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

Silencing of levB, the second structural gene of the tricistronic levansucrase operon encoding the endolevanase LevB, decreases the level of levansucrase expression in Bacillus subtilis. Conversely, independent expression of levB greatly stimulates operon expression. This autogenous effect is mediated by the levB transcript, which carries an internal sequence (5'-AAAGCAGGCAA-3') involved in the enhancing effect.

Bacillus subtilis alpha-amylase: interactions of a partially folded conformer with small unilamellar vesicles.

We studied the interactions between conformers of exocellular alpha-amylase and small unilamellar vesicles (SUV) composed of the major membrane lipids of Bacillus subtilis under physiological conditions of pH, temperature and ionic strength. Using fluorescence spectroscopy, surface plasmon resonance (SPR) and phase separation, we show that the native alpha-amylase has no affinity for the SUV, whereas a partially folded form, displaying structural properties in common with the competent state for secretion, binds to the vesicles (KA approximately 10(5) M(-1)). This association prevented its subsequent folding.

Purification and characterization of YfkN, a trifunctional nucleotide phosphoesterase secreted by Bacillus subtilis.

YfkN isolated from the culture supernatant of Bacillus subtilis in the exponential phase of growth is a protein of 143.5 kDa that derives from a putative large precursor of 159.6 kDa processed at both the N- and C-terminal ends.

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.

Transcripts of the genes sacB, amyE, sacC and csn expressed in Bacillus subtilis under the control of the 5' untranslated sacR region display different stabilities that can be modulated.

When Bacillus subtilis levanase (SacC), alpha-amylase (AmyE) and chitosanase (Csn) structural genes were expressed under the regulated control of sacR, the inducible levansucrase (SacB) leader region in a degU32(Hy) mutant, it was observed that the production yields of the various extracellular proteins were quite different. This is mainly due to differences in the stabilities of their corresponding mRNAs which lead to discrepancies between the steady-state level of mRNA of sacB and csn on the one hand and amyE and sacC on the other. In contrast to levansucrase mRNA, the decay curves of alpha-amylase and levanase mRNAs obtained by Northern blotting analysis did not match the decay curves of their functional mRNA.

Kinetics of the secretion of Bacillus subtilis levanase overproduced during the exponential phase of growth.

The Bacillus subtilis levanase structural gene sacC was expressed under the regulated control of sacR, the inducible levansucrase leader region, in a degU32(Hy) strain. In this genetic context, exocellular levanase is overproduced (0.5% of total protein) during the exponential phase of growth upon induction by sucrose at 37 degrees C and pH 7.

Characterization of the rate-limiting step of the secretion of Bacillus subtilis alpha-amylase overproduced during the exponential phase of growth.

The Bacillus subtilis alpha-amylase gene, amyE, was expressed under the regulated control of sacR, the levansucrase leader region. The gene fusion including the complete amyE coding sequence with the signal peptide sequence was integrated into the chromosome of a degU32(Hy) strain deleted of the sacB DNA fragment. In this genetic contex, alpha-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.

Molecular characterization of the levansucrase gene from the endophytic sugarcane bacterium Acetobacter diazotrophicus SRT4.

The Acetobacter diazotrophicus SRT4 gene encoding levansucrase (EC 2.4.1.