RESUMO
The complete decipherment of the functions and interactions of the elements of the riboflavin biosynthesis operon (rib operon) of Bacillus subtilis are necessary for the development of superproducers of this important vitamin. The function of its terminal ribT gene has not been established to date. In this work, a search for homologs of the hypothetical amino acid sequence of the gene product through databases, as well as an analysis of the homolgs, was performed; the distribution of secondary structure elements was theoretically predicted; and the tertiary structure of the RibT protein was proposed. The ribT gene nucleotide sequence was amplified and cloned into the standard high-copy expression vector pET15b and then expressed after induction with IPTG in E. coli BL21 (DE3) strain cells containing the inducible phage T7 RNA polymerase gene. The ribT gene expression was confirmed by SDS-PAGE. The protein product of the expression was purified by affinity chromatography. Therefore, the real possibility of RibT protein production in quantities sufficient for further investigation of its structure and functional activity was demonstrated.
RESUMO
The transcription start sites of two internal promoters, the P2 and P3 promoters, in the Bacillus subtilis riboflavin biosynthesis operon were identified by primer extension. Putative -35 and -10 sequences that are recognized by the vegetative delta(70) subunit of RNA polymerase have been found upstream of the P2 and P3 transcription start sites. The relative strengths of the P1, P2, and P3 promoters were determined by cloning these promoters into the pDG268 expression vector. It was shown that the transcriptional activity of the P3 promoter is approximately fivefold higher as compared with P1, the major promoter, whereas P2 promoter activity is lower by almost two orders of magnitude. Real-time PCR demonstrated that unlike the P1 promoter, P2- and P3-driven expression is not regulated by flavins.
Assuntos
Bacillus subtilis/genética , RNA Polimerases Dirigidas por DNA , Regiões Promotoras Genéticas , Riboflavina , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Óperon/genética , Riboflavina/biossíntese , Riboflavina/genética , Sítio de Iniciação de TranscriçãoRESUMO
The nucleotide sequence of the ribC gene encoding the synthesis ofbifunctional flavokinase/flavine adenine nucleotide (FAD) synthetase in Bacillus subtilis have been determined in a family of riboflavin-constitutive mutants. Two mutations have been found in the proximal region of the gene, which controls the transferase (FAD synthase) activity. Three point mutations and one double mutation have been found (in addition to the two mutations that were detected earlier) in the distal region of the gene, which controls the flavokinase (flavin mononucleotide (FMN) synthase) activity. On the basis of all data known to date, it has been concluded that the identified mutations affect riboflavin and ATP binding sites. No mutations have been found in the PTAN conserved sequence, which forms the magnesium and ATP common binding site and is identical for organisms of all organizational levels, from bacteria too humans.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Genes Bacterianos/genética , Mutação , Nucleotidiltransferases/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Análise Mutacional de DNA , Nucleotidiltransferases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismoRESUMO
Among Bacillus subtilis riboflavin-resistant mutants we identified one, which differed from other regulatory mutants by overproduction of riboflavin and simultaneous upregulation of the rib C gene encoding flavokinase/FAD-synthase. Genetic and biochemical analysis showed that the ribU1 mutation determines a trans-acting factor that simultaneously regulates activity of riboflavin and truB-ribC-rpsO operons. Regulatory activity of the ribU1 mutation comprises about 10% of Rfn element activity on interaction with flavins. The ribUl mutation can be presumably ascribed to a gene of the transcriptional regulators family.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Mutação , Nucleotidiltransferases/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Riboflavina/genética , Fatores de Transcrição/genética , Bacillus subtilis/enzimologia , Proteínas de Bactérias/biossíntese , Regulação Bacteriana da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/genética , Nucleotidiltransferases/biossíntese , Óperon/genética , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Fármacos Fotossensibilizantes/farmacologia , Riboflavina/biossíntese , Riboflavina/farmacologia , Fatores de Transcrição/metabolismoRESUMO
Insertion and deletion mutagenesis of the leader region of the Bacillus subtilis rib operon encoding FMN-specific sensor RNA was conducted. Insertions of different structure and length in the conservative motif of the leader sequence (Rfn-element) were shown to cause partially constitutive expression of the operon resulted in an increased accumulation of riboflavin. At the same time, introducing into the genome of insertion mutants an additional ribC mutation blocking FMN synthesis leads to an increase in riboflavin production. Deletion of the main Rho-dependent transcription terminator gives rise to the same effect. These results indicate that some additional FMN-dependent regulation is involved in rib-operon control, which is, however, still connected with the primary and secondary structure of the leader region.
Assuntos
Bacillus subtilis/fisiologia , Óperon , Riboflavina/biossíntese , Bacillus subtilis/genética , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Mutação , Estrutura Secundária de Proteína , Riboflavina/genéticaRESUMO
Analysis of the phenotypic manifestation of inactivation of several genes from the ytlI-ytnM operon containing the ribR gene and results of Northern hybridization showed that the ribR gene does not have the self promoter and is transcribed from the main promoter of the ytlI-ytnM operon. Two sites of single nucleotide substitutions leading to derepresson of ribR gene were identified between the putative main promoter and transcription start of the ytlI-ytnM operon regulatory region.
Assuntos
Bacillus subtilis/genética , Regulação Bacteriana da Expressão Gênica , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Regiões Promotoras Genéticas/genética , Sequência de Bases , Inativação Gênica , Dados de Sequência Molecular , Mutação/genética , Óperon/genética , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Riboflavina/biossíntese , Riboflavina/genética , Sítio de Iniciação de Transcrição , Transcrição Gênica , Regulação para Cima/genéticaRESUMO
Sequence analysis of several Bacillus subtilis mutants with increased activity of flavokinase/FAD-synthase and the results of Northern hybridization showed that the TTGCCG-17n-TACATT motif localized to the C-end of the truB gene is a regulatory region that controls the ribC gene at the level of transcription.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias , Genes Bacterianos , Nucleotidiltransferases/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Sequência de Bases , Northern Blotting , DNA Bacteriano , Dados de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico , Transcrição GênicaRESUMO
After inactivation of the ypaA gene in Bacillus subtilis, the phenotypic pattern obtained showed that this gene controls a system for active flavin transport and, possibly, riboflavin excretion under the conditions of constitutive synthesis.
Assuntos
Bacillus subtilis/genética , Inativação Gênica , Bacillus subtilis/metabolismo , Transporte Biológico , Fenótipo , Riboflavina/biossíntese , Riboflavina/metabolismoRESUMO
A point mutation (C-->A substitution) in the -35 region of a putative promoter-operator site TTGCCG-17n-TACATT results in a more than 25-fold increase in the activity of ribC gene encoding the bifunctional enzyme--flavokinase/FAD-synthase--in Bacillus subtilis.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Nucleotidiltransferases/genética , Regiões Operadoras Genéticas , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Sequência de Bases , DNA Bacteriano , Dados de Sequência Molecular , Regiões Promotoras GenéticasRESUMO
A 3.5 kb EcoRI-BamHI fragment of Bacillus subtilis chromosomal DNA carrying the ribR gene, involved in regulation of the B. subtilis riboflavin operon, was cloned in the B. subtilis-Escherichia coli shuttle vector pCB20. DNA sequence analysis of this fragment revealed several ORFs, one of which encodes a polypeptide of 230 amino acids with up to 45% sequence identity with FAD synthetases from a number of micro-organisms, such as Corynebacterium ammoniagenes, E. coli and Pseudomonas fluorescens, and also to the ribC gene product of B. subtilis. The ribR gene was amplified by PCR, cloned and expressed in E. coli. Measurement of flavokinase activity in cell extracts demonstrated that ribR encodes a monofunctional flavokinase which converts riboflavin into FMN but not to FAD, and is specific for the reduced form of riboflavin.
Assuntos
Bacillus subtilis/genética , Regulação Bacteriana da Expressão Gênica , Óperon , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Riboflavina/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Escherichia coli/genética , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Genes Bacterianos/genética , Dados de Sequência Molecular , Mutação , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Fases de Leitura Aberta/genética , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Riboflavina/análogos & derivados , Riboflavina/química , Riboflavina/genética , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The PCR copy of the ribR gene of Bacillus subtilis was subcloned in Escherichia coli cells under the control of the phage T7 inducible promoter. The polypeptide of 26 kDa corresponding to the 690-bp gene is the product of the ribR gene. The protein encoded by the ribR gene is flavokinase, and the riboflavin-reduced form is the substrate for it.
Assuntos
Bacillus subtilis/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Sequência de Aminoácidos , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA Recombinante , Genes Bacterianos , Dados de Sequência Molecular , Nucleotidiltransferases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Reação em Cadeia da Polimerase , Riboflavina/metabolismo , Especificidade por SubstratoRESUMO
A 13.0-kb EcoRI fragment of Bacillus subtilis DNA carrying an additional regulatory ribR gene of riboflavin operon was cloned on the basis of resistance to 7, 8-dimethyl-10 (O-methylacetoxym)-isoalloxasin. The cloned fragment was trans-dominant with regard to ribC constitutive mutations that block the overproduction of riboflavin but inactive relative to constitutive mutations in the rib(O) regulatory region.
Assuntos
Bacillus subtilis/genética , Genes Reguladores , Óperon , Riboflavina/genética , Clonagem Molecular , DNA Bacteriano , Desoxirribonuclease EcoRI/metabolismo , Resistência Microbiana a Medicamentos , Flavinas/farmacologia , Genes BacterianosRESUMO
Assessment of specific activity of riboflavin synthase and the level of riboflavin accumulation in strains with a 110-nucleotide deletion in the regulatory region of the riboflavin operon showed that this deletion specified semi-constitutive expression of the operon. This was assumed to be connected with the elimination of three nucleotides from a potential transcription antiterminator.
