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  • 1
    Electronic Resource
    Electronic Resource
    Glucose kinase of Streptomyces coelicolor A3(2): large-scale purification and biochemical analysis (2000)
    Springer
    Antonie van Leeuwenhoek 78 (2000), S. 253-261 
    Details
    ISSN: 1572-9699
    Keywords: carbon catabolite repression ; glucose kinase ; Streptomyces
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Glucose kinase of Streptomyces coelicolor A3(2) is essential for glucose utilisation and is required for carbon catabolite repression (CCR) exerted through glucose and other carbon sources. The protein belongs to the ROK-family, which comprises bacterial sugar kinases and regulators. To better understand glucose kinase function, we have monitored the cellular activity and demonstrated that the choice of carbon sources did not significantly change the synthesis and activity of the enzyme. The DNA sequence of the Streptomyces lividans glucose kinase gene glkA was determined. The predicted gene product of 317 amino acids was found to be identical to S. coelicolor glucose kinase, suggesting a similar role for this protein in both organisms. A procedure was developed to produce pure histidine-tagged glucose kinase with a yield of approximately 10 mg/l culture. The protein was stable for several weeks and was used to raise polyclonal antibodies. Purified glucose kinase was used to explore protein-protein interaction by surface plasmon resonance. The experiments revealed the existence of a binding activity present in S. coelicolor cell extracts. This indicated that glucose kinase may interact with (an)other factor(s), most likely of protein nature. A possible cross-talk with proteins of the phosphotransferase system, which are involved in carbon catabolite repression in other bacteria, was investigated.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1010234916745
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    Paper (German National Licenses)
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  • 2
    Electronic Resource
    Electronic Resource
    Construction and characterisation of a series of multi-copy promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase gene from Tn5 as indicator (1986)
    Springer
    Molecular genetics and genomics 203 (1986), S. 468-478 
    Details
    ISSN: 1617-4623
    Keywords: Streptomyces ; Promoter-probes ; Transcription ; Gene expression ; fd terminator
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Several versatile, multi-copy, promoter-probe plasmid vectors have been constructed that replicate in a wide range of Streptomyces species. Transcriptional activity is detected by the expression of a promoter-less aminoglycoside phosphotransferase gene (neo) derived from the transposon Tn5; expression of this gene confers kanamycin and neomycin resistance on Streptomyces lividans. An efficient transcriptional terminator from E. coli phage fd has been inserted upstream of the neo coding region to prevent significant transcriptional read-through from vector promoters. A translational stop codon situated downstream from the site(s) used for cloning and preceding and in frame with the ATG start codon of the neo gene ensures the detection of transcriptional, rather than translational, fusions. Relative promoter strengths can be determined by gradient plate assays of kanamycin resistance, by measuring the amount of aminoglycoside phosphotransferase produced or by estimating neo mRNA synthesised. The high copy number of the vectors facilitates the rapid isolation and characterisation of promoter-active fragments and convenient restriction sites are available for DNA sequencing and S1 mapping of cloned inserts. Some derivatives contain a poly-linker that facilitates the insertion, excision and analysis of cloned fragments and which enhances the use of these plasmids as general cloning vectors.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00422072
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    Paper (German National Licenses)
    Fulltext
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