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The signal peptidase (SPase) SipS of Bacillus subtilis is responsible for the processing of precursors of secreted proteins. It differs from the SPases of Gram-negative bacteria in structure and specificity. To assay the activity of SipS in vitro, two efficient transcription-translation systems for the synthesis of radio-labelled precursors were developed. The systems were completely derived from B. subtilis. Post-translational in vitro processing of pre-staphylokinase by SipS was demonstrated. SipS activity was stimulated in vitro by several non-ionic detergents, whereas it was not affected by a large variety of proteinase inhibitors. SipS shares the latter property with other SPases.

A method for transformation of whole Bacillus amyloliquefaciens cells by electroporation was developed. The procedure is as efficient as the protoplast transformation method, resulting in up to 10(5) transformants/micrograms plasmid DNA, but requires less effort and time. Cells for electroporation were grown to late exponential phase in a rich medium supplemented with 0.25 M sucrose, washed with and resuspended in 0.25 M sucrose, 1 mM HEPES, 1 mM MgCl2, 10% (v/v) glycerol, pH 7.0, at 3-5 x 10(10) cells/ml for storage at -80 degrees C. The highest transformation frequency was obtained at 7.5 kV/cm with a 25 microF capacitor. The transformation efficiency increased linearly with DNA concentration at least over the range 10 ng-12.5 micrograms/ml. Transformations with ligated DNA and of industrial strains were also successful. In addition, B. subtilis cells treated as above could be transformed by electroporation, resulting in 10(4) transformants/micrograms DNA at 12.5 kV/cm.