Transfer and expression of recombinant plasmids carrying pneumococcal mal genes in Bacillus subtilis.

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.