RESUMEN
To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximately 4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.
Asunto(s)
Bacillus subtilis/genética , Genes Bacterianos , Bacillus subtilis/citología , Bacillus subtilis/metabolismo , División Celular/genética , Membrana Celular/genética , Coenzimas/genética , Coenzimas/metabolismo , Metabolismo Energético/genética , Genoma Bacteriano , Mutación , Nucleótidos/genética , Nucleótidos/metabolismo , FilogeniaRESUMEN
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
The ykzB and ykoL genes encode two peptides, of 51 and 60 amino acids, the functions of which are unknown. The ykzB and tnrA genes are contiguous and transcribed divergently. Expression of ykzB and ykoL is induced by glutamate and is under the control of the TnrA global regulator of nitrogen utilization. TnrA regulated its own synthesis in glutamate minimal medium. Two DNA sequences (TnrAB1 and TnrAB2) homologous to the TnrA binding site are present in the region between tnrA and ykzB. Deletion mapping indicated that the TnrAB2 binding site was involved in activation of the ykzB promoter. In addition, transcription of tnrA depends on the presence of the TnrAB1 binding site. The ykzB and ykoL genes are probably in the same transcriptional unit. A single promoter involved in transcription in the presence of glutamate was mapped by primer extension. ykoL expression was induced by phosphate limitation and depended on the PhoP-PhoR two-component regulatory system. Its promoter was mapped to the region between ykoL and ykzB. Four boxes similar to the PhoP binding site are present upstream from the ykoL promoter. These boxes are probably recognized by PhoP approximately P during the activation of transcription in phosphate limitation conditions.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/fisiología , Genes Bacterianos/genética , Operón/genética , Proteínas Represoras , Factores de Transcripción/fisiología , Secuencia de Bases , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Ácido Glutámico/farmacología , Operón Lac/genética , Datos de Secuencia Molecular , Fosfatos/farmacología , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/genética , Transcripción Genética , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
A new gene, bkdR (formerly called yqiR), encoding a regulator with a central (catalytic) domain was found in Bacillus subtilis. This gene controls the utilization of isoleucine and valine as sole nitrogen sources. Seven genes, previously called yqiS, yqiT, yqiU, yqiV, bfmBAA, bfmBAB, and bfmBB and now referred to as ptb, bcd, buk, lpd, bkdA1, bkdA2, and bkdB, are located downstream from the bkdR gene in B. subtilis. The products of these genes are similar to phosphate butyryl coenzyme A transferase, leucine dehydrogenase, butyrate kinase, and four components of the branched-chain keto acid dehydrogenase complex: E3 (dihydrolipoamide dehydrogenase), E1alpha (dehydrogenase), E1beta (decarboxylase), and E2 (dihydrolipoamide acyltransferase). Isoleucine and valine utilization was abolished in bcd and bkdR null mutants of B. subtilis. The seven genes appear to be organized as an operon, bkd, transcribed from a -12, -24 promoter. The expression of the bkd operon was induced by the presence of isoleucine or valine in the growth medium and depended upon the presence of the sigma factor SigL, a member of the sigma 54 family. Transcription of this operon was abolished in strains containing a null mutation in the regulatory gene bkdR. Deletion analysis showed that upstream activating sequences are involved in the expression of the bkd operon and are probably the target of bkdR. Transcription of the bkd operon is also negatively controlled by CodY, a global regulator of gene expression in response to nutritional conditions.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/fisiología , Proteínas de Unión al ADN/fisiología , Isoleucina/metabolismo , Factor sigma/metabolismo , Transactivadores/fisiología , Factores de Transcripción , Valina/metabolismo , Secuencia de Aminoácidos , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Secuencia de Bases , ADN Bacteriano , Proteínas de Unión al ADN/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Proteína Reguladora de Respuesta a la Leucina , Datos de Secuencia Molecular , Nitrógeno , Operón , Fenotipo , Regiones Promotoras Genéticas , Transactivadores/genéticaRESUMEN
As a soil bacterium also found in estuarine and marine habitats, Bacillus subtilis has evolved various sensing and adaptation systems in order to face salt stress conditions. Among these regulatory mechanisms is the DegS-DegU signal transduction system, which was previously shown to be stimulated by high salt concentrations. A search for promoters regulated in response to salt stress led to the identification of wapA, encoding a wall-associated protein, which is strongly expressed at low salt concentrations and almost completely repressed in the presence of 0.7 M disodium succinate. Repression of wapA transcription by salt stress was shown to require the phosphorylated form of DegU. Moreover, DegU-mediated repression of wapA occurred only in high-salt medium. Alignment between the control region of wapA and other DegU-regulated promoters allowed the identification of a putative DegU target sequence, AGAAN(11)TTCAG. Mutation/deletion analyses of the wapA promoter region confirmed the role of the putative DegU control site in repression of wapA transcription at high salt concentrations and revealed a second site of repression located downstream from the transcription start site. Since residual negative control was observed at this second site in the absence of DegU, it seems likely that an additional repressor acts on the wapA control region to further downregulate wapA transcription under salt stress conditions.
Asunto(s)
Antígenos Bacterianos , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Genes Bacterianos , Regulón , Cloruro de Sodio/farmacología , Bacillus subtilis/efectos de los fármacos , Secuencia de Bases , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas , Relación Estructura-Actividad , Transcripción GenéticaRESUMEN
To gain insight into the mode of action of mesentericin Y105, a bacteriocin bactericidal agent against Listeria monocytogenes, we undertook to identify the listerial factors mediating this susceptibility by using a genetic approach. Transposon mutants resistant to the bacteriocin were obtained. One of them corresponded to a transposon insertion in a gene (rpoN) encoding a putative protein (447 amino acids) with strong homologies to alternative transcriptional sigma54 factors, including that of Bacillus subtilis (38% identity). Complementation experiments with the wild-type rpoN gene demonstrated that the insertion in rpoN was responsible for the resistance phenotype in L. monocytogenes. Moreover, expression of the L. monocytogenes rpoN gene in an rpoN mutant strain of B. subtilis promoted transcription of a sigma54-dependent operon in the presence of the associated regulator. These results demonstrate that the L. monocytogenes rpoN gene encodes a new sigma54 factor.
Asunto(s)
Antibacterianos/farmacología , Bacteriocinas/farmacología , Proteínas de Unión al ADN , ARN Polimerasas Dirigidas por ADN/genética , Listeria monocytogenes/efectos de los fármacos , Péptidos , Factor sigma/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , ARN Polimerasas Dirigidas por ADN/clasificación , Farmacorresistencia Microbiana , Prueba de Complementación Genética , Leuconostoc , Listeria monocytogenes/genética , Datos de Secuencia Molecular , Mutagénesis Insercional , ARN Polimerasa Sigma 54 , Homología de Secuencia de Aminoácido , Factor sigma/clasificaciónRESUMEN
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
Expression of the sacPA and sacB genes of Bacillus subtilis is positively modulated by transcriptional regulatory proteins encoded by the sacT and sacY genes, respectively. Previous genetic studies led to the suggestion that SacT and SacY function as nascent mRNA binding proteins preventing early termination of transcription at terminators located in the leader regions of the corresponding genes. Here we report the overproduction, purification to near homogeneity, and characterization of the two antiterminators, SacT and SacY. Using mRNA band migration retardation assays and a reconstituted transcriptional antitermination system, the mRNA binding functions and antitermination activities of purified SacT and SacY are demonstrated under in vitro conditions. The results establish for the first time that members of the BglG family of antiterminators function in antitermination in the absence of other proteins in vitro. Purified SacT is shown to be phosphorylated by phosphoenolpyruvate in a phosphotransferase-catalyzed reaction dependent on Enzyme I and HPr. Unexpectedly, the purified SacT is shown to be functional in mRNA binding and in transcriptional antitermination independently of its phosphorylation state.
Asunto(s)
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Factores de Transcripción , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , Cartilla de ADN/genética , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Datos de Secuencia Molecular , Operón , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas de Unión al ARN/metabolismo , Transcripción GenéticaRESUMEN
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
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
Bacillus subtilis can use ammonium and various amino acids as sole nitrogen sources. The utilization of arginine or ornithine is abolished in a sigma L-deficient strain of B. subtilis, indicating that one or several genes involved in this pathway are transcribed by a sigma L-RNA polymerase holoenzyme. Three B. subtilis genes, called rocA, rocB, and rocC, which seem to form an operon, were found near the sacTPA locus (P. Glaser, F. Kunst, M. Arnaud, M.-P. Coudart, W. Gonzales, M.-F. Hullo, M. Ionescu, B. Lubochinsky, L. Marcelino, I. Moszer, E. Presecan, M. Santana, E. Schneider, J. Schweizer, A. Vertes, G. Rapport, and A. Danchin, Mol. Microbiol. 10:371-384, 1993). The expression of this putative operon is induced by arginine and is sigma L dependent. Mutants impaired in the transcription of rocA were obtained. One of these mutants was used as recipient to clone and sequence a new regulatory gene, called rocR. This gene encodes a polypeptide of 52 kDa which belongs to the NtrC/NifA family of transcriptional activators. Upstream activating sequences highly similar to those of NtrC in Escherichia coli were also identified upstream from the rocABC genes. A B. subtilis strain containing a rocR null mutation is unable to use arginine as the sole nitrogen source, indicating that RocR is a positive regulator of arginine catabolism. After LevR, RocR is the second example of an activator stimulating sigma 54-dependent promoters in gram-positive bacteria.
Asunto(s)
Arginina/metabolismo , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Genes Bacterianos , Transactivadores/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Bacillus subtilis/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Clonación Molecular , Secuencia de Consenso , Cartilla de ADN , Escherichia coli , Proteínas de Escherichia coli , Genotipo , Datos de Secuencia Molecular , Operón , Proteínas PII Reguladoras del Nitrógeno , Proteínas Recombinantes de Fusión/biosíntesis , Mapeo Restrictivo , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Transactivadores/biosíntesis , Transactivadores/metabolismo , Factores de Transcripción/biosíntesisRESUMEN
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
The sacT gene which controls the sacPA operon of Bacillus subtilis encodes a polypeptide homologous to the B. subtilis SacY and the Escherichia coli BglG antiterminators. Expression of the sacT gene is shown to be constitutive. The DNA sequence upstream from sacP contains a palindromic sequence which functions as a transcriptional terminator. We have previously proposed that SacT acts as a transcriptional antiterminator, allowing transcription of the sacPA operon. In strains containing mutations inactivating ptsH or ptsI, the expression of sacPA and sacB is constitutive. In this work, we show that this constitutivity is due to a fully active SacY antiterminator. In the wild-type sacT+ strain or in the sacT30 mutant, SacT requires both enzyme I and HPr of the phosphotransferase system (PTS) for antitermination. It appears that the PTS exerts different effects on the sacB gene and the sacPA operon. The general proteins of the PTS are not required for the activity of SacY while they are necessary for SacT activity.
Asunto(s)
Bacillus subtilis/genética , Regulación Bacteriana de la Expresión Génica/genética , Genes Reguladores/genética , Operón/genética , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Proteínas Bacterianas/genética , Secuencia de Bases , Análisis Mutacional de ADN , Operón Lac/genética , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas de Unión al ARN/genética , Proteínas Recombinantes/análisis , Mapeo Restrictivo , Regiones Terminadoras Genéticas , beta-Galactosidasa/análisisRESUMEN
The levanase operon in Bacillus subtilis is expressed from a -12, -24 promoter and transcription is stimulated by the regulator LevR, which contains a domain homologous with the central domain of the NifA and NtrC family of regulators. We isolated mutants defective in the expression of the levanase operon. These strains contain mutations that define a gene, called sigL, located between cysB and sacB on the genetic map. The sigL gene was cloned and sequenced. It encodes a polypeptide containing 436 residues with a molecular weight of 49,644. The amino acid sequence of SigL is homologous with all sigma 54 factors from Gram-negative bacteria, including Rhizobium meliloti (32% identity) and Klebsiella pneumoniae (30% identity). B. subtilis sigL mutants have a pleiotropic phenotype: (i) the transcription of the levanase operon is strongly reduced and (ii) in minimal medium lacking ammonia, sigL mutants cannot grow when arginine, ornithine, isoleucine, or valine is the sole nitrogen source. These results indicate that the sigL gene encodes an equivalent of the sigma 54 factor in B. subtilis, to our knowledge, the first of this type to be identified in Gram-positive bacteria.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas , Genes Bacterianos , Bacterias Gramnegativas/genética , Factor sigma/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Genes Reguladores , Glicósido Hidrolasas/genética , Datos de Secuencia Molecular , Operón , Plásmidos , Regiones Promotoras Genéticas , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Transducción Genética , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoAsunto(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
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.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/genética , Genes Reguladores , Glicósido Hidrolasas/genética , Operón , Factores de Transcripción/genética , Secuencia de Aminoácidos , Bacillus subtilis/enzimología , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/genética , Escherichia coli/genética , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/metabolismo , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
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 AbiertaRESUMEN
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
The expression of the Bacillus subtilis sacPA operon is induced by sucrose. A DNA fragment containing the upstream region of this operon was cloned. This fragment contains a promoter from which the operon is expressed. This upstream region also contains a palindromic DNA sequence very similar to the transcriptional terminator which regulates the induction of the B. subtilis sacB gene. Of 37 nucleotides in a region partially overlapping the sacP palindromic sequence, 34 were identical to the corresponding region of the sacB gene. A similar motif is also present in the bgl operon of Escherichia coli. The sacT locus controlling sacPA expression had been identified by a single constitutive mutation sacT30 which mapped close to the sacPA operon. DNA fragments containing the sacT+ and sacT30 alleles were cloned and sequenced. The sacT gene product is very similar to the B. subtilis sacY and to the E. coli bglG gene products. The constitutive sacT30 mutation was identified. It corresponds to a Asp-96-to-Tyr missense mutation located in a highly conserved region in SacT and SacY. These results strongly suggest that sacT is a specific regulatory gene of the sacPA operon.
Asunto(s)
Bacillus subtilis/genética , Genes Bacterianos , Genes Reguladores , Operón , Sacarosa/metabolismo , Regiones Terminadoras Genéticas , Transcripción Genética , Secuencia de Aminoácidos , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/enzimología , Secuencia de Bases , Clonación Molecular/métodos , Elementos Transponibles de ADN , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Genotipo , Datos de Secuencia Molecular , Plásmidos , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Sacarosa/farmacologíaRESUMEN
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.