Increasing the stability of sacB transcript improves levansucrase production in Bacillus subtilis.

Aims: To develop a strategy to increase the stability of transcripts of structural genes expressed under the control of sacR, the leader region of Bacillus subtilis levansucrase gene. Methods and Results: Insertion of Shine Dalgarno like sequences in the 5'-untranslated sacR region controlling the expression of sacB. Depending on the number of stabilizing sequences inserted and the position of these sequences with respect to the translation start codon, it was observed that the mRNA stability and the final protein production could be increased or decreased. Conclusions: This mRNA stabilization can be used to increase exocellular protein production in the degU32 (Hy) mutant. Significance and Impact of the Study: This approach can be applied to the expression of heterologous genes of biotechnological interest

Autogenous modulation of the Bacillus subtilis sacB-levB-yveA levansucrase operon by levB transcript.

Silencing of levB, the second structural gene of the tricistronic levansucrase operon encoding the endolevanase LevB, decreases the level of levansucrase expression. Conversely, independent expression of levB greatly stimulates operon expression in Bacillus subtilis. This autogenous effect is mediated by the levB transcript, which carries an internal sequence (5'-AAAGCAGGCAA-3') involved in the enhancing effect. In vitro, the levB transcript displays an affinity to the N-terminal fragment of SacY (KD 0.2 µM), the regulatory protein that prevents transcription termination of levansucrase operon. This positive feed back loop leads to an increase in the operon expression when B. subtilis is growing in the presence of high sucrose concentrations. Under these conditions, extracellular levan synthesized by the fructosyl polymerase activity of levansucrase can be degraded mainly into levanbiose by the action of LevB. Levanbiose is neither taken up nor metabolized by the bacteria. This work modifies the present view of the status of levansucrase in B. subtilis physiology

L'opéron lévane saccharase de Bacillus subtilis (régulation transcriptionnelle et post-transcriptionnelle)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

In vitro analysis of aggregation-disaggregation of the folding intermediate of Bacillus subtilis α-amylase.

The refolding intermediate of Bacillus subtilis α-amylase is prone to aggregate at 37°C and pH 7 when the protein concentration is relatively high (≥ 1µM). Low concentrations of 2,2,2 trifluoroethanol greatly increased the rate of aggregation. Aggregation made the folding intermediate resistant to proteases and there was kinetic competition between aggregation and proteolytic degradation. Analysis by Fourier transform infrared spectroscopy indicated that the secondary structure of the refolding intermediate is sightly different under soluble or aggregated states. Aggregates were readily solubilized by guanidium chloride (D1/2 = 1.25 M), but disaggregation was slow when aggregates were resuspended in solutions of various foreign native proteins under physiological conditions of pH and temperature. This destabilizing effect resulting from protein-protein interactions may make the aggregation process reversible in vivo

Calcium triggers the refolding of Bacillus subtilis chitosanase.

We characterized the reversible folding-unfolding transition of Bacillus subtilis exocellular chitosanase from either thermal or urea denaturation of the protein. The transitions were monitored in each case by intrinsic fluorescence changes and resistance to proteolysis. Unfolding and refolding kinetics and differential scanning calorimetry analysis suggested a two-state equilibrium. The equilibrium between the folded and unfolded states was rapidly displaced towards the folded state in the presence of a low concentration of calcium (2-20 mM). The binding titration curve indicated that chitosanase possesses one weak Ca(2+)-binding site (with an equilibrium affinity constant, K (A), of 0.3x10(3) M(-1)). These results support the hypothesis that this metal ion, which is accumulated in the cell wall environment of B. subtilis, is an effector that influences folding and stability of newly translocated proteins

Differential Dependence of Levansucrase and α-Amylase Secretion on SecA (Div) during the Exponential Phase of Growth of Bacillus subtilis

SecA, the translocation ATPase of the preprotein translocase, accounts for 0.25% of the total protein in a degU32(Hy) Bacillus subtilis strain in logarithmic phase. The SecA level remained constant irrespective of the demand for exoprotein production but dropped about 12-fold during the late stationary phase. Modulation of the level of functional SecA during the exponential phase of growth affected differently the secretion of levansucrase and α-amylase overexpressed under the control of the sacB leader region. The level of SecA was reduced in the presence of sodium azide and in the div341 thermosensitive mutant at nonpermissive temperatures. Overproduction of SecA was obtained with a multicopy plasmid bearing secA. The gradual decrease of the SecA level reduced the yield of secreted levansucrase with a concomitant accumulation of unprocessed precursor in the cells, while an increase in the SecA level resulted in an elevation of the production of exocellular levansucrase. In contrast, α-amylase secretion was almost unaffected by high concentrations of sodium azide or by very low levels of SecA. Secretion defects were apparent only under conditions of strong SecA deprivation of the cell. These data demonstrate that the α-amylase and levansucrase precursors markedly differ in their dependency on SecA for secretion. It is suggested that these precursors differ in their binding affinities for SecA