RESUMEN
Silencing of levB, the second structural gene of the tricistronic levansucrase operon encoding the endolevanase LevB, decreases the level of levansucrase expression in Bacillus subtilis. Conversely, independent expression of levB greatly stimulates operon expression. This autogenous effect is mediated by the levB transcript, which carries an internal sequence (5'-AAAGCAGGCAA-3') involved in the enhancing effect. In vitro, the levB transcript displays an affinity for the N-terminal fragment of SacY (K(D) 0.2 microM), the regulatory protein that prevents transcription termination of the levansucrase operon. This positive-feedback loop leads to an increase in the operon expression when B. subtilis is growing in the presence of high sucrose concentrations. Under these conditions, extracellular levan synthesized by the fructosyl polymerase activity of levansucrase can be degraded mainly into levanbiose by the action of LevB. Levanbiose is neither taken up nor metabolized by the bacteria. This work modifies the present view of the status of levansucrase in B. subtilis physiology.
Asunto(s)
Bacillus subtilis/genética , Regulación Bacteriana de la Expresión Génica , Hexosiltransferasas/genética , Operón , Sistemas de Transporte de Aminoácidos Acídicos/biosíntesis , Sistemas de Transporte de Aminoácidos Acídicos/genética , Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Disacáridos/metabolismo , Fructanos/metabolismo , Silenciador del Gen , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Hexosiltransferasas/biosíntesis , Hexosiltransferasas/metabolismo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Bacteriano/análisis , ARN Bacteriano/metabolismo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Sacarosa/metabolismo , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
We studied the interactions between conformers of exocellular alpha-amylase and small unilamellar vesicles (SUV) composed of the major membrane lipids of Bacillus subtilis under physiological conditions of pH, temperature and ionic strength. Using fluorescence spectroscopy, surface plasmon resonance (SPR) and phase separation, we show that the native alpha-amylase has no affinity for the SUV, whereas a partially folded form, displaying structural properties in common with the competent state for secretion, binds to the vesicles (KA approximately 10(5) M(-1)). This association prevented its subsequent folding. The complex was destabilized in the presence of PrsA, a major peripheric lipoprotein of B. subtilis which displays a strong affinity for SUV (KA approximately 1.5x10(8) M(-1)). Vesicles coated with PrsA lost their ability to bind the partially folded conformer. The approach in vitro, in which our aim was to mimic the last stage of alpha-amylase translocation, indicates that PrsA possibly helps, in vivo, the secreted protein to acquire its native conformation by modulating the interaction between the latter and the lipid polar heads on the trans side of the cytoplasmic membrane.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas , Membrana Dobles de Lípidos/química , alfa-Amilasas/química , Distribución en Contracorriente , Lipoproteínas/farmacología , Lipoproteínas/fisiología , Proteínas de la Membrana/farmacología , Proteínas de la Membrana/fisiología , Fosfolípidos/química , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Espectrometría de Fluorescencia , Resonancia por Plasmón de Superficie , alfa-Amilasas/antagonistas & inhibidores , alfa-Amilasas/metabolismoRESUMEN
YfkN isolated from the culture supernatant of Bacillus subtilis in the exponential phase of growth is a protein of 143.5 kDa that derives from a putative large precursor of 159.6 kDa processed at both the N- and C-terminal ends. Pulse-chase experiments indicated that the release occurs slowly with a half-time longer than 30 min, suggesting that the event is coupled with wall turnover. YfkN exhibits 2',3' cyclic nucleotide phosphodiesterase, 2' (or 3') nucleotidase and 5' nucleotidase activities. In vitro the protein is reduced by subtilisin digestion to a shorter polypeptide (68 kDa), displaying phosphodiesterase activity but devoid of any 5'nucleotidase activity. This proteolytic processing led us to localize the potential active sites of the various nucleotidase activities. When bacteria were grown in low phosphate medium, the exocellular production of the enzyme was enhanced, suggesting that it plays a role in phosphate metabolism. Comparison with nucleotidase databases suggests that yfkN resulted from gene fusion.
Asunto(s)
2',3'-Nucleótido Cíclico Fosfodiesterasas/aislamiento & purificación , 2',3'-Nucleótido Cíclico Fosfodiesterasas/metabolismo , Bacillus subtilis/enzimología , Nucleotidasas/aislamiento & purificación , Nucleotidasas/metabolismo , Secuencia de Aminoácidos , Bacillus subtilis/metabolismo , Cinética , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Subtilisina/metabolismoRESUMEN
We characterized the reversible folding-unfolding transition of Bacillus subtilis exocellular chitosanase from either thermal or urea denaturation of the protein. The transitions were monitored in each case by intrinsic fluorescence changes and resistance to proteolysis. Unfolding and refolding kinetics and differential scanning calorimetry analysis suggested a two-state equilibrium. The equilibrium between the folded and unfolded states was rapidly displaced towards the folded state in the presence of a low concentration of calcium (2-20 mM). The binding titration curve indicated that chitosanase possesses one weak Ca(2+)-binding site (with an equilibrium affinity constant, K (A), of 0.3x10(3) M(-1)). These results support the hypothesis that this metal ion, which is accumulated in the cell wall environment of B. subtilis, is an effector that influences folding and stability of newly translocated proteins.
Asunto(s)
Bacillus subtilis/enzimología , Calcio/metabolismo , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Secuencia de Aminoácidos , Bacillus subtilis/crecimiento & desarrollo , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Sitios de Unión , Calcio/química , División Celular/fisiología , Durapatita/metabolismo , Estabilidad de Enzimas , Fluorescencia , Glicósido Hidrolasas/aislamiento & purificación , Cinética , Datos de Secuencia Molecular , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Volumetría , Urea/químicaRESUMEN
When Bacillus subtilis levanase (SacC), alpha-amylase (AmyE) and chitosanase (Csn) structural genes were expressed under the regulated control of sacR, the inducible levