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Salmonella infections continue to cause gastrointestinal and systemic disease throughout the world. Salmonella typhimurium DT104 further poses a major health concern due to its acquisition of resistance to multiple antibiotics. The rapid detection of multiresistant S. typhimurium DT104 would facilitate strategies aimed at controlling this pathogen. We developed a specific and sensitive polymerase chain reaction (PCR) assay that amplifies a segment of DNA that is conserved in multiresistant S. typhimurium DT104. To provide further specificity for this PCR-based diagnostic test, we amplified two other gene fragments that are present in S. typhimurium DT104. A multiplex PCR containing primers for targeted sequences resulted in the amplification of predicted size fragments from S. typhimurium DT104 exhibiting the ACSSuT (ampicillin, chloramphenicol, streptomycin, sulphamethoxazole and tetracycline) or ASSuT resistance phenotypes. A minor modification of the multiplex PCR enabled the detection of other related multiresistant Salmonella such as S. typhimurium U302. To augment the detection process, we also designed a fluorogenic PCR assay that can detect the DNA of multiresistant S. typhimurium DT104 in the presence of excess contaminating bacterial DNA. These results provide a method by which multiresistant S. typhimurium DT104, or potentially the next emerging multiresistant Salmonella, can be accurately detected in only 3-4 h.
Mol Cell Probes 1999 Jun
PMID:Detection of multiresistant Salmonella typhimurium DT104 using multiplex and fluorogenic PCR. 1036 47

Myxococcus xanthus is a gram-negative bacterium that develops in response to starvation on a solid surface. The cells assemble into multicellular aggregates in which they differentiate from rod-shaped cells into spherical, environmentally resistant spores. Previously, we have shown that the induction of beta-lactamase is associated with starvation-independent sporulation in liquid culture (K. A. O'Connor and D. R. Zusman, Mol. Microbiol. 24:839-850, 1997). In this paper, we show that the chromosomally encoded beta-lactamase of M. xanthus is autogenously induced during development. The specific activity of the enzyme begins to increase during aggregation, before spores are detectable. The addition of inducers of beta-lactamase in M. xanthus, such as ampicillin, D-cycloserine, and phosphomycin, accelerates the onset of aggregation and sporulation in developing populations of cells. In addition, the exogenous induction of beta-lactamase allows M. xanthus to fruit on media containing concentrations of nutrients that are normally too high to support development. We propose that the induction of beta-lactamase is an integral step in the development of M. xanthus and that this induction is likely to play a role in aggregation and in the restructuring of peptidoglycan which occurs during the differentiation of spores. In support of this hypothesis, we show that exogenous induction of beta-lactamase can rescue aggregation and sporulation of certain mutants. Fruiting body spores from a rescued mutant are indistinguishable from wild-type fruiting body spores when examined by transmission electron microscopy. These results show that the signal transduction pathway leading to the induction of beta-lactamase plays an important role in aggregation and sporulation in M. xanthus.
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PMID:Induction of beta-lactamase influences the course of development in Myxococcus xanthus. 1051 21

The two-dimensional membrane topology of the Escherichia coli and Staphylococcus aureus MraY transferases, which catalyse the formation of the first lipid intermediate of peptidoglycan synthesis, was established using the beta-lactamase fusion system. All 28 constructed mraY-blaM fusions produced hybrid proteins. Analysis of the ampicillin resistance of the strains with hybrids led to a common topological model possessing 10 transmembrane segments, five cytoplasmic domains and six periplasmic domains including the N- and C-terminal ends. The agreement between the topologies of E. coli and S. aureus, their agreement to a fair extent with predicted models and a number of features arising from the comparative analysis of 25 orthologue sequences strongly suggested the validity of the model for all eubacterial MraYs. The primary structure of the 10 transmembrane segments diverged among orthologues, but they retained their hydrophobicity, number and size. The similarity of the sequences and distribution of the five cytoplasmic domains in both models, as well as their conservation among the MraY orthologues, clearly suggested their possible involvement in substrate recognition and in the catalytic process. Complementation tests showed that only fusions with untruncated mraY restored growth. It was noteworthy that S. aureus MraY was functional in E. coli. An increased MraY transferase activity was observed only with the untruncated hybrids from both organisms.
Mol Microbiol 1999 Nov
PMID:Topological analysis of the MraY protein catalysing the first membrane step of peptidoglycan synthesis. 1056 98

A new set of broad-host-range promoter-probe vectors has been constructed. One subset contains the pVS1 and p15a replicons and confers resistance to either gentamicin or kanamycin. The other set contains the broad-host-range replicon from pBBR1 and confers resistance to kanamycin, tetracycline, ampicillin, or spectinomycin/streptomycin. Both plasmid sets are highly stable and are maintained without selection for more than 30 generations in several bacterial taxa. Each plasmid contains a promoter-probe cassette that consists of a multicloning site, containing several unique restriction sites, and gfp or inaZ as a reporter gene. The cassette is bound by transcriptional terminators to permit the insertion of strong promoters and to insulate the cassette from external transcription enabling the detection of weak or moderate promoters. The vector suite was augmented with derivatives of the kanamycin-resistant gfp promoter-probe plasmids that encode Gfp variants with different half-life times.
Mol Plant Microbe Interact 2000 Nov
PMID:Improved gfp and inaZ broad-host-range promoter-probe vectors. 1105 91

We constructed several versatile sets of vectors that can be used to introduce any gene into the pgl/picA locus of the Agrobacterium tumefaciens C58 chromosome without affecting T-DNA transfer. One set contains a fragment containing the lacIq and lacZ genes and a multiple cloning site from pBluescriptII SK(+) inserted into a PstI site between the pgl and picA genes on an incPalpha plasmid. The resulting plasmid contains eight unique restriction endonuclease sites and the ability to use blue-white screening for the presence of an insert. A second plasmid also contains a beta-lactamase gene within this locus and provides a convenient ampicillin-carbenicillin resistance marker for the selection of genes integrated into the chromosome following double homologous recombination (homogenotization). A third plasmid contains, in addition to the lacZ, lacIq, and beta-lactamase genes within the pgl/picA locus, a sacRB gene cassette within the vector to counterselect against the presence of the vector within A. tumefaciens. To test this system, we introduced a wild-type virD2 gene into the A. tumefaciens chromosome at the pgl/picA locus. When a Ti plasmid harboring a deletion of virD2 was in this strain, the integrated virD2 gene complemented the virD2 deletion and the resulting transformation phenotype was identical to that resulting from A. tumefaciens strains harboring a wild-type virD2 gene located on a replicating plasmid.
Mol Plant Microbe Interact 2001 Apr
PMID:Novel constructions to enable the integration of genes into the Agrobacterium tumefaciens C58 chromosome. 1131 Jul 46

Single-chain Fv (scFv) antibody libraries were constructed from mice immunized with an ampicillin-bovine serum albumin conjugate. Several antibodies with specificity for intact ampicillin were selected by phage display and characterized. The antibody scFv fragment aL2 binds to intact ampicillin and shows no detectable cross-reactivity with hydrolyzed ampicillin. We determined the X-ray structures of two crystal forms of w.t. aL2, which differ mainly in the side-chain conformation of Trp H109 (according to a new consensus nomenclature Kabat residue number H95) in the extremely short (three residues) CDR H3 and the presence or absence of a well-resolved molecule of 2-methyl-pentane-2,4-diol in the bottom of the binding pocket. Attempts to co-crystallize aL2 with its antigen or to diffuse ampicillin into the wild-type aL2 crystals were unsuccessful, since crystal contacts obstruct the binding pocket. However, a mutant with two point mutations near the N terminus (Gln H6 replaced by Glu and Ala H10 (Kabat H9) replaced by Gly) crystallized in a form compatible with antigen-binding. Although the mutations affect the conformation of framework I, the conformations of the binding pocket of the uncomplexed wild-type aL2 and of the mutant complex were almost identical. The structure explains the specificity of the antibody for intact ampicillin and the degree of cross-reactivity of aL2 with a wide variety of ampicillin analogs. This antibody system will be very useful as a diagnostic reagent for antibiotics use and abuse, as a model for the effect of expression of antibiotic binding molecules in Escherichia coli, and for directed evolution towards high antibiotic resistance.
J Mol Biol 2001 Jun 08
PMID:Selection, characterization and x-ray structure of anti-ampicillin single-chain Fv fragments from phage-displayed murine antibody libraries. 1139 88

