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  • 1
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The consequences of using anti-microbial agents in a complex ecosystem like the animal intestine can be difficult to predict. We have looked at effects of modulations in growth of competing intestinal bacteria on transfer and establishment of new genetic elements in the intestinal microflora. For this purpose, we used tetracycline, which gradually reduces the growth rate of tetracycline-sensitive bacteria, as the concentration of this drug is increased. The effect of tetracycline on transfer and establishment of the plasmid RP4, which encodes resistance to this drug, in populations of Escherichia coli BJ4 colonizing the intestine was investigated. A tetracycline-sensitive E. coli BJ4 strain was allowed to establish in the gastrointestinal tract of mice, where after an isogenic E. coli BJ4 carrying RP4 was given to the mice per os. Tetracycline in the drinking water given to the animals was kept in concentrations that allowed the sensitive recipient strain to colonize the gut. A given ‘window’ between the highest and the lowest antibiotic doses tested was shown to be optimal for the establishment of transconjugants in the intestine. These observations are important for the evaluation of the effect of a given drug on the intestinal ecosystem. A reduced potential for growth of a given bacterial species, caused by the presence of sub-inhibitory concentrations of a bacteriostatic antibiotic, will facilitate establishment of competing (i.e. closely related) organisms, which have acquired resistance genes and therefore grow well in the presence of the drug.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology ecology 42 (2002), S. 0 
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: An efficient approach for the insertion of fluorescent marker genes with sequence specificity into conjugative plasmids in Escherichia coli is described. For this purpose, homologous recombination of linear double-stranded targeting DNA was mediated by the bacteriophage λ recombination functions using very short regions of homology. Initial manipulation of the IncFII target plasmids R1 and R1drd19 indicated that the linear targeting DNA should be devoid of all extraneous homologies to the target molecule for optimal insertion specificity. Indeed, a simple recombination assay proved that in the presence of additional homologous regions in the targeting DNA, strand exchanges occurred exclusively within the longest regions of homology. A versatile panel of vectors was created to facilitate convenient PCR amplification of targeting DNAs containing various combinations of different antibiotic resistance genes and fluorescent markers. The choice of 5′ non-homologous extensions in primer pairs used for amplifying the marker cassettes determines the site specificity of the targeting DNA. This methodology is applicable to the modification of all plasmids that replicate in E. coli and is not restricted by plasmid size.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology ecology 26 (1998), S. 0 
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: PCR amplification of 16S rDNA was found to be highly biased, so that the rDNA from one species out of four was preferentially amplified. We present evidence that the observed PCR bias most likely occurs because the genomic DNA of some species contains segments outside the amplified sequence that inhibit the initial PCR steps. Attempts to overcome this bias by use of a ‘touch down’ PCR procedure or by performing PCR in the presence of denaturants or cosolvents such as acetamide, DMSO, or glycerol were unsuccessful. Since the PCR inhibiting interference from template flanking DNA segments evidently is dependent on the position of the primer sites, we suggest that community diversity analysis based on PCR amplification of 16S rDNA can be improved by extending the procedure from comparative analysis of 16S rDNA amplified by use of only one primer set to a procedure involving at least two different 16S rDNA PCR amplifications performed with different primer sets.
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  • 4
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The potential of the green fluorescent protein (GFP) as a marker gene for ecological investigations of an activated sludge community was assessed. By inserting the hybrid transposon mini-Tn5 gfp into the chromosome of Pseudomonas putida KT2442 a strongly fluorescent mutant was obtained. This strain was used for in vivo tracking of individual cells after introduction into a simple sludge microcosm. It is demonstrated that the observed reduction of introduced bacteria from sewage is mainly the result of predation by protozoa. The feasibility of combining detection of GFP fluorescence with whole cell hybridization employing fluorescently labeled, rRNA-targeted oligonucleotides in paraformaldehyde fixed samples is demonstrated.
