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A host-vector system for an Arthrobacter species.

P C Shaw1, B S Hartley

  • 1Centre for Biotechnology, Imperial College of Science and Technology, London, UK.

Journal of General Microbiology
|April 1, 1988
PubMed
Summary
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A novel host-vector system was developed for Arthrobacter sp. NRRL B3728, enabling efficient genetic manipulation for industrial applications like glucose isomerase production. This system utilizes hybrid vectors for high transformation frequencies.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Biotechnology

Background:

  • Industrial strains of Arthrobacter sp. are crucial for producing enzymes like glucose isomerase.
  • Efficient genetic tools are needed for strain improvement and metabolic engineering in Arthrobacter.
  • Existing host-vector systems may lack efficiency or applicability for specific Arthrobacter strains.

Purpose of the Study:

  • To develop an efficient host-vector system for the industrial Arthrobacter sp. strain NRRL B3728.
  • To construct and characterize novel hybrid vectors for transformation of Arthrobacter protoplasts.
  • To assess the transformation efficiency, stability, and utility of the developed system for genetic manipulation.

Main Methods:

  • Protoplast generation from Arthrobacter sp. NRRL B3728 using lysozyme and osmotic stabilization with sodium succinate.

Related Experiment Videos

  • Construction of hybrid vectors (pBL2100, pCG1100, pCG2100) by combining E. coli plasmid pBR322, a kanamycin-resistance gene, and cryptic plasmids.
  • Transformation of Arthrobacter protoplasts with hybrid vectors and screening for kanamycin resistance.

    • Main Results:

    • Successfully generated and regenerated Arthrobacter protoplasts with approximately 30% efficiency.
    • Developed three hybrid vectors capable of transforming Arthrobacter protoplasts and conferring kanamycin resistance.
    • Achieved high transformation frequencies ranging from 10^5 to 10^6 transformants per microgram of plasmid DNA.
    • Demonstrated structural stability of plasmids pBL2100 and pCG2100 in Arthrobacter cells, though maintenance required selective pressure.
    • Identified a deletion mutant (pCG2120) capable of transforming Arthrobacter protoplasts.

    Conclusions:

    • The developed host-vector system provides an efficient platform for genetic engineering of industrial Arthrobacter sp. NRRL B3728.
    • The hybrid vectors offer unique restriction sites for cloning foreign DNA, facilitating further genetic studies and applications.
    • This system enhances the potential for strain improvement and the development of novel biotechnological processes using Arthrobacter.