Expression of bacillar glutamyl endopeptidase genes in Bacillus subtilis by a new mobilizable single-replicon vector pLF.

The pLF1311 natural plasmid from Lactobacillus fermentum 1311 was used to construct a single-replicon vector suitable for rapid cloning in a wide range of gram-positive hosts and Escherichia coli. The new vector is capable of conjugative mobilization from E. coli to various hosts by conjugal transfer. The final vector (3.4 kb) showed a high segregational and structural stability and a high copy number. Glutamyl endopeptidase genes from Bacillus licheniformis (gseBL) and B. intermedius (gseBI) were cloned in both pLF9 and pLF14 vectors and introduced to B. subtilis. The yield of enzymes in the pLF-derived producers was 6- to 30-fold more than in the natural producers and reached 100-150 mg/L of mature protease.

[Structure of the beta-galactosidase gene from Streptococcus diacetylactis 144 and its expression in Escherichia coli and Streptococcus diacetylactis cells]. / Struktura gena beta-galaktozidazy Streptococcus diacetylactis 144 i egoékspressiia v kletkakh Escherichia coli i Streptococcus diacetylactis.

The ability of industrial strains of mesophylic Streptococcus diacetylactis to synthesize the enzyme beta-galactosidase has been studied. Among the 22 studied strains 8 were found to synthesize the enzyme. Plasmid DNA was isolated from the Streptococcus diacetylactis strain 144 possessing the highest level of beta-galactosidase activity. The cells of the strain harbour the 35, 40 and 60 kb plasmids. The alpha-galactosidase genes from this strain was cloned in Escherichia coli cells. The gene is located on the BglIII DNA fragment of the total plasmid DNA from Streptococcus diacetylactis the size of 2.8 kb. Following the Sau3A restriction endonuclease digestion the gene was subcloned on a birepliconed vector plasmid pCB20. The latter is capable of replication in the Gram-negative as well as Gram-positive microorganisms. The pCB20 derivatives carrying the different length fragments with the beta-galactosidase gene were isolated. DNA of an obtained plasmid was used for transformation of Streptococcus diacetylactis cells. The presence of the recombinant plasmid in streptococcus strain 144 results in the 1.8 fold increase in beta-galactosidase production.

[Conjugation transfer of the plasmid pAMbeta1 into various Bacillus species]. / Kon''iugatsionnyi perenos plazmidy pAMbeta1 v batsilly raznykh vidov.

Streptococcal broad host range plasmid pAM beta 1 was transferred by a conjugation-like process from Streptococcus faecalis to 13 strains of different Bacilli species. In intraspecies matings the frequencies of transfer of pAM beta 1 varied from 2.10(-5) to 1.10(-8). As it was shown by comparative analysis the frequency of transfer and stability of the maintainance of plasmid pAM beta 1 in Bacilli were not connected. Molecular weight and restriction pattern of pAM beta 1 DNA isolated from Bacilli were the same as those of pAM beta 1 DNA from Streptococcal donor strain.