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  • 1
    Online Resource
    Online Resource
    Stable Transformation of the Xylella fastidiosa Citrus Variegated Chlorosis Strain with oriC Plasmids
    American Society for Microbiology ; 2001
    In:  Applied and Environmental Microbiology Vol. 67, No. 5 ( 2001-05), p. 2263-2269
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 67, No. 5 ( 2001-05), p. 2263-2269
    Abstract: Xylella fastidiosa is a gram-negative, xylem-limited bacterium affecting economically important crops (e.g., grapevine, citrus, and coffee). The citrus variegated chlorosis (CVC) strain of X. fastidiosa is the causal agent of this severe disease of citrus in Brazil and represents the first plant-pathogenic bacterium for which the genome sequence was determined. Plasmids for the CVC strain of X. fastidiosa were constructed by combining the chromosomal replication origin ( oriC ) of X. fastidiosa with a gene which confers resistance to kanamycin (Kan r ). In plasmid p16KdAori, the oriC fragment comprised the dnaA gene as well as the two flanking intergenic regions, whereas in plasmid p16Kori the oriC fragment was restricted to the dnaA - dnaN intergenic region, which contains dnaA -box like sequences and AT-rich clusters. In plasmid p16K, no oriC sequence was present. In the three constructs, the promoter region of one of the two X. fastidiosa rRNA operons was used to drive the transcription of the Kan r gene to optimize the expression of kanamycin resistance in X. fastidiosa . Five CVC X. fastidiosa strains, including strain 9a5c, the genome sequence of which was determined, and two strains isolated from coffee, were electroporated with plasmid p16KdAori or p16Kori. Two CVC isolates, strains J1a12 and B111, yielded kanamycin-resistant transformants when electroporated with plasmid p16KdAori or p16Kori but not when electroporated with p16K. Southern blot analyses of total DNA extracted from the transformants revealed that, in all clones tested, the plasmid had integrated into the host chromosome at the promoter region of the rRNA operon by homologous recombination. To our knowledge, this is the first report of stable transformation in X. fastidiosa . Integration of oriC plasmids into the X. fastidiosa chromosome by homologous recombination holds considerable promise for functional genomics by specific gene inactivation.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.67.5.2263-2269.2001
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2001
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 2
    Online Resource
    Online Resource
    Emergence of Atypical Mycoplasma agalactiae Strains Harboring a New Prophage and Associated with an Alpine Wild Ungulate Mortality Episode
    American Society for Microbiology ; 2012
    In:  Applied and Environmental Microbiology Vol. 78, No. 13 ( 2012-07), p. 4659-4668
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 78, No. 13 ( 2012-07), p. 4659-4668
    Abstract: The bacterium Mycoplasma agalactiae is responsible for contagious agalactia (CA) in small domestic ruminants, a syndrome listed by the World Organization for Animal Health and responsible for severe damage to the dairy industry. Recently, we frequently isolated this pathogen from lung lesions of ibexes during a mortality episode in the French Alps. This situation was unusual in terms of host specificity and tissue tropism, raising the question of M. agalactiae emergence in wildlife. To address this issue, the ibex isolates were characterized using a combination of approaches that included antigenic profiles, molecular typing, optical mapping, and whole-genome sequencing. Genome analyses showed the presence of a new, large prophage containing 35 coding sequences (CDS) that was detected in most but not all ibex strains and has a homolog in Mycoplasma conjunctivae , a species causing keratoconjunctivitis in wild ungulates. This and the presence in all strains of large integrated conjugative elements suggested highly dynamic genomes. Nevertheless, M. agalactiae strains circulating in the ibex population were shown to be highly related, most likely originating from a single parental clone that has also spread to another wild ungulate species of the same geographical area, the chamois. These strains clearly differ from strains described in Europe so far, including those found nearby, before CA eradication a few years ago. While M. agalactiae pathogenicity in ibexes remains unclear, our data showed the emergence of atypical strains in Alpine wild ungulates, raising the question of a role for the wild fauna as a potential reservoir of pathogenic mycoplasmas.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.00332-12
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2012
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 3
    Online Resource
    Online Resource
    P‐Glycoprotein preferentially effluxes anthracyclines containing free basic versus charged amine
    Wiley ; 2001
    In:  European Journal of Biochemistry Vol. 268, No. 6 ( 2001-03-15), p. 1561-1567
    Details
    In: European Journal of Biochemistry, Wiley, Vol. 268, No. 6 ( 2001-03-15), p. 1561-1567
