucoid strains.
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PMID:Involvement of the alginate algT gene and integration host factor in the regulation of the Pseudomonas aeruginosa algB gene. 832 Feb 29

The lasR gene of Pseudomonas aeruginosa is required for transcription of the genes for elastase (lasB) and LasA protease (lasA), two proteases associated with virulence. We report here that the alkaline protease gene (apr) also requires the lasR gene for transcription. Alkaline protease mRNA was absent in the lasR mutant PAO-R1 and present when an intact lasR gene was supplied in trans as determined by Northern (RNA) analysis. The lasR gene also enhances exotoxin A production. Exotoxin A activity in supernatants of PAO-R1 were 30% less than in supernatants of the parental strain, PAO-SR. Multiple copies of lasR in trans in PAO-R1 in increased toxin A activity to twice the parental levels. Analysis of PAO-R1 containing the toxA promoter fused to beta-galactosidase suggests that LasR acts at the toxA promoter or at upstream toxA mRNA sequences. beta-Galactosidase activity was approximately 40% lower in PAO-R1 than in the parental strain, PAO-SR. Furthermore, the effect of LasR on the toxA promoter is not due to the stimulation of transcription of regA, a transcriptional activator of toxA. No difference in chloramphenicol acetyltransferase (CAT) activity was noted between PAO-SR and PAO-R1 containing transcriptional regA promoter-CAT gene fusions. These results broaden the regulatory dominion of lasR and suggest that the lasR gene plays a global role in P. aeruginosa pathogenesis.
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PMID:LasR of Pseudomonas aeruginosa is a transcriptional activator of the alkaline protease gene (apr) and an enhancer of exotoxin A expression. 845 22

We report the construction of two cloning vectors that are based on the Pseudomonas-Escherichia shuttle vector, pUCP19. The new vectors, pUCPKS and pUCPSK, contain a significantly expanded multiple cloning site (MCS) with an adjacent T7 promoter sequence. In conjunction with specifically engineered host strains encoding an inducible T7 RNA polymerase, these vectors allow the controlled production of plasmid-encoded proteins in both Escherichia coli and Pseudomonas aeruginosa to analyse the spectrum of products encoded by cloned segments of DNA. The usefulness of these vectors was demonstrated by expressing the chloramphenicol acetyltransferase (CAT)-encoding gene.
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PMID:Construction of improved vectors for protein production in Pseudomonas aeruginosa. 865 79

In Pseudomonas aeruginosa, expression of the lasB gene which codes for the metalloprotease, elastase, depends on small diffusible N-acylhomoserine lactones. lasB expression is regulated through the interactions of N-3-oxododecanoyl-L-homoserine lactone and N-butanoyl-L-homoserine lactone with the transcriptional activators LasR and VsmR(RhlR), respectively. To investigate lasB expression further, we first located the transcriptional start site to a position 141 bp upstream from the translational start site. Using this information, we constructed a series of plasmids containing consecutive 5' deletions of the upstream region of lasB fused to a promoterless chloramphenicol acetyltransferase reporter gene. The results obtained indicate that three regions are required for efficient transcription of lasB; a 35 bp palindromic sequence located at +26 to +60 bp upstream from the translation start site, and two regions located upstream of the transcription start site, at -135 to -85 bp and -63 to -26 bp, respectively. Deletion of the latter region results in the loss of both N-butanoyl-L-homoserine lactone- and N-3-oxododecanoyl-L-homoserine lactone-mediated stimulation of lasB expression and provides further support for the role of this operator site as a target for either or both LasR and VsmR.
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PMID:Dissection of the promoter/operator region and evaluation of N-acylhomoserine lactone mediated transcriptional regulation of elastase expression in Pseudomonas aeruginosa. 901 Oct 52

Expression of ExsC, ExsB, and ExsA (the exoenzyme S trans-regulatory locus) of Pseudomonas aeruginosa was analyzed by using complementation, RNase protection, translational fusion, and T7-directed protein expression analyses. T7 expression analyses in E. coli hosts demonstrated that ExsC, ExsA, and a truncated form of ExsD (a partial open reading frame located 3' of ExsA) were translated; however, a product corresponding to ExsB was undetectable. T7-mediated transcription and translation of the antisense strand resulted in production of a 18.5-kDa product, termed ExsB', which overlapped the predicted ExsB product. In complementation experiments, deletion of the region encoding ExsB and most of ExsB' severely reduced exoenzyme S production. Site-specific mutagenesis of the start codons for ExsB and ExsB', however, did not affect exoenzyme S production. RNase protection studies were initiated to examine the hypothesis that RNA encoded within the ExsB/ExsB' region exerted a regulatory effect. RNA encoding ExsB' was not detectable from chromosomal genes or complementation constructs, indicating that ExsB' was not expressed in P. aeruginosa. To determine the pattern of translation, a chloramphenicol acetyltransferase gene (cat) reporter was fused in frame with ExsB and with ExsA in the context of the entire locus or in the absence of the exsB region. These experiments indicated that exsB was not translated but that deletion of the exsB region affected the translation of ExsA-CAT. RNase protection assays further suggested that deletion of exsB resulted in a processing of ExsA mRNA. Our data indicate that the untranslated exsB region of the trans-regulatory locus mRNA mediates either the stability or the translation of exsA. Complementation analysis further suggests that ExsC may play a role in the translation or stability of ExoS.
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PMID:Functional analysis of exsC and exsB in regulation of exoenzyme S production by Pseudomonas aeruginosa. 904 25

