Mario W. Chen

Global Gene Expression Profiles of Bacillus subtilis Grown under Anaerobic Conditions

Bacillus subtilis can grow under anaerobic conditions, either with nitrate or nitrite as the electron acceptor or by fermentation. A DNA microarray containing 4,020 genes from this organism was constructed to explore anaerobic gene expression patterns on a genomic scale. When mRNA levels of aerobic and anaerobic cultures during exponential growth were compared, several hundred genes were observed to be induced or repressed under anaerobic conditions. These genes are involved in a variety of cell functions, including carbon metabolism, electron transport, iron uptake, antibiotic production, and stress response. Among the highly induced genes are not only those responsible for nitrate respiration and fermentation but also those of unknown function. Certain groups of genes were specifically regulated during anaerobic growth on nitrite, while others were primarily affected during fermentative growth, indicating a complex regulatory circuitry of anaerobic metabolism.

Isolation of Brachymonas petroleovorans CHX, a novel cyclohexane‐degrading β‐proteobacterium

A new bacterium that grows aerobically on cyclohexane was isolated from the wastewater plant of a petroleum refinery. This strain grows on a range of light hydrocarbons (C5-C10) as well as on some aromatic compounds such as toluene and m-cresol. Growth on hydrocarbons requires the presence of yeast extract and other complex media components that are not substrates for growth themselves. Strain CHX is resistant to cyclohexane and grows at concentrations up to 2 g l(-1). Strain CHX branches deeply within the Comamonadeae family of beta-proteobacteria and is tentatively assigned to the Brachymonas genus as Brachymonas petroleovorans CHX.

Modular expression and secretion vectors for Bacillus subtilis

A modular vector system has been developed for the extracellular production of heterologous proteins in Bacillus subtilis. This modular vector system consists of four secretion vectors which are based upon the genes encoding the Bacillus amyloliquefaciens extracellular alkaline protease, neutral protease, barnase and levansucrase. The modular vectors contain compatible restriction sites downstream from the signal peptide-coding region. Three reporter proteins (staphylococcal protein A, levansucrase and Escherichia coli alkaline phosphatase) that offer complementary advantages for cloning, genetic manipulations and media optimization have been fused to the various signal peptides. These secretion vectors function in E. coli and hence can be used to compare the mechanisms of protein secretion in E. coli and B. subtilis.

The roles of signal peptide and mature protein in RNase (barnase) export from Bacillus subtilis

Barnase, an extracellular RNAse from Bacillus amyloliquefaciens is secreted post-translationally from B. subtilis. The rate of secretion of barnase from B. subtilis was improved by replacement of the barnase signal peptide with a heterologous signal peptide. However, the barnase signal peptide exported Escherichia coli alkaline phosphatase faster than mature barnase. Heat shock of B. subtilis cells did not significantly alter the export of barnase using the barnase signal peptide. The slow rate of export of barnase from B. subtilis is due to both the signal peptide and the mature protein sequence rather than either alone.