Thermal regulation of membrane lipid fluidity by a two-component system in Bacillus subtilis.

This article describes a simple and robust laboratory exercise on the regulation of membrane unsaturated fatty acid composition in bacteria by a decrease in growth temperature. We take advantage of the well characterized Des pathway of Bacillus subtilis, composed of a Δ5-desaturase (encoded by the des gene) and the canonical two-component system DesK-DesR, to study the transcriptional regulation of des during cold shock. Students analyze the expression of a reporter transcriptional fusion between the des promoter and the bacterial lacZ gene in a wild-type B. subtilis strain and in des or desK-desR mutants grown under different culture conditions. Measurements of ß-galactosidase activity allow them to investigate how the Des pathway works and to assess the role of each component of this regulatory system.

Transcriptional control of the sulfur-regulated cysH operon, containing genes involved in L-cysteine biosynthesis in Bacillus subtilis.

The molecular mechanisms of regulation of the genes involved in the biosynthesis of cysteine are poorly characterized in Bacillus subtilis and other gram-positive bacteria. In this study we describe the expression pattern of the B. subtilis cysH operon in response to sulfur starvation. A 6.1-kb polycistronic transcript which includes the cysH, cysP, ylnB, ylnC, ylnD, ylnE, and ylnF genes was identified. Its synthesis was induced by sulfur limitation and strongly repressed by cysteine. The cysH operon contains a 5' leader portion homologous to that of the S box family of genes involved in sulfur metabolism, which are regulated by a transcription termination control system. Here we show that induction of B. subtilis cysH operon expression is dependent on the promoter and independent of the leader region terminator, indicating that the operon is regulated at the level of transcription initiation rather than controlled at the level of premature termination of transcription. Deletion of a 46-bp region adjacent to the -35 region of the cysH promoter led to high-level expression of the operon, even in the presence of cysteine. We also found that O-acetyl-L-serine (OAS), a direct precursor of cysteine, renders cysH transcription independent of sulfur starvation and insensitive to cysteine repression. We propose that transcription of the cysH operon is negatively regulated by a transcriptional repressor whose activity is controlled by the intracellular levels of OAS. Cysteine is predicted to repress transcription by inhibiting the synthesis of OAS, which would act as an inducer of cysH expression. These novel results provide the first direct evidence that cysteine biosynthesis is controlled at a transcriptional level by both negative and positive effectors in a gram-positive organism.

Monomeric selectively S-alkylated derivatives of seminal ribonuclease: preparation and properties.

Procedures are described for preparing monomeric selectively S-carboxamido-methylated and S-aminoethylated derivatives of seminal ribonuclease. The main properties of the derivatives, including their extinction coefficients, have been determined. Their catalytic activities and that of the S-carboxymethyl derivative have been tested. On double-stranded RNA as a substrate the monomeric derivatives are less active than the native dimeric enzyme, but much more active than pancreatic ribonuclease. On yeast RNA as a substrate the amino-ethyl derivative is found to be less active (80%) than the native enzyme, while the other two are over 30 percent more active. The monomers are stable in solution and when lyophilized from acetic acid solution do not associate to the same extent as pancreatic or native seminal ribonucleases.