RESUMEN
Three genes called rocD, rocE and rocF were found near the rocR gene in B. subtilis. The product of rocD is similar to eukaryotic ornithine aminotransferases. The product of rocE shares similarity with the product of B. subtilis rocC and with the product of E. coli lysP. The rocE gene may encode an arginine permease. The rocF gene encodes a polypeptide similar to several arginases. Heterologous expression in E. coli indicated that rocD encodes an ornithine aminotransferase and that rocF encodes an arginase. Arginine utilization was abolished in both rocD and rocF mutants of B. subtilis confirming the role of these genes in arginine catabolism. The rocDEF genes form an operon transcribed from a -12, -24 promoter almost identical to the -12, -24 promoter of the rocABC operon. The expression of the rocDEF operon was induced by the presence of arginine, ornithine or proline in the growth medium and depended on the presence of the sigma factor SigL. Transcription of this operon was also abolished in a B. subtilis strain containing a null mutation in the regulatory gene rocR. Two tandemly repeated upstream activating sequences very similar to those previously identified in the rocABC system were found centered at positions -120 and -70, respectively, upstream from the transcription start site of rocDEF. Deletion analysis showed that at least one upstream activating sequence is involved in the expression of the rocDEF operon. These sequences are probably the target of RocR. Analysis of a rocR'-'lacZ fusion strain showed that the expression of rocR is not induced by arginine and is negatively autoregulated.
Asunto(s)
Arginina/metabolismo , Bacillus subtilis/metabolismo , Genes Bacterianos , Secuencia de Aminoácidos , Arginasa/genética , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Clonación Molecular , Cartilla de ADN/química , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica , Genes Reguladores , Datos de Secuencia Molecular , Operón , Ornitina-Oxo-Ácido Transaminasa/genética , Regiones Promotoras Genéticas , ARN Bacteriano/genética , ARN Mensajero/genética , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
The malA region of Escherichia coli contains one of the three maltose operons, namely malPQ, and the positive regulatory gene, malT. Gene malT and the malPQ operon are transcribed in opposite directions, in a divergent manner. The distance separating the transcription start-points in the two directions was previously shown to be 513 base-pairs. We are now presenting a deletion analysis of this unexpectedly long intergenic region. Two sets of deletions were created in vitro, by using exonuclease BAL31. One set comprised deletions centered on a HincII restriction site located in the malPQ promoter, and extending towards gene malT. The other set was centered on an EcoRI site, which had been introduced close to the beginning of the malT cistron, and extended towards gene malP. These deletions, initially created on plasmids, were transferred onto the bacterial chromosome. By studying the phenotype resulting from the presence of these deletions, we concluded that: (1) all of the DNA sequences required for expression of malT and malPQ are within 100 base-pairs of the respective transcription start-points for these genes; (2) a sequence located more than 120 base-pairs upstream from the malT transcription start-point plays a role in limiting malT expression; and (3) a remaining DNA segment, 150 to 300 base-pairs in length, and centrally located in the inter-promoter region, seems to play no role in the expression of malT or malPQ.
Asunto(s)
Deleción Cromosómica , Mapeo Cromosómico , Cromosomas Bacterianos , Endodesoxirribonucleasas , Escherichia coli/genética , Maltosa/genética , Secuencia de Bases , ADN Bacteriano , Exodesoxirribonucleasas , Genes Bacterianos , Genes Reguladores , Mutación , Operón , Transcripción GenéticaRESUMEN
The levanase gene (sacC) of Bacillus subtilis is the distal gene of a fructose-inducible operon containing five genes. The complete nucleotide sequence of this operon was determined. The first four genes levD, levE, levF and levG encode polypeptides that are similar to proteins of the mannose phosphotransferase system of Escherichia coli. The levD and levE gene products are homologous to the N and C-terminal part of the enzyme IIIMan, respectively, whereas the levF and levG gene products have similarities with the enzymes IIMan. Surprisingly, the polypeptides encoded by the levD, levE, levF and levG genes are not involved in mannose uptake, but form a fructose phosphotransferase system in B. subtilis. This transport is dependent on the enzyme I of the phosphotransferase system (PTS) and is abolished by deletion of levF or levG and by mutations in either levD or levE. Four regulatory mutations (sacL) leading to constitutive expression of the lavanase operon were mapped using recombination experiments. Three of them were characterized at the molecular level and were located within levD and levE. The levD and levE gene products that form part of a fructose uptake PTS act as negative regulators of the operon. These two gene products may be involved in a PTS-mediated phosphorylation of a regulator, as in the bgl operon of E. coli.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica , Glicósido Hidrolasas/genética , Operón , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Secuencia de Aminoácidos , Bacillus subtilis/enzimología , Secuencia de Bases , Transporte Biológico , Fructosa/metabolismo , Glicósido Hidrolasas/metabolismo , Manosa/metabolismo , Datos de Secuencia Molecular , Mutación , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoRESUMEN
Transcription of the levanase operon of Bacillus subtilis is controlled by LevR, an activator of the NifA/NtrC family of regulators. An upstream activating sequence (UAS) located in a 16 bp palindromic structure has previously been characterized. LevR was overproduced in B. subtilis and interaction between the activator and the UAS was demonstrated by gel shift and footprint experiments. The LevR protein specifically binds to the two-halves of the palindromic structure centered at -125 bases upstream from the transcriptional start site. In addition, footprint analysis suggests that LevR interacts with a third DNA region located at positions -90 to -80. To investigate the function of the different domains of the LevR activator, stop codons were introduced at various positions in the levR gene. The ability of the truncated LevR polypeptides to activate transcription, to respond to the inducer or to interact with the UAS was tested. The results obtained suggest that LevR is a multidomain protein. The amino-terminal part of the protein is required for DNA binding whereas the central domain allows the activation of transcription. The carboxy-terminal region is involved in the modulation of the LevR activity by the inducer.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/genética , Glicósido Hidrolasas/genética , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Electroforesis en Gel de Poliacrilamida , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Datos de Secuencia Molecular , Plásmidos , Mapeo Restrictivo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The levanase operon of Bacillus subtilis is controlled by RNA polymerase associated with sigma 54 factor and by the LevR activator that is homologous to the NifA/NtrC family of regulators. A "-12, -24" promoter is present at the appropriate distance from the transcription start site. The drastic down effect of base substitutions in the TGGCAC, TTGCA consensus sequence on the expression of the levanase operon confirmed the involvement of the "-12, -24" region in promoter function. Deletion derivatives of the upstream sequence of the operon promoter were constructed using translational levD'-'lacZ fusions and were integrated as single copies at the amyE locus of the B. subtilis chromosome. A cis-acting DNA sequence that is required for activation of the operon promoter by LevR was identified. This regulatory sequence is about 50 base-pairs long and is centered 125 base-pairs upstream from the transcription start site in a region containing a 16 base-pair palindromic structure. This region of dyad symmetry functions as a regulatory element when placed up to at least 600 base-pairs upstream from the "-12, -24" promoter, although the efficacy of activation is lowered. Thus, in common with most sigma 54-dependent promoters, an upstream activating sequence (UAS) is involved in the control of expression of the levanase operon. The isolation and characterization of eight mutations in the UAS region confirmed the importance of the palindromic structure in promoter activation. Moreover, the expression of the levanase operon was inhibited by placing the UAS in trans on a multicopy plasmid, probably through titration of the LevR polypeptide. In conclusion, the levanase promoter region can be divided into two regulatory sequences: the "-12, -24" promoter recognized by the sigma 54 RNA polymerase holoenzyme and the UAS, an inverted repeat sequence that is probably the LevR binding site.
Asunto(s)
Bacillus subtilis/genética , Regulación Bacteriana de la Expresión Génica/genética , Glicósido Hidrolasas/genética , Operón/genética , Regiones Promotoras Genéticas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Datos de Secuencia Molecular , Mutagénesis/genética , Plásmidos/genética , Proteínas Recombinantes de Fusión/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Factores de Transcripción/metabolismoRESUMEN
We describe an immunological method which allows the in situ colorimetric detection of translated DNA fragments in bacteria. In the absence of lysis only cell surface proteins are detected. For cytoplasmic proteins, lysis is required. The procedure comprises the following steps: bacteria are lysed, the proteins are transferred onto a disc of nitrocellulose sheet, the remaining protein sites are blocked, the disc is successively soaked in a solution of antibodies specific for the protein to be detected and in a solution of peroxidase-labelled anti-IgG antibody solution. Finally, the immune complexes are made visible by enzyme substrate incubation. We describe the application of this method to the detection of the LamB protein, the LacZ protein, and a LamB-polio VP1 chimera translated from cloned DNA fragment in E. coli.
