The rate of exocellular levansucrase synthesis in an overproducing (sacUh) strain of Bacillus suhtilis was shown to be directly proportional to the amount of two different transient forms of this enzyme located within the membrane fraction of the cells. The apparent M , of the larger membrane form was 53000, and that of the smaller form 50000; the half-life time of each form was estimated in vivo to be 4-6 s and 32-42 s, respectively. Ethanol treatment of the cells lead to the accumulation of the 53 000-M, form which may represent 1.5% of total membrane proteins. This latter form, partially purified, was transformed in vitro into the 50000-M, form by the action of the Escherichia coli leader peptidase. These enzyme forms were quite different from the exocellular levansucrase since they showed a weak affinity for hydroxyapatite and needed complexed iron to display enzyme activity. Assuming the membrane forms were precursors of exocellular levansucrase, we propose a two-step mechanism for the secretion process of levansucrase. The number of exoprotein synthesis/secretion sites in a B. suhtilk cell is estimated to 2.5 x lo4. [4] the sacUh strain QBl12, when fully induced, produces only exocellular levansucrase (about 8% of total proteins) and, moreover, this enzyme became the unique protein secreted in the culture medium during the exponential phase of growth. Thus, this strain seems to contain optimal characteristics for a biochemical study of levansucrase secretion.The existence of a 53000-M, precursor form of levansucrase was demonstrated by the expression of the cloned structural gene of this enzyme in a minicell-producing strain of Escherichia coli; the nucleotide sequence of the signal peptide was determined [5, 61. The levansucrase precursor form synthesized in minicells in the presence of phenethyl alcohol was found in the outer membrane fraction [7]. However, the yield of the precursor form accumulated in such conditions (less than 0.02% of total protein) was too low for purification. Furthermore, characterization of the other components of a presumed B. subtilis export machinery is not possible using this approach. In order to prepare a large amount of the precursor of exocellular levansucrase, we investigated the conditions of its synthesis and accumulation in

The refolding of levansucrase denatured by urea was studied as a possible model for the second step of the secretion pathway of this protein. The folding-unfolding transition was monitored by measuring intrinsic fluorescence and resistance to proteolysis. Both methods provided the same estimation for the unfolding free energy of levansucrase, delta GD, which was 30.1 +/- 1.7 kJ.mol-1 (7.2 +/- 0.4 kcal.mol-1) at pH 7 in 0.1 M-potassium phosphate buffer. The rate of refolding was greatly enhanced by Fe3+, whereas the Fe3+ chelator EDTA prevented correct refolding. Fe3+ allowed the protein to reach its folded form in medium in which the dielectric constant had been lowered by ethanol. The efficiency in vivo of the export of levansucrase bearing an amino acid modification which blocks the second step of the translocation pathway was greatly increased by high concentrations of Fe3+ in the culture medium. Assuming that the protein folding governs the second step of the secretion process of levansucrase, we discuss from an irreversible thermodynamic point of view the possible role of Fe3+ in the efficient coupling of the two events.

Studies of the equilibrium between native and denatured forms of wild-type levansucrase showed that the denatured form was predominant at 37 degrees C and pH 7 in the absence of free metal. The shift to the native form was promoted by metal ions such as Fe3+ or Ca2+. This metal-dependent refolding process was not observed in levansucrase variants bearing the amino acid substitution Gly-366----Asp or Gly-366----Val. These variants were only slightly secreted by Bacillus subtilis although their signal sequences were normally cleaved and their exocellular forms stable. In contrast, the Gly-366----Ser variant was secreted at near-normal levels and shared a part of the in vitro refolding properties of the wild-type protein. These differential properties might be related to the ability of the altered region to form a beta-turn structure. We discuss the possible role of metal ions in the coupling of protein folding and secretion.