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  • 5
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 148 (1997), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Serratia liquefaciens secretes a broad spectrum of hydrolytic enzymes to the surrounding medium and possesses the ability to differentiate into specialized swarmer cells capable of rapid surface motility. Control of exoenzyme production and swarming motility is governed by similar regulatory components, including a quorum-sensing mechanism and the flagellar master operon flhDC.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 149 (1997), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Exponential phase cells of Pseudomonas putida KT2442 rapidly lost viability when incubated at 0°C without entering a viable but non-culturable state. The majority of dead cells retained their cellular integrity and contained DNA. However, their cellular rRNA content was substantially reduced. By employing a luciferase-marked derivative of P. putida KT2442 in combination with a highly sensitive low-light imaging system, live and dead cells could be distinguished.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Several bacterial species possess the ability to differentiate into highly motile swarmer cells capable of rapid surface colonization. In Serratia liquefaciens, we demonstrate that initiation of swarmer-cell differentiation involves diffusible signal molecules that are released into the growth medium. Using high-performance liquid chromatography (HPLC), high resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy, we identified N-butanoyl-l-homoserine lactone (BHL) and N-hexanoyl-l-homoserine lactone (HHL) in cell-free Serratia culture supernatants. BHL and HHL are present in a ratio of approximately 10:1 and their structures were unequivocally confirmed by chemical synthesis. The swrlswarmer initiation) gene, the predicted translation product of which exhibits substantial homology to the Luxl family of putative Nacyl homoserine lactone (AHL) synthases is responsible for directing synthesis of both BHL and HHL. In an swrl mutant, swarming motility is abolished but can be restored by the addition of an exogenous AHL. These results add swarming motility to the rapidly expanding list of phenotypes known to be controlled through quorum sensing.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Single-cell localization and activity of Pseudomonas fluorescens F113, colonizing alfalfa roots, were monitored using fusions of the Escherichia coli rrnBP1 ribosomal promoter and gfp genes encoding green fluorescent protein (Gfp) of different stability. The monitoring systems permitted non-destructive in situ detection of F113rifpcb cells on the entire root system grown in both the presence and absence of 3-chlorobiphenyl (PCB-2). The root tip and sites of lateral root emergence were found to be hotspots for fast-growing cells. In addition, a reporter strain of P. fluorescens F113rifpcb for degradation of chlorinated biphenyl was constructed, using another gfp fusion with the meta-pathway Pm promoter from Pseudomonas putida (TOL plasmid). Expression of this promoter, which is strongly induced by the PCB-2 degradation product, 3-chlorobenzoate, was tested in vitro and subsequently monitored in vivo on alfalfa roots using the P. fluorescens F113rifpcb reporter. A small but distinct fraction of the introduced bacteria activated the Pm promoter and thus appeared to sense a PCB-2 degradation product in the alfalfa rhizosphere. The degrading cells, which by design were identical to the sensing cells, were located in distinct microcolonies on the root surface or in intercellular crevices between the root epidermal cells. However, PCB-degrading cells were not observed in the root areas containing fast-growing cells, indicating that PCB degradation was not linked to high cellular activity.
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  • 9
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology ecology 34 (2000), S. 0 
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Recombinant derivatives of Pseudomonas putida strain KT2440 are of potential interest as microbial inoculants to be deliberately released for agricultural applications. To facilitate tracking of this strain and its derivatives after introduction into the environment, a mini-Tn5-′luxAB transposon was introduced into the chromosome of P. putida KT2440, yielding strain P. putida S1B1. Sequencing of the DNA region located upstream of the ′luxAB genes and similarity search with the P. putida KT2440 genome sequence, localized the transposon within a 3021-bp open reading frame (ORF), whose translated sequence showed significant similarity with the hypothetical YdiJ proteins from Escherichia coli and Haemophilus influenzae. A second ORF adjacent to and divergent from the ydiJ sequence was also found and showed significant homology with various LysR-type transcriptional activator proteins from several bacteria. Disruption of the ydiJ locus in P. putida S1B1 did not affect the survival of the strain in unvegetated or vegetated soils. Bioluminescent detection of P. putida S1B1 cells enriched in selective media directly from soil allowed detection of culturable cells in soil samples over a period of at least 8 months. The addition of the luxAB biomarker facilitates tracking in the root system of several plant species grown under sterile and non-sterile conditions. The correlation of the bioluminescent phenotype with the growth activity of P. putida S1B1 cells colonizing the root system of barley and corn plants was estimated by monitoring ribosomal contents using quantitative hybridization with fluorescence-labeled ribosomal RNA probes. A correlation between inoculum density, light output, and ribosomal contents was found for P. putida cells colonizing the root system of barley seedlings grown under sterile conditions. Although ribosomal contents, and therefore growth activity, of P. putida S1B1 cells extracted from the rhizosphere of corn plants grown in non-sterile soil were similar to those found in starved cells, the luminescent system permitted non-destructive in situ detection of the strain in the upper root system.
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  • 10
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 142 (1996), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract Heat treatment of Salmonella typhimurium results in cell death, which coincides with a significant reduction of the cellular content of 16S ribosomal RNA. It is suggested that the degradation of ribosomal RNA is a direct cause of cell death. This conclusion is based on the observations of carbon-starved and magnesium-supplemented cells, which survive heat treatment much better, and which also maintain stable levels of ribosomal RNA.
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