RESUMO
The Bacillus subtilis rhiLFGN-rhgR-yesTUVWXYZ (formerly yesOPQRSTUVWXYZ) gene cluster includes genes for metabolizing rhamnogalacturonan type I (RG-I), a major pectin constituent, and the rhgR gene encoding an AraC/XylS transcriptional activator. The yesL-rhgKL (formerly yesLMN) operon, adjacent to the rhiL gene, includes the rhgKL genes encoding a two-component regulatory system. The reporter analyses showed that 3 promoters immediately upstream of the rhiL, yesW, and yesL genes were induced by RG-I and repressed by glucose in the medium. The reporter analyses also showed that RhgL and RhgR contribute to the RG-I-dependent induction of the rhiL promoter and that CcpA mediates the catabolite repression of the rhiL and yesL promoters. The in vitro experiments demonstrated that the RhgL response regulator and the CcpA complex bind to each site in the rhiL promoter region. The RT-PCR analysis and the different properties of the rhiL and yesW promoters suggested the rhiLFGN-rhgR-yesTUV genes as an operon.
Assuntos
Bacillus subtilis , Ramnogalacturonanos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica , Glucose/metabolismo , Óperon/genética , Proteínas Repressoras/genéticaRESUMO
The Bacillus subtilis ilv-leu operon involved in the biosynthesis of branched-chain amino acids is under negative regulation mediated by TnrA and CodY, which recognize and bind to their respective cis-elements located upstream of the ilv-leu promoter. This operon is known to be under CcpA-dependent positive regulation. We have currently identified a catabolite-responsive element (cre) for this positive regulation (bases -96 to -82; +1 is the ilv-leu transcription initiation base) by means of DNase I-footprinting in vitro, and deletion and base-substitution analyses of cre. Under nitrogen-rich growth conditions in glucose-minimal medium supplemented with glutamine and amino acids, CcpA and CodY exerted positive and negative regulation of ilv-leu, respectively, but TnrA did not function. Moreover, CcpA and CodY were able to function without their counteracting regulation of each other, although the CcpA-dependent positive regulation did not overcome the CodY-dependent negative regulation. Furthermore, under nitrogen-limited conditions in glucose-minimal medium with glutamate as the sole nitrogen source, CcpA and TnrA exerted positive and negative regulation, respectively, but CodY did not function. This CcpA-dependent positive regulation occurred without the TnrA-dependent negative regulation. However, the TnrA-dependent negative regulation did not occur without the CcpA-dependent positive regulation, raising the possibility that this negative regulation might decrease the CcpA-dependent positive regulation. The physiological role of this elaborate transcription regulation of the B. subtilis ilv-leu operon in overall metabolic regulation in this organism is discussed.
Assuntos
Aminoácidos de Cadeia Ramificada/biossíntese , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Óperon , Transcrição Gênica , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Sequência de Bases , Pegada de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Isoleucina/biossíntese , Leucina/biossíntese , Dados de Sequência Molecular , Nitrogênio/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Elementos de Resposta/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Valina/biossínteseRESUMO
Solanesyl diphosphate (SPP) is regarded as the precursor of the side-chains of both plastoquinone and ubiquinone in Arabidopsis thaliana. We previously analyzed A. thaliana SPP synthase (At-SPS1) (Hirooka et al., Biochem. J., 370, 679-686 (2003)). In this study, we cloned a second SPP synthase (At-SPS2) gene from A. thaliana and characterized the recombinant protein. Kinetic analysis indicated that At-SPS2 prefers geranylgeranyl diphosphate to farnesyl diphosphate as the allylic substrate. Several of its features, including the substrate preference, were similar to those of At-SPS1. These data indicate that At-SPS1 and At-SPS2 share their basic catalytic machinery. Moreover, analysis of the subcellular localization by the transient expression of green fluorescent protein-fusion proteins showed that At-SPS2 is transported into chloroplasts, whereas At-SPS1 is likely to be localized in the endoplasmic reticulum in the A. thaliana cells. It is known that the ubiquinone side-chain originates from isopentenyl diphosphate derived from the cytosolic mevalonate pathway, while the plastoquinone side-chain is synthesized from isopentenyl diphosphate derived from the plastidial methylerythritol phosphate pathway. Based on this information, we propose that At-SPS1 contributes to the biosynthesis of the ubiquinone side-chain and that At-SPS2 supplies the precursor of the plastoquinone side-chain in A. thaliana.