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  • 1
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: An improved method for allele replacement in Pseudomonas aeruginosa was developed. The two main ingredients of the method are: (i) novel ColE1-type cloning vectors derived from pBR322 and pUC19; and (ii) a family of cassettes containing a portable oriT, the sacB gene from Bacillus subtilis as a counter-selectable marker, and a chloramphenicol-resistance gene allowing positive selection of both oriT and sacB. Introduction of plasmid-borne DNA into the chromosome was achieved in several steps. The DNA to be exchanged was first cloned into the new ColE1-type vectors. After insertion of the oriT and sacB sequences, these plasmid were conjugally transferred into P. aeruginosa and plasmid integrants were selected. Plating on sucrose-containing medium allowed positive selection for both plasmid excision and curing since Pseudomonas aeruginosa strains containing the sacB gene in single- or multiple copy were highly sensitive to 5% sucrose in rich medium. This procedure was successfully used to introduce an agmR mutation into P. aeruginosa wild-type strain PA01 and should allow the exchange of any DNA segment into any non-essential regions of the P. aeruginosa chromosome.
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  • 2
    ISSN: 1617-4623
    Keywords: ugp operon, gene regulation ; Regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The gene products of the ugp operon of Escherichia coli are responsible for the uptake of sn-glycerol-3-phosphate and certain glycerophosphodiesters. The regulation of ugp is mainly phoBR-dependent. Significant expression, however, can be observed even in the presence of high concentrations of phosphate, a condition which normally completely represses pho expression. Pho-independent ugp expression was found to be derepressed during the late logarithmic growth phase due to carbon starvation. Among different carbon sources tested, glucose caused the most complete repression. Addition of cAMP prevented glucose repression, indicating that a cAMP-CRP control mechanism may be directly or indirectly involved in the carbon-starvation response. This conclusion is supported by the fact that pho-independent ugp expression correlated with the presence of the cya and crp gene products.
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  • 3
    ISSN: 1617-4623
    Keywords: Gene regulation ; tdc operon ; Escherichia coli ; tdc activator
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Efficient in vivo expression of the biodegradative threonine dehydratase (tdc) operon of Escherichia coli is dependent on a regulatory gene, tdcR. The tdcR gene is located 198 base pairs upstream of the tdc operon and is transcribed divergently from this operon. The nucleotide sequence of tdcR and two unrelated reading frames has been determined. The deduced amino acid sequence of TdcR indicates that is is a polypeptide of Mr 12000 with 99 amino acid residues and contains a potential helix-turnhelix DNA binding motif. Deletion analysis and minicell expression of the tdcR gene suggest that TdcR may serve as a trans-acting positive activator for the tdc operon.
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  • 4
    ISSN: 1617-4623
    Keywords: Escherichia coli ; tdc operon ; Mapping ; Restriction analysis ; Transposable element
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The physical and genetic structure of 37 kilobases of DNA encopassing the tdc region at 68.3 min of the Escherichia coli chromosome was determined by DNA sequence analysis and restriction mapping. Reexamination of new data concerning the direction of transcription of the tdc operon revealed that in strain W3110 the tdc region is located on a transposable segment of DNA.
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  • 5
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 202 (2001), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Triclosan is the active ingredient in a multitude of health care and consumer products with germicidal properties, which have flooded the market in recent years in response to the public's fear of communicable bacteria. Although originally thought to kill bacteria by attacking multiple cellular targets, triclosan was recently shown to target a specific bacterial fatty acid biosynthetic enzyme, enoyl-[acyl-carrier protein] reductase, in Gram-negative and Gram-positive bacteria, as well as in the Mycobacteria. Triclosan resistance mechanisms include target mutations, increased target expression, active efflux from the cell, and enzymatic inactivation/degradation. These are the same types of mechanisms involved in antibiotic resistance and some of them account for the observed cross-resistance with antibiotics in laboratory isolates. Therefore, there is a link between triclosan and antibiotics, and the widespread use of triclosan-containing antiseptics and disinfectants may indeed aid in development of microbial resistance, in particular cross-resistance to antibiotics.
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  • 6
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The contribution of efflux pumps to multidrug resistance in 12 Pseudomonas aeruginosa isolates from various animal sources was assessed. Western immunoblot analyses demonstrated that all twelve isolates expressed significant levels of the MexAB–OprM efflux system whereas two isolates simultaneously expressed the MexEF–OprN or MexXY systems, respectively. One strain contained a single mutation in mexR, a regulator of mexAB–oprM expression, that did not adversely affect the MexR amino acid sequence, and three isolates contained the same, single base change in the mexA–mexR intergenic region. The MexXY-expressing strain contained two base substitutions in its mexZ regulatory gene which did not alter the MexR sequence.
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  • 7
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The LasI/LasR quorum-sensing system plays a pivotal role in virulence gene regulation of the opportunistic human pathogen, Pseudomonas aeruginosa. Here we report the crystal structure of the acyl-homoserine lactone (AHL) synthase LasI that produces 3-oxo-C12-AHL from the substrates 3-oxo-C12-acyl-carrier protein (acyl-ACP) and S-adenosyl-L-methionine. The LasI six-stranded beta sheet platform, buttressed by three alpha helices, forms a V-shaped substrate-binding cleft that leads to a tunnel passing through the enzyme that can accommodate the acyl-chain of acyl-ACP. This tunnel places no apparent restriction on acyl-chain length, in contrast to a restrictive hydrophobic pocket seen in the AHL-synthase EsaI. Interactions of essential conserved N-terminal residues, Arg23, Phe27 and Trp33, suggest that the N-terminus forms an enclosed substrate-binding pocket for S-adenosyl-L-methionine. Analysis of AHL-synthase surface residues identified a binding site for acyl-ACP, a role that was supported by in vivo reporter assay analysis of the mutated residues, including Arg154 and Lys150. This structure and the novel explanation of AHL-synthase acyl-chain-length selectivity promise to guide the design of Pseudomonas aeruginosa-specific quorum-sensing inhibitors as antibacterial agents.
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