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[Establishment of a host-vector system in Mycobacterium bovis BCG]
Y Mizuguchi1, H Taniguchi, T Udou
1Department of Microbiology, University of Occupational and Environmental Health, Kitakyushu, Japan.
Abstract:
The recombinant plasmids, pYT72 and pYT92, were generated from a mycobacterial plasmid, pMSC262, and a Escherichia coli plasmid, pACYC177. These plasmids were capable of replication, and of stable maintainance in Mycobacterium bovis BCG when introduced by electroporation technique. Efficiency of transformation was about 10(4) transformants/micrograms DNA, and was the highest in the phage sensitive mutants (S-10, S-20) isolated from BCG Tokyo strain. We have also isolated transformable mutants from rapidly growing bacterium, M. smegmatis strains Jucho and TMC1533. By isolating deletion mutants from pYT72/92, we could determine the location of replication region of pMSC262 within a 2.3 kb Pst I-Hind III fragment. Using this fragment, we constructed "mini" shuttle plasmid pYT937 (5.9 kb in size) which possesses kanamycin and ampicillin resistance markers and replicates in both E. coli and Mycobacterium. Nucleotide sequence analysis of the replication region revealed that there are 2 potential coding regions which contain more than 200 amino acids. The largest one (ORF1) which codes 311 amino acids, however, lacks Shine-Dalgarno like sequence in the upstream and therefore may not be functional. The other coding region (ORF2) contains 260 amino acids and was preceded by Shine-Dalgarno like sequence. Upstream of the ORF2, there were several repeat sequences which may be important in the plasmid replication. GC content of the 2.3 kb fragment was 69.8%.
Insights
Researchers developed new shuttle plasmids for Mycobacterium species, enabling stable replication in both Mycobacterium bovis BCG and Escherichia coli. These plasmids facilitate genetic manipulation in mycobacteria, aiding in the study of these important bacterial pathogens.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Context:
- Mycobacterial plasmids are crucial for genetic studies but often lack efficient replication and maintenance systems in diverse hosts.
- Developing shuttle vectors that replicate in both Escherichia coli and Mycobacterium species is essential for mycobacterial research.
Purpose:
- To construct and characterize novel shuttle plasmids capable of replication and stable maintenance in Mycobacterium bovis BCG and Escherichia coli.
- To identify and analyze the minimal replication region of a mycobacterial plasmid for shuttle vector construction.
Summary:
- Recombinant plasmids pYT72 and pYT92 were created from mycobacterial and E. coli plasmids, demonstrating stable replication in Mycobacterium bovis BCG via electroporation.
- Transformation efficiency was highest in specific phage-sensitive mutants. A 2.3 kb fragment containing the replication region was identified, leading to the construction of the mini shuttle plasmid pYT937.
- Sequence analysis revealed two potential coding regions within the replication fragment, with ORF2 showing characteristics for functionality.
Impact:
- The developed shuttle plasmid pYT937 serves as a valuable tool for genetic engineering in mycobacteria, including Mycobacterium tuberculosis.
- This research enhances the ability to study mycobacterial genetics, potentially leading to new therapeutic strategies against mycobacterial infections.
- The findings contribute to the understanding of mycobacterial plasmid replication mechanisms.