Deacetoxycephalosporin C synthase from Streptomyces clavuligerus catalyses the conversion of the five-membered penicillin ring to the unsaturated six-membered cephem ring of deacetoxycephalosporin C. The effects on enzyme activity of the penicillin substrate sidechain and various cofactors were investigated using a continuous spectrophotometric assay. The conversion of penicillin G to phenylacetyl-7-aminodeacetoxycephalo sporanic acid (G-7-ADCA) was confirmed, and further details of the reaction were elucidated. The conversion of ampicillin to cephalexin was faster than that of acetyl-6-APA to acetyl-7-ADCA kcat = 0.120 +/- 0.001 s(-1) versus 0.035 +/- 0.001 s(-1), but they had similar Km values: 4.86 +/- 0.12 and 3.28 +/- 0.26 mM, respectively. Amoxycillin and penicillin V were also converted at low levels. Conversion was not detected for penicillanate, 6-aminopenicillanate, carbenicillin, temocillin, ticarcillin or benzylpenicilloic acid, suggesting that the enzyme has a relatively strict selectivity for the sidechain of the penicillin substrate.
Cell Mol Life Sci 2001 May
PMID:Probing the penicillin sidechain selectivity of recombinant deacetoxycephalosporin C synthase. 1143 42

DNA microarrays provide a global view of the physiological state of the cell by parallel analysis of the expression levels of all the genes in an organism. The effects of four bactericidal agents on the expression pattern of Escherichia coli MG1655 were assessed. Compounds were chosen on the basis of their different mechanisms of action and included inhibitors of DNA replication and recombination, translation, transcription and cell wall biosynthesis. The addition of rifampin resulted in increased expression of the target, rpoB, as well as several genes involved in nucleotide salvage and purine biosynthesis. The addition of ampicillin resulted in overall changes in gene expression that showed some similarity to changes induced by rifampin. The addition of the antibiotics kanamycin or norfloxacin resulted in the induction of unique gene expression signatures: a heat shock response to kanamycin and an SOS response to norfloxacin. Several genes of unknown function showed expression profiles similar to the genes associated with the SOS or the heat shock response. Thus, these profiles define families of genes with similar expression phenotypes that can be tested for related function.
J Mol Microbiol Biotechnol 2003
PMID:Comparison of the changes in global gene expression of Escherichia coli induced by four bactericidal agents. 1273 33

Because peptide nucleic acids (PNAs) are capable of blocking amplification of deoxyribonucleic acid (DNA) by Taq DNA polymerase in vitro, we postulated that PNAs might be able to block replication in vivo. To explore this possibility, we assessed the ability of PNA to specifically block the replication of pUC19 plasmids by allowing a PNA, directed against segments of the Ampr sequence to bind to pUC19 prior to electroporation into Escherichia coli, strain DH10B. Colonies produced by this maneuver not only remained sensitive to ampicillin but were also incapable of blue color production on X-gal-containing media, thus demonstrating true blockade of pUC19 replication, rather than antisense activity. The ability of the PNA to prevent pUC19 replication in these experiments was shown to be dose related. Attempts to prevent the replication of E. coli using a PNA directed against a portion of the lac Z sequence found within the bacterial genome were not uniformly successful. Subsequent experiments showed that the electroporated PNA did not consistently enter a sufficient number of cells for an effect to be demonstrated in the assays used. Nonetheless, this is the first demonstration of in vivo complete replication blockade by a PNA and opens up the potential for new forms of specific antibiosis in both prokaryotic and eukaryotic cells.
Mol Biotechnol 2003 Nov
PMID:Blockade of plasmid replication mediated by peptide nucleic acids. 1466 37

Two allosteric enzymes have been created by the covalent linkage of non-interacting, monomeric proteins with the prerequisite effector-binding and catalytic functionalities, respectively. This was achieved through a combinatorial process called random domain insertion. The fragment of the TEM-1 beta-lactamase gene coding for the mature protein lacking its signal sequence was randomly inserted into the Escherichia coli maltose-binding protein (MBP) gene to create a domain insertion library. This library's diversity derived both from the site of insertion and from a distribution of tandem duplications or deletions of a portion of the MBP gene at the insertion site. From a library of approximately 2 x 10(4) in-frame fusions, approximately 800 library members conferred a phenotype to E.coli cells that was consistent with the presence of bifunctional fusions that could hydrolyze ampicillin and transport maltose in E.coli. Partial screening of this bifunctional sublibrary resulted in the identification of two enzymes in which the presence of maltose modulated the rate of nitrocefin hydrolysis. For one of these enzymes, the presence of maltose increased k(cat) by 70% and k(cat)/K(m) by 80% and resulted in kinetic parameters that were almost identical to TEM-1 beta-lactamase. Such an increase in activity was only observed with maltooligosaccharides whose binding to MBP is known to induce a conformational change. Modulation of the rate of nitrocefin hydrolysis could be detected at maltose concentrations less than 1 microM. Intrinsic protein fluorescence studies were consistent with a conformational change being responsible for the modulation of activity.
J Mol Biol 2004 Feb 06
PMID:Creation of an allosteric enzyme by domain insertion. 1474 Dec 21


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