The Bacillus subtilis a-amylase gene, amyE, was expressed under the regulated control of sacR, the levansucrase leader region. The gene fusion including the complete 0myE coding sequence with the signal peptide sequence was Tour 43-2, place Jussieu, 75251 Paris Cedex 05, France integrated into the chromosome of a degU32(Hy) strain deleted of the sac6 DNA fragment. In this genetic context, a-amylase is produced in the culture supernatant at a high level (2% of total protein) during the exponential phase of growth upon induction by sucrose. Pulse-chase experiments showed that the rate-limiting step (tin = 120 s) of the secretion process is the release of a cell-associated precursor form whose signal peptide has been cleaved. The efficiency of this ultimate step of secretion decreased dramatically in the presence of a metal chelator (EDTA) or when the cells were converted to protoplasts. The hypothesis that this step is tightly coupled with the folding process of a-amylase occurring within the cell wall environment was substantiated by in vitm folding studies. The unfolding-folding transition, monitored by the resistance to proteolysis, was achieved within the same time range (tin = 60 s) and required the presence of calcium. This metal requirement could possibly be satisfied in vivo by the integrity of the cell wall. The tlR of the a-amylase release step is double that of levansucrase, although their folding rates are similar. This perhaps indicates that the passage through the cell wall may depend on parietal properties (e.g. metal ion binding and porosity) and on certain intrinsic properties of the protein (molecular mass and folding properties).

Single-crossover homologous integration in Lactobacillus sake was studied. Integration was conducted with nonreplicative delivery vector pRV300. This vector is composed of a pBluescript SK ؊ replicon for propagation in Escherichia coli and an erythromycin resistance marker. Random chromosomal DNA fragments of L. sake 23K ranging between 0.3 and 3.4 kb were inserted into pRV300. The resulting plasmids were able to integrate into the chromosome by homologous recombination as single copies and were maintained stably. The single cross-over integration frequency was logarithmically proportional to the extent of homology between 0.3 and 1.2 kb and reached a maximum value of 1.4 ؋ 10 3 integrants/g of DNA. We used this integration strategy to inactivate the ptsI gene, encoding enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system, and the lacL gene, which is one of the two genes required for the synthesis of a functional ␤-galactosidase. The results indicated that our method facilitates genetic analysis of L. sake.

Bacillus subtilis exocellular a-amylase is reversibly refolded after denaturation by guanidine hydrochloride at pH 7 and 37 "C. The unfolding-folding transition monitored by intrinsic fluorescence changes and resistance to proteolysis was resolved into a two-state transition. The first step (ti,* < 1 s) led from D, the totally unfolded state, to C, a stable partially structured state of the protein. This folding intermediate was devoid of any enzyme activity and partially resistant to protease degradation. Calcium was required for the transition from C to N, the native state. This metal did not remain associated with the native form and could be replaced by barium or strontium, but not by magnesium. We discuss the hypothesis that C, the folding intermediate whose further transformation is under kinetic control, is the competent state involved in the secretion process of a-amylase.