A chloramphenicol acetyltransferase from Pseudomonas aeruginosa genomic DNA has been overexpressed, refolded, purified, and crystallized. Crystals suitable for a three-dimensional x-ray structure determination were obtained from solutions of polyethyleneglycol methyl ether 2000 containing NiCl2 at pH 8.5. These crystals belong to the cubic space group P4(1/3)32 (a = 154.8 A) and diffract x-rays to approximately 3.2 A resolution.
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PMID:Purification and crystallization of Pseudomonas aeruginosa chloramphenicol acetyltransferase. 918 47

In the DNA of bacteriophage phiCTX, the attachment site (attP), the cohesive end site (cos), and the gene (ctx) encoding the pore-forming cytotoxin, are clustered. Deletion variants and PCR-generated fragments of the DNA were cloned into the Pseudomonas aeruginosa and Escherichia coli vector pHA10. Recombinant plasmids carrying the chloramphenicol acetyltransferase gene, cat, were used for promoter studies. Two promoters responsible for ctx expression are located between attP and cos and between cos and ctx, the promoter upstream of cos being stronger than the one downstream. The promoters and the ribosomal binding site are recognized by both the P. aeruginosa and E. coli transcriptional systems. The results indicate that chromosomal integration of phiCTX DNA, which requires cos site ligation, is necessary for high ctx expression in phiCTX-infected P. aeruginosa strains.
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PMID:Control of effective expression of the phage phiCTX-encoded ctx gene in Pseudomonas aeruginosa by a promoter upstream of the cos site. 952 Feb 58

A Tn5-derived mobile element has been constructed to identify genes and promoters related to pathogenesis and virulence in Pseudomonas syringae pv. phaseolicola. To enhance the rate of mutation this Tn5 derivative was constructed carrying a mutant transposase which was placed in cis to the transposable element, but just outside the inverted repeats, therefore eliminating secondary transposition and increasing the stability of the insertion. The new element also contains a promoterless cat (chloramphenicol acetyltransferase) gene as reporter to allow for positive selection of promoters being expressed under specific conditions. To facilitate cloning and manipulations in Escherichia coli, a ColE1 origin of replication has been included within the transposable element as well as the Mob region from the broad-host-range plasmid RP4, which allows this element to be efficiently mobilized by a triparental mating or by using an E. coli strain such as S17-1 to provide the tra functions. Sites for the rare cutters PacI and PmeI have also been included to facilitate locating the insertions on a PacI and/or PmeI physical map. This construction combines the properties of both a mobilizable plasmid and a transposon and therefore has been termed pTn5cat. It is almost the same size as the wild-type Tn5, 5877 bp, and has successfully been tested in P.s. phaseolicola and Xanthomonas campestris pv. campestris.
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PMID:pTn5cat: a Tn5-derived genetic element to facilitate insertion mutagenesis, promoter probing, physical mapping, cloning, and marker exchange in phytopathogenic and other gram-negative bacteria. 957 Nov 37

The crystal structure of the xenobiotic acetyltransferase from Pseudomonas aeruginosa PA103 (PaXAT) has been determined, as well as that of its complex with the substrate chloramphenicol and the cofactor analogue desulfo-coenzyme A. PaXAT is a member of the large hexapeptide acyltransferase family of enzymes that display tandem repeated copies of a six-residue hexapeptide repeat sequence motif encoding a left-handed parallel beta helix (L betaH) structural domain. The xenobiotic acetyltransferase class of hexapeptide acyltransferases is composed of microbial enzymes that utilize acetyl-CoA to acylate a variety of hydroxyl-bearing acceptors. The active site of trimeric PaXAT is a short tunnel into which chloramphenicol and the cofactor analogue desulfo-CoA project from opposite ends. This tunnel is formed by the flat parallel beta sheets of two separate L betaH domains and an extended 39-residue loop. His 79 of the extended loop forms hydrogen bonds from its imidazole NE2 atom to the 3-hydroxyl group of chloramphenicol and from its ND1 group to the peptide oxygen of Thr 86. The interactions of this histidine residue are similar to those found in the structurally unrelated type III chloramphenicol acetyltransferase and suggest that His 79 of PaXAT may be similarly positioned and tautomerically stabilized to serve as a general base catalyst.
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PMID:Structure of the hexapeptide xenobiotic acetyltransferase from Pseudomonas aeruginosa. 957 52

The location and environment of the acquired blaIMP gene, which encodes the IMP-1 metallo-beta-lactamase, were investigated in a Japanese Pseudomonas aeruginosa clinical isolate (isolate 101/1477) that produced the enzyme. In this isolate, blaIMP was carried on a 36-kb plasmid, and similar to the identical alleles found in Serratia marcescens and Klebsiella pneumoniae clinical isolates, it was located on a mobile gene cassette inserted into an integron. The entire structure of this integron, named In31, was determined. In31 is a class 1 element belonging to the same group of defective transposon derivatives that originated from Tn402-like ancestors such as In0, In2, and In5. The general structure of In31 appeared to be most closely related to that of In5 from pSCH884, suggesting a recent common phylogeny for these two elements. In In31, the blaIMP cassette is the first of an array of five gene cassettes that also includes an aacA4 cassette and three original cassettes that have never been described in other integrons. The novel cassettes carry, respectively, (i) a new chloramphenicol acetyltransferase-encoding allele of the catB family, (ii) a qac allele encoding a new member of the small multidrug resistance family of proteins, and (iii) an open reading frame encoding a protein of unknown function. All the resistance genes carried on cassettes inserted in In31 were found to be functional in decreasing the in vitro susceptibilities of host strains to the corresponding antimicrobial agents.
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PMID:Structure of In31, a blaIMP-containing Pseudomonas aeruginosa integron phyletically related to In5, which carries an unusual array of gene cassettes. 1010 96


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