AdeR, a PucR-type transcription factor, activates expression of L-alanine dehydrogenase and is required for sporulation of Bacillus subtilis.
ABSTRACT The Bacillus subtilis ald gene encodes L-alanine dehydrogenase, which catalyzes the NAD(+)-dependent deamination of L-alanine to pyruvate for the generation of energy and is required for normal sporulation. The transcription of ald is induced by alanine, but the mechanism underlying alanine induction remains unknown. Here we report that a gene (formerly yukF and now designated adeR) located upstream of ald is essential for the basal and alanine-inducible expression of ald. The disruption of the adeR gene caused a sporulation defect, whereas the complementation of an adeR mutation with an intact adeR gene restored the sporulation ability. adeR expression was not subject to autoregulation and alanine induction. Deletion and mutation analyses revealed that an inverted repeat, centered at position -74.5 relative to the transcriptional initiation site of ald, was required for ald expression and also likely served as a ρ-independent transcription terminator. Electrophoretic mobility shift assays showed that purified His-tagged AdeR was a specific DNA-binding protein and that this inverted repeat was required for AdeR binding. AdeR shows no significant amino acid sequence similarity to the known transcriptional activators of ald genes from other bacteria. AdeR appears to be a member of the PucR family of transcriptional regulators. Its orthologs of unknown function are present in some other Bacillus species. Collectively, these findings support the notion that AdeR is a transcriptional activator which mediates ald expression in response to alanine availability and is important for normal sporulation in B. subtilis.
Article: A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions.[show abstract] [hide abstract]
ABSTRACT: Specific, end-labeled DNA fragments can be simply and rapidly prepared using the polymerase chain reaction (PCR). Such fragments are suitable for use in DNase I protection footprint assays, chemical sequencing reactions, and for the production and analysis of paused RNA polymerase transcription complexes. Moreover, a general means of introducing a specific mutation at any position along the length of such PCR-generated fragments is described. These procedures, which can circumvent the need for large-scale phage or plasmid growths, preparative gel-electrophoresis and the screening of molecular clones, can facilitate the rapid study of sequence-specific interactions of proteins and DNA. A rapid means of removing excess oligonucleotide primers from completed PCRs is also described.Nucleic Acids Research 09/1988; 16(15):7351-67. · 8.03 Impact Factor
Article: Characterization of plasmid transformation in Bacillus subtilis: kinetic properties and the effect of DNA conformation.[show abstract] [hide abstract]
ABSTRACT: Transformation of competent cells of Bacillus subtilis with antibiotic resistance plasmid DNA has shown that (a) competence for plasmid and chromosomal DNA develops with similar kinetics; (b) DNA linearized with a variety of restriction endonucleases does not transform; (c) CCC plasmid DNA is inactivated for transformation by a single nick; (d) T4 ligase restores transforming activity to both nicked and linearized DNA; (E) CCC relaxed DNA is fully active in transformation; (f) the DNA concentration-dependence of plasmid transformation is first order; and (g) plasmid transformation proceeds with a low efficiency, requiring the uptake of 10(3) to 10(4) DNA molecules per transformant. Based on this information, a model for the processing of chromosomal, plasmid and transfecting DNA is proposed.MGG - Molecular and General Genetics 02/1979; 167(3):251-8.
Article: Indirect repression by Bacillus subtilis CodY via displacement of the activator of the proline utilization operon.[show abstract] [hide abstract]
ABSTRACT: Proline is an efficient source of both carbon and nitrogen for many bacterial species. In Bacillus subtilis, the proline utilization pathway, encoded by the putBCP operon, is inducible by proline. Here, we show that this induction is mediated by PutR, a proline-responsive transcriptional activator of the PucR family. When other amino acids are present in the medium, proline utilization is prioritized through transient repression by CodY, a global transcriptional regulator in Gram-positive bacteria that responds to amino acid availability. CodY-mediated repression of the putBCP operon has two novel features. First, repression requires the cooperative binding of CodY to at least two adjacent motifs. Second, though CodY binds to the region that overlaps the putB promoter, repression is due to displacement of PutR rather than competition with RNA polymerase.Journal of Molecular Biology 08/2011; 413(2):321-36. · 4.00 Impact Factor