A Bacillus subtilis fusion protein system to produce soybean Bowman-Birk protease inhibitor.

A fusion protein based expression system was developed in the Gram-positive bacterium Bacillus subtilis to produce the soybean Bowman-Birk protease inhibitor (sBBI). The N-terminus of the mature sBBI was fused to the C-terminus of the 1st cellulose binding domain linker (CBD linker) of the BCE103 cellulase (from an alkalophilic Bacillus sp.).

An in vitro assay for (1 --> 6)-beta-D-glucan synthesis in Saccharomyces cerevisiae.

(1 --> 6)-beta-D-glucan is a key cell wall component of Saccharomyces cerevisiae and Candida albicans. Many genes are known to affect the levels or structure of this glucan, but their roles and a molecular description of the synthesis of (1 --> 6)-beta-D-glucan remain to be established and a method to measure (1 --> 6)-beta-D-glucan synthase activity in vitro would provide an enabling tool. Here, conditions for the detection of in vitro synthesis of this polymer are described.

Characterization of the transcriptional response to cell wall stress in Saccharomyces cerevisiae.

The cell wall perturbants Calcofluor white and Zymolyase activate the Pkc1-Rho1-controlled Slt2p MAP kinase pathway in Saccharomyces cerevisiae. A downstream transcription factor of this pathway, Rlm1p, is known to control expression of about 20 cell wall-related genes. Global transcript analysis of Calcofluor white and Zymolyase treatment was performed to determine whether cell wall stress affects transcription of these and other genes.

The protein kinase Kic1 affects 1,6-beta-glucan levels in the cell wall of Saccharomyces cerevisiae.

KIC1 encodes a PAK kinase that is involved in morphogenesis and cell integrity. Both over- and underexpressing conditions of KIC1 affected cell wall composition. Kic1-deficient cells were hypersensitive to the cell wall perturbing agent calcofluor white and had less 1,6-beta-glucan.

Cell wall perturbation in yeast results in dual phosphorylation of the Slt2/Mpk1 MAP kinase and in an Slt2-mediated increase in FKS2-lacZ expression, glucanase resistance and thermotolerance.

The protein kinase C (PKC1) pathway is essential for maintaining cell integrity in yeast. Here it is shown that various forms of cell wall damage result in activation of the downstream MAP kinase Slt2/Mpk1. Several cell wall mutants displayed enhanced FKS2-lacZ expression, a known output of Slt2 activation.