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Transfer and expression of recombinant plasmids carrying pneumococcal mal genes in Bacillus subtilis
Abstract:
The pneumococcal mal recombinant plasmid pLS70, which carries two strong promoters for transcription, could not be transferred and maintained intact in Bacillus subtilis. Although it could be established at low frequency, pLS70 was unstable and was rapidly replaced by deleted forms of the plasmid. A deleted derivative plasmid, pLS69, could be transferred at high frequency and maintained intact. In pLS69 the deletion reduces function of both the malM (amylomaltase) and malX (X-fragment) promoters. This mutant mal plasmid still codes for an intact amylomaltase, and the enzyme is produced in both S. pneumoniae and B. subtilis. The amylomaltase, which is inducible by maltose in S. pneumoniae, is synthesized constitutively in B. subtilis and is localized in the cytosol. Although pLS69 enables S. pneumoniae to grow with maltose, the plasmid did not enhance the ability of B. subtilis to use this sugar, presumably because the latter does not transport free maltose into the cell. Minicells of B. subtilis containing pLS69 synthesized the amylomaltase polypeptide but no X-fragment. In S. pneumoniae carrying pLS69, production of the X-fragment is also reduced more than the amylomaltase, when compared to cells carrying pLS70, which produce equal amounts of the two proteins. Inasmuch as the down promoter mutation leaves unchanged both structural genes, their ribosome-binding sites and -10 and -35 promoter sequences, the unequal effect is attributed to differential reduction in AT composition proximal to the promoters. Vector proteins were revealed in minicells as several bands, all located in the cytosol except for an Mr 35000 polypeptide located in the membrane.
Insights
A modified plasmid, pLS69, enables stable transfer and maintenance in Bacillus subtilis, unlike its predecessor. This engineered plasmid facilitates amylomaltase production in bacteria, though its utility for maltose utilization in B. subtilis is limited.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- The pneumococcal mal recombinant plasmid pLS70 exhibits instability and poor maintenance in Bacillus subtilis.
- Deletions in pLS70 lead to unstable forms, hindering its effective use in B. subtilis.
Purpose of the Study:
- To investigate the transfer and maintenance characteristics of a modified mal recombinant plasmid (pLS69) in Bacillus subtilis.
- To analyze the expression and function of amylomaltase and the X-fragment from pLS69 in both Streptococcus pneumoniae and Bacillus subtilis.
Main Methods:
- Construction and characterization of the deleted plasmid derivative pLS69.
- Assessment of plasmid transfer frequency and stability in Bacillus subtilis.
- Analysis of amylomaltase and X-fragment production in minicells and whole cells of S. pneumoniae and B. subtilis.
- Investigation of maltose utilization in B. subtilis strains harboring pLS69.
Main Results:
- The deleted plasmid pLS69 demonstrated high-frequency transfer and stable maintenance in Bacillus subtilis.
- pLS69 encodes an intact amylomaltase, constitutively produced in the cytosol of B. subtilis and inducibly in S. pneumoniae.
- While pLS69 supports maltose growth in S. pneumoniae, it does not enhance maltose utilization in B. subtilis due to transport limitations.
- Differential expression of amylomaltase and X-fragment was observed, attributed to promoter down mutations and AT composition.
Conclusions:
- The engineered plasmid pLS69 is stable and efficiently maintained in Bacillus subtilis.
- The study elucidates the expression patterns and functional limitations of mal genes in different bacterial hosts.
- Plasmid stability and gene expression are influenced by promoter strength, sequence composition, and host-specific factors.