Assuntos
Bacillus subtilis/genética , Óperon , Sequências Reguladoras de Ácido Nucleico/genética , Riboflavina/genética , Deleção de Sequência , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Sequência de Bases , DNA Bacteriano , Dados de Sequência Molecular , Fenótipo , Riboflavina Sintase/metabolismo , Transcrição Gênica/genéticaAssuntos
Bacillus/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Riboflavina/biossíntese , Sequência de Bases , Cromossomos Bacterianos , Clonagem Molecular , DNA Bacteriano , Dados de Sequência Molecular , Mutagênese , Sequências Reguladoras de Ácido Nucleico , Homologia de Sequência do Ácido NucleicoAssuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Genes Bacterianos , Fosfotransferases (Aceptor do Grupo Álcool) , Sequência de Aminoácidos , Proteínas de Bactérias/química , Clonagem Molecular , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/biossíntese , Dados de Sequência Molecular , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Óperon , Mapeamento por Restrição , Homologia de Sequência de AminoácidosRESUMO
The sequencing of a 3.5 kb EcoRI-BamHI fragment located in the 236 degrees region of the Bacillus subtilis chromosome revealed a 690-bp long open reading frame, partly similar in amino acid composition to flavokinases/FAD synthases from several microorganisms. The discovered sequence can be identified with the gene ribR, an auxiliary regulatory gene of the riboflavin operon of Bacillus subtilis.
Assuntos
Bacillus subtilis/genética , Mapeamento Cromossômico , Genes Bacterianos , Genes Reguladores , Óperon , Riboflavina/genética , Sequência de Aminoácidos , Sequência de Bases , Dados de Sequência MolecularRESUMO
Two mutations designated ribR5 and ribR6, which effectively decrease the constitutive expression of the Bacillus subtilis riboflavin operon, were mapped in the 236 degrees region of the B. subtilis genetic map based on linkage analysis with respect to the apt6 marker. Cotransfer indices for the ribR5 and ribR6 markers and apt6 suggest that both ribR mutations are alleles of exactly the same genetic locus. Total results of transductional and transformational crosses indicate the following marker order: pheA-apt6-ribR-azlB-aroD4. Phenotypic peculiarities of the ribR mutants allow us to assume that they belong to an additional subunit of the regulatory protein of the riboflavin operon.
Assuntos
Bacillus subtilis/genética , Mapeamento Cromossômico , Genes Bacterianos , Genes Reguladores , Óperon , Riboflavina/genética , Cruzamentos Genéticos , Ligação Genética , Marcadores Genéticos , Mutação , Fenótipo , Transdução Genética , Transformação GenéticaRESUMO
Seventeen cis-dominant mutations leading to riboflavin overproduction in Bacillus subtilis were localized to the region between nucleotides +37 and +159 relative to the transcription initiation site of the riboflavin operon. This region displays an unusual structure for regulatory sequences. The main part of it represents clusters of A/T and G/C-rich sequences that symmetrically blank a short inverted repeat.
Assuntos
Bacillus subtilis/genética , Óperon/genética , Sequências Reguladoras de Ácido Nucleico/genética , Sequências Repetitivas de Ácido Nucleico/genética , Riboflavina/genética , Bacillus subtilis/enzimologia , Sequência de Bases , Dados de Sequência Molecular , Mutagênese/genética , Conformação de Ácido Nucleico , Riboflavina/biossíntese , Riboflavina Sintase/metabolismoRESUMO
Influence of the recE1, recB2, recB3, recB19, recF15, recF18, recL16, recM13 and recM27 mutations of the induction of the SOS-like system component, i. e. the RecE protein of Bacillus subtilis was studied by RIA-dot-blot method in UV-irradiated or treated by nalidixic acid cells. These agents caused a significant increase in the wild type (rec+) cells but did not stimulate the RecE synthesis in the rec mutants tested. The two exceptions were recB2 and recF18 mutants treated by nalidixic acid. The tsi23 mutation caused thermoinduction of phi 105 bacteriophage in the rec+ genetic background while no prophage particles were induced in the recE, recF, recL, recM mutants. The data suggest that the genetic damage of several rec genes including recB, recE, recF, recL and recM can block induction of the SOS-like system of Bacillus subtilis.
Assuntos
Bacillus subtilis/genética , Proteínas de Escherichia coli , Exodesoxirribonucleases/genética , Mutação , Resposta SOS em Genética , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/efeitos da radiação , Ácido Nalidíxico/farmacologia , Temperatura , Raios UltravioletaRESUMO
Seven mutations leading to riboflavin overproduction in Bacillus subtilis were found to be linked to the marker dnaF133 (145 degrees on the B. subtilis genetic map) by transformation. Cotransfer indexes (42.5%-61.7%) suggest that the ribC mutations are alleles of the same locus. Results of transduction and transformation crosses suggest the following order of markers:pyrD26-ts-6-dnaF133-ribC-recA1.