    Abstract: The multidrug resistant (MDR) tumor phenotype, characterized by a decreased cellular drug accumulation is achieved by ATP‐dependent extrusions of drugs from cells by P‐glycoprotein (P‐gp) and/or by multidrug resistance protein (MRP1). Despite the huge amount of research that has been performed on the mechanisms of P‐gp‐mediated efflux of drug, it is not yet known what the molecular parameters are required for a molecule to be recognized and pumped out by P‐gp. Anthracyclines are weak bases and, depending on the pH, can exist either in the neutral or in the positively charged form. The aim of the work reported here was to determine which molecular form is actively pumped out by P‐gp (the neutral form, the protonated form, or both), and if both, the relative efficiencies of pumping. We used spectrofluorometric methods to determine the efflux of anthracyclines in K562/Adr cells, at different intracellular and extracellular pH levels. Using 3′‐deamino, 3′‐hydroxyl doxorubicin (OH‐DOX), which is permanently neutral, we first verified that our methodologies were accurate and that the P‐gp‐mediated efflux of OH‐DOX would not depend on the pH being in the range 6.6–8.4. The P‐gp‐mediated efflux of daunorubicin (DNR) and 3′‐hydroxy‐4‐amino (WP608) was determined at different pH values. These two drugs were chosen because: (a) the lipophilicity of the neutral forms of these two molecules is so similar that any difference in the P‐gp‐mediated efflux cannot be assigned to lipohilicity variation, and (b) their p K a values are different (8.4 and 7.7 for DNR and WP608, respectively), which makes it easy to obtain a large variation in the proportions of the neutral and positively charged forms. Our data show that both forms are recognized by P‐gp but the neutral form is pumped about three times more efficiently than the charged form. This is corroborated by results showing the active efflux (checked at pH i 7.3 only) of five other anthracycline containing a basic center. We interpret these data to mean that: (a) the positive charge of anthracycline is not a necessary requirement for P‐gp recognition, but that (b) the presence of a protonable basic nitrogen facilitates the processing of these compounds by MDR efflux system.
    Type of Medium: Online Resource
    ISSN: 0014-2956 , 1432-1033
    URL: Article
    DOI: 10.1046/j.1432-1327.2001.01989.x
    RVK:
    WA 15000
    Language: English
    Publisher: Wiley
    Publication Date: 2001
    detail.hit.zdb_id: 1398347-7
    detail.hit.zdb_id: 2172518-4
    SSG: 12
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  • 4
    Online Resource
    Online Resource
    Diversity of Shiga Toxin-Producing Escherichia coli (STEC) O26:H11 Strains Examined via stx Subtypes and Insertion Sites of Stx and EspK Bacteriophages
    American Society for Microbiology ; 2015
    In:  Applied and Environmental Microbiology Vol. 81, No. 11 ( 2015-06), p. 3712-3721
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 81, No. 11 ( 2015-06), p. 3712-3721
    Abstract: Shiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen that may be responsible for severe human infections. Only a limited number of serotypes, including O26:H11, are involved in the majority of serious cases and outbreaks. The main virulence factors, Shiga toxins (Stx), are encoded by bacteriophages. Seventy-four STEC O26:H11 strains of various origins (including human, dairy, and cattle) were characterized for their stx subtypes and Stx phage chromosomal insertion sites. The majority of food and cattle strains possessed the stx 1a subtype, while human strains carried mainly stx 1a or stx 2a . The wrbA and yehV genes were the main Stx phage insertion sites in STEC O26:H11, followed distantly by yecE and sbcB . Interestingly, the occurrence of Stx phages inserted in the yecE gene was low in dairy strains. In most of the 29 stx -negative E. coli O26:H11 strains also studied here, these bacterial insertion sites were vacant. Multilocus sequence typing of 20 stx -positive or stx -negative E. coli O26:H11 strains showed that they were distributed into two phylogenetic groups defined by sequence type 21 (ST21) and ST29. Finally, an EspK-carrying phage was found inserted in the ssrA gene in the majority of the STEC O26:H11 strains but in only a minority of the stx -negative E. coli O26:H11 strains. The differences in the stx subtypes and Stx phage insertion sites observed in STEC O26:H11 according to their origin might reflect that strains circulating in cattle and foods are clonally distinct from those isolated from human patients.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.00077-15
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2015
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 5
    Online Resource
    Online Resource
    P-Glycoprotein preferentially effluxes anthracyclines containing free basic versus charged amine
    Wiley ; 2001
    In:  European Journal of Biochemistry Vol. 268, No. 6 ( 2001-03), p. 1561-1567
    Details
    In: European Journal of Biochemistry, Wiley, Vol. 268, No. 6 ( 2001-03), p. 1561-1567
    Type of Medium: Online Resource
    ISSN: 0014-2956 , 1432-1033
    URL: Article
    DOI: 10.1046/j.1432-1033.2001.01989.x
    RVK:
    WA 15000
    Language: English
    Publisher: Wiley
    Publication Date: 2001
    detail.hit.zdb_id: 1398347-7
    detail.hit.zdb_id: 2172518-4
    SSG: 12
    Library Location Call Number Volume/Issue/Year Availability
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    Online Resource
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