Asunto(s)
Antígenos Bacterianos/análisis , Antígenos de Superficie/análisis , Proteínas Bacterianas/análisis , Técnicas Bacteriológicas , Técnicas para Inmunoenzimas , Proteínas de la Membrana Bacteriana Externa , Colodión , Colorimetría , Escherichia coli/análisis , Estudios de Evaluación como Asunto , Poliovirus/análisis , Porinas , Receptores Virales/análisis , Proteínas Recombinantes/análisis , Proteínas Virales/análisis , Proteínas Estructurales ViralesRESUMEN
Within the frame of an attempt to sequence the whole Bacillus subtilis genome, a region of 5.5 kbp of the B. subtilis chromosome near the sacS locus has been sequenced. It contains five complete coding sequences, including the sequence of sacY, three unknown CDS and a sequence coding for a tyrosine tRNA synthetase. That the corresponding CDS encodes a functional synthetase has been demonstrated by complementation of an Escherichia coli mutant possessing a thermosensitive tRNA synthetase. Insertion of a kanamycin resistance cassette in the B. subtilis chromosome at the corresponding locus resulted, however, in no apparent phenotype, demonstrating that this synthetase is dispensable. Finally phylogenetic relationships between known tyrosine and tryptophan tRNA synthetases are discussed.
Asunto(s)
Bacillus subtilis/genética , Genoma Bacteriano , Tirosina-ARNt Ligasa/genética , Secuencia de Aminoácidos , Bacillus subtilis/enzimología , Secuencia de Bases , Mapeo Cromosómico , ADN Bacteriano , Ligamiento Genético , Datos de Secuencia Molecular , Sistemas de Lectura AbiertaAsunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica/fisiología , Genes Bacterianos/genética , Hexosiltransferasas/genética , Sacarasa/genética , Bacillus subtilis/enzimología , Glicósido Hidrolasas/genética , Técnicas In Vitro , Operón/genética , Sacarosa/metabolismoRESUMEN
Some of the previously described malT-lacZ fusion strains (Débarbouillé and Schwartz, 1979) produce very low amounts of beta-galactosidase activity and hence grow poorly on lactose. Spontaneous mutants growing faster on lactose have been isolated. Some of the mutations map in, or close to, the promoter of the hybrid gene. They lead to an increased production of the hybrid proteins, which then become detectable on polyacrylamide gels. This effect is cis dominant. When the mutations, called malTq, are transduced into a malT+ background the resulting transductants express the three maltose operons in a partially constitutive way. The malTq mutations therefore represent a new type of constitutive mutation. Their existence provides further evidence for the previously proposed model of positive regulation in the maltose regulon. In addition they should facilitate the purification of the malT product, and the identification of the malT promoter on the DNA.
Asunto(s)
Escherichia coli/genética , Maltosa/genética , Mutación , Operón , Operón Lac , Recombinación GenéticaRESUMEN
The malPQ operon, one of the three operons of the maltose regulon, is positively controlled by the product of gene malT. The starting point for malPQ transcription was deduced from experiments which involved a hybridization of in vivo-synthesized malPQ mRNA with adequate DNA probes, followed either by a digestion of nonhybridized DNA (S1 nuclease mapping) or by an extension of the hybridized probe (reverse transcriptase mapping). In the wild-type strain, this starting point was 37 nucleotides upstream from the initiation codon for malP. This analysis was also performed on a double mutant which contained both a 13-base pair deletion and a 3-base pair insertion in the promoter region. This double mutant expressed the malPQ operon exactly as the wild-type strain did, in a maltose-inducible manner. In this strain, the starting point for malPQ transcription was shifted 11 nucleotides downstream from the wild-type location. An analysis of these results suggests that (i) the binding site for the malT product is located upstream from the region which is severely altered in the double mutant, i.e., upstream from position -31; and (ii) the 30-base pair sequence which precedes the transcription starting point contains very few positions which are essential for promoter activity.
Asunto(s)
Escherichia coli/genética , Regulación de la Expresión Génica , Operón , Secuencia de Bases , Enzimas de Restricción del ADN/metabolismo , ADN Bacteriano/análisis , Desoxirribonucleasa HpaII , Regulación de la Expresión Génica/efectos de los fármacos , Maltosa/farmacología , Transcripción GenéticaRESUMEN
In Bacillus subtilis, genes involved in arginine and ornithine catabolism constitute two operons, rocABC and rocDEF. Inducible expression of these two operons is SigL-dependent and requires the transcriptional activator RocR. RocR is a member of the NtrC/NifA family of regulators. To study the molecular mechanisms leading to the activation of RocR, we constructed a series of mutants affected in various steps of arginine catabolism. Results obtained using these mutants strongly suggest that the true inducer is ornithine or citrulline. Constitutive mutants of rocR containing either missense mutations, frameshift mutations resulting from deletions, or in-frame deletions leading to the synthesis of N-terminal truncated RocR polypeptides were obtained. Analysis of these mutants indicates that the N-terminal part of RocR is an intramolecular repressor domain. AhrC is a second positive regulatory protein of the rocABC and rocDEF operons. Two missense mutations modifying the N-terminal domain of RocR led to high constitutive expression of the Roc regulon in the absence of AhrC. Constitutive RocR proteins still require the presence of UAS1 and therefore probably bending of the DNA region located between the UAS1 and the promoter, suggesting that AhrC is not involved in DNA bending which facilitates interaction between RocR and sigma54-RNA polymerase. We suggest that the positive role of AhrC involves protein-protein interaction with RocR.
Asunto(s)
Arginina/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/fisiología , Regulación Bacteriana de la Expresión Génica , Transactivadores/fisiología , Arginasa/genética , ADN Bacteriano/genética , Proteínas de Unión al ADN/genética , Genes Bacterianos , Operón , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Factor sigma/fisiología , Transducción de Señal , Transcripción Genética , Activación TranscripcionalRESUMEN
The order of 802 base pairs was established in a DNA segment containing the promoter for malPQ which is one of the three maltose operons, and the promoter for malT, the positive regulator gene of the maltose regulon. The determination of the amino-terminal sequence of the MalT protein allowed us to identify the beginning of the malT gene on the sequence. The position of the malP gene was deduced from the published amino-terminal sequence of maltodextrin phosphorylase. A total of 611 base pairs separate the initiation codons for these two genes, which are transcribed in opposite directions. This large intergenic region does not code for any polypeptide of significant size. The main features of this sequence are discussed in terms of the regulation known to operate on malT and malPQ expression.
Asunto(s)
Cromosomas Bacterianos/ultraestructura , Proteína Receptora de AMP Cíclico , Escherichia coli/genética , Proteínas Bacterianas/biosíntesis , Secuencia de Bases , Proteínas Portadoras/fisiología , AMP Cíclico/fisiología , ADN Bacteriano/análisis , Regulación de la Expresión Génica , Genes Reguladores , Operón , Biosíntesis de ProteínasRESUMEN
A 5,800-bp (base pair) HindIII-EcoRI DNA fragment containing malT, the positive regulator gene of the maltose regulon, and most of malP, the structural gene for maltodextrin phosphorylase, was cloned into pBR322. A sequence of 802 bp was established in a DNA segment containing the promotor for malPQ and the promoter for malT. A total of 611 bp separates the initiation codons for these two genes, which are transcribed in opposite directions. The malT product was identified as a 94,000 dalton polypeptide.
Asunto(s)
Clonación Molecular , Escherichia coli/genética , Genes Bacterianos , Glucosiltransferasas/genética , Secuencia de Bases , Codón/biosíntesis , Genes Reguladores , Peso Molecular , OperónRESUMEN
We previously obtained strains of Escherichia coli in which the beginning of gene lacZ, which codes for beta-galactosidase, is replaced by the beginning of gene lamB, which codes for a maltose-inducible outer membrane protein. In some of these strains the induction (with maltose) of lamB-lacZ hybrid protein synthesis was lethal because of membrane damage resulting from an incomplete export of this protein to the outer membrane. We describe here a class of maltose-resistant mutants obtained from one such strain. Mutants in this class fail to produce the lamB-lacZ hybrid protein but retain the ability to express lacY, which is located distal to the hybrid gene. Some of the mutants carry deletions within the hybrid gene. The others carry point mutations which most probably affect the initiation of translation at the beginning of the hybrid gene. One of these is located in the sequence that codes for the presumed ribosome interaction site on the mRNA. Three others, of which two are located in the coding region (sixth codon), are believed to result in an alteration of mRNA secondary structure such that the accessibility of the ribosome interaction site is reduced.
Asunto(s)
Escherichia coli/genética , Galactosidasas/genética , Genes , Mutación , Operón , beta-Galactosidasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cruzamientos Genéticos , Enzimas de Restricción del ADN , Hibridación Genética , Hibridación de Ácido NucleicoRESUMEN
The Escherichia coli LamB protein is located in the outer membrane. It is both a component of the maltose and maltodextrin transport system, and the receptor for phages lambda and K10. It is a trimer composed of three identical polypeptide chains, each containing 421 residues. Six independent mutants have been isolated, in which the LamB protein is altered in its interaction with one or more monoclonal antibodies specific for regions of the protein that are exposed at the cell surface. Some of the mutations also altered the binding site for phage lambda. All of the mutations were clustered in the same region of the lamB gene, corresponding to residues 333-394 in the polypeptide. This and previous results strongly suggest that a rather large segment of the LamB polypeptide, extending from residue 315 to 401, is exposed at the outer face of the outer membrane. This segment would bear the epitopes for the four available anti-LamB monoclonal antibodies that react with the cell surface, and part of the binding site for phage lambda.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Epítopos/análisis , Escherichia coli/genética , Genes Bacterianos , Genes , Mutación , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/inmunología , Secuencia de Bases , Especificidad de la EspecieRESUMEN
Bacillus subtilis grown in media containing amino acids or glucose secretes acetate, pyruvate, and large quantities of acetoin into the growth medium. Acetoin can be reused by the bacteria during stationary phase when other carbon sources have been depleted. The acoABCL operon encodes the E1alpha, E1beta, E2, and E3 subunits of the acetoin dehydrogenase complex in B. subtilis. Expression of this operon is induced by acetoin and repressed by glucose in the growth medium. The acoR gene is located downstream from the acoABCL operon and encodes a positive regulator which stimulates the transcription of the operon. The product of acoR has similarities to transcriptional activators of sigma 54-dependent promoters. The four genes of the operon are transcribed from a -12, -24 promoter, and transcription is abolished in acoR and sigL mutants. Deletion analysis showed that DNA sequences more than 85 bp upstream from the transcriptional start site are necessary for full induction of the operon. These upstream activating sequences are probably the targets of AcoR. Analysis of an acoR'-'lacZ strain of B. subtilis showed that the expression of acoR is not induced by acetoin and is repressed by the presence of glucose in the growth medium. Transcription of acoR is also negatively controlled by CcpA, a global regulator of carbon catabolite repression. A specific interaction of CcpA in the upstream region of acoR was demonstrated by DNase I footprinting experiments, suggesting that repression of transcription of acoR is mediated by the binding of CcpA to the promoter region of acoR.
Asunto(s)
Acetoina Deshidrogenasa/genética , Acetoína/metabolismo , Bacillus subtilis/genética , Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica , Factor sigma/metabolismo , Acetoina Deshidrogenasa/metabolismo , Bacillus subtilis/crecimiento & desarrollo , Bacillus subtilis/metabolismo , Secuencia de Bases , Medios de Cultivo , Huella de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Glucosa/metabolismo , Datos de Secuencia Molecular , Operón , Plásmidos , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factor sigma/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Levanase expression in Bacillus subtilis was studied by using transcriptional and translational fusions. It was shown that the degradative products of levan or inulin and low concentrations of fructose were able to induce levanase expression. In the wild-type strain and in a constitutive overproducing sacL mutant, levanase synthesis was repressed by glucose or fructose. This catabolite repression was partially abolished in the derepressed alpha-amylase gra-26 mutant. The levanase gene (sacC) appears to be the distal gene of an operon transcribed from a fructose-inducible promoter. Deletion mapping experiments and primer extension analysis revealed a transcriptional start point located 2.7 kilobases upstream from the sacC gene. Two constitutive sacL mutations were shown to be closely linked by transformation to the sacC gene. The sacL6 and sacL8 mutations were mapped in the promoter-proximal region of the operon.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas , Glicósido Hidrolasas/biosíntesis , Secuencia de Aminoácidos , Bacillus subtilis/genética , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Análisis Mutacional de ADN , Represión Enzimática , Fructosa/metabolismo , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , Proteínas Recombinantes de Fusión/genética , Transcripción GenéticaRESUMEN
The regulatory gene levR of the levanase operon of Bacillus subtilis was cloned and sequenced. It encodes a polypeptide of Mr 106,064 with two domains homologous to members of two families of bacterial activators. One domain in LevR is homologous with one region of bacterial regulators including SacT and SacY of B. subtilis and BglG from Escherichia coli. Another domain of LevR is homologous to one part of the central domain of NifA and NtrC, which control nitrogen assimilation in Gram-negative bacteria. The levanase promoter contains two regions almost identical to the -12, -24 consensus regions present in sigma 54-dependent promoters. The expression of the levanase operon in E. coli was strongly dependent on sigma 54. Taken together, these results suggest that the operon is expressed from a -12, -24 promoter regulated by a sigma 54-like-dependent system in B. subtilis.