RESUMO
An expression vector for systematic protein overproduction in Bacillus subtilis has been constructed. It derives from pDG148 and combines the main property of this vector, i.e. conditional expression of the gene in response to isopropylbeta-D-thiogalactopyranoside, with (i) rapid orientated cloning by a ligation independent procedure and (ii) a ribosome binding site of high translational efficiency. When used for overproduction of several proteins in B. subtilis, this vector gave good levels of protein synthesis.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Clonagem Molecular/métodos , Regulação Bacteriana da Expressão Gênica , Expressão Gênica , Vetores Genéticos , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Sequência de Bases , Isopropiltiogalactosídeo/farmacologia , Dados de Sequência Molecular , Transformação BacterianaRESUMO
The production of Desulfovibrio vulgaris Hildenborough cytochrome c(3) (M(r) 13000), which is a tetraheme cytochrome, in Escherichia coli was examined. This cytochrome was successfully produced in an E. coli strain co-expressing the ccmABCDEFGH genes involved in the cytochrome c maturation process. The apocytochrome c(3) was matured in either anaerobic or aerobic conditions, but aerobic growth in the presence of delta-aminolevulinic acid was found to be best for cytochrome c(3) production. Site-directed mutagenesis was performed to investigate the effect of the presence of four amino acids in between the two cysteines of the heme binding sites 2 and 4 on the maturation of holocytochrome c(3) in E. coli.
Assuntos
Grupo dos Citocromos c/genética , Escherichia coli/genética , Ácido Aminolevulínico/farmacologia , Sítios de Ligação , Grupo dos Citocromos c/biossíntese , Desulfovibrio vulgaris/genética , Escherichia coli/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Heme/química , Mutagênese Sítio-Dirigida , Mutação , Periplasma/enzimologia , PlasmídeosRESUMO
The nucleotide sequence of the Bacillus subtilis clpP gene was determined. The predicted protein shows very high similarity to members of the ClpP family of proteolytic subunits (68% amino acid sequence identity with that of Escherichia coli). We show that ClpP plays an essential role in stationary phase adaptive responses. Indeed, a delta clpP mutant was constructed and shown to display a pleiotropic phenotype, including a deficiency in both sporulation initiation and competence for DNA uptake. The delta clpP mutant has a highly filamentous morphology and appears to be non-motile, as judged by swarm plate assays. Expression of clpP is strongly induced under heat shock conditions, and ClpP is shown to be essential for growth of B. subtilis at high temperature. The role of ClpP in the sporulation and competence regulatory pathways was investigated. ClpP is required for expression of the spollA and spollG operons, encoding the sigmaF and sigmaE sporulation-specific sigma factors. ClpP is also necessary for the expression of the comK gene, encoding a positive transcriptional regulator of competence genes. ComK-dependent transcription of sacB, encoding the exocellular degradative enzyme levansucrase, was found to be abolished in the delta clpP mutant. MecA has been characterized previously as a negative regulator of comK expression, whose overproduction inhibits both sporulation and competence development. Expression of a mecA'-'lacZ translational fusion is shown to be increased in the delta clpP mutant. We suggest that ClpP is involved in controlling MecA levels in the cell through proteolysis. Increased levels of MecA in the absence of ClpP are at least partly responsible for the observed pleiotropic phenotype of the delta clpP mutant.
Assuntos
Adenosina Trifosfatases/genética , Bacillus subtilis/genética , Proteínas de Choque Térmico/genética , Serina Endopeptidases/genética , Adenosina Trifosfatases/química , Adenosina Trifosfatases/fisiologia , Sequência de Aminoácidos , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endopeptidase Clp , Galactosidases/metabolismo , Deleção de Genes , Genes Bacterianos , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/fisiologia , Hexosiltransferases/genética , Dados de Sequência Molecular , Mutação , Fenótipo , Plasmídeos , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Serina Endopeptidases/química , Serina Endopeptidases/fisiologia , Esporos Bacterianos , Especificidade por Substrato , Temperatura , Transformação BacterianaRESUMO
Few systematic studies have been devoted to investigating the role of Ca2+ as an intracellular messenger in prokaryotes. Here we report an investigation on the potential involvement of Ca2+ in signalling in Bacillus subtilis, a Gram-positive bacterium. Using aequorin, it is shown that B. subtilis cells tightly regulate intracellular Ca2+ levels. This homeostasis can be changed by an external stimulus such as hydrogen peroxide, pointing to a relationship between oxidative stress and Ca2+ signalling. Also, B. subtilis growth appears to be intimately linked to the presence of Ca2+, as normal growth can be immediately restored by adding Ca2+ to an almost non-growing culture in EGTA containing Luria broth medium. Addition of Fe2+ or Mn2+ also restores growth, but with 5-6 h delay, whereas Mg2+ did not have any effect. In addition, the expression of alkyl hydroperoxide reductase C (AhpC), which is strongly enhanced in bacteria grown in the presence of EGTA, also appears to be regulated by Ca2+. Finally, using 45Ca2+ overlay on membrane electrotransferred two-dimensional gels of B. subtilis, four putative Ca2+ binding proteins were found, including AhpC. Our results provide strong evidence for a regulatory role for Ca2+ in bacterial cells.