Restriction on Conjugational Transfer of pLS20 in Bacillus subtilis 168

Conjugational transfer of pLS20 in Bacillus subtilis Marburg 168 is restricted by the BsuM restriction-modification system. Restriction efficiency was measured using pLS20 derivatives possessing various numbers of XhoI sites, which are known to be recognized by BsuM. An increase in XhoI sites clearly reduced the conjugational efficiency of pLS20 as compared with that of pUB110 plasmid lacking XhoI.     

A system for the inducible secretion of proteins from Bacillus subtilis during logarithmic growth

Abstract A Bacillus subtilis-Escherichia coli shuttle vector was constructed containing the B. subtilis levansucrase gene promoter and region encoding its signal sequence.
A site for the restriction enzyme Nae I was included to facilitate precise translational fusions to the DNA encoding the levansucrase signal sequence. Fusions of TEM β-lactamase to this construct displayed sucrose-inducible expression and secretion of B. subtilis .     

Cloning in Bacillus subtilis of temperature-dependent promoter fragments from Bacillus stearothermophilus and Bacillus subtilis

Abstract Using promoter-probe plasmids, more than 200 promoter-containing fragments from Bacillus stearothermophilus and Bacillus subtilis were cloned in B. subtilis . Among these, 15 promoter fragments were highly temperature-dependent in activity compared to the promoter sequence (TTGAAA for the −35 region, TATAAT for the −10 region) of the amylase gene, amyT , from B. stearothermophilus . Some fragments exhibited higher promoter activities at elevated temperature (48°C), others showed higher activities at lower temperature (30°C). Active promoter fragments at higher and lower temperatures were obtained mainly from the thermophile ( B. stearothermophilus ) and the mesophile ( B. subtilis ), respectively. A promoter fragment active at high temperature was sequenced, and the feature of the putative promoter region was discussed.     

Prochymosin expression in Bacillus subtilis

Prochymosin (PC) sequence was cloned in Bacillus subtilis using two kinds of plasmid constructions. In plasmid pSM316 the cDNA was inserted to obtain the intracellular expression of the enzyme. The enzyme turned out to be expressed in an insoluble form which could be converted to native enzyme under proper denaturing and refolding conditions. The levels of intracellular expression of PC were further enhanced by modifying the 5' region of the gene in a way that a two-cistron expression system was created. For the PC secretion, the cDNA was fused to the subtilisin leader sequence and expressed under the control of the B. subtilis neutral protease promoter. A properly folded PC was secreted by the cells, although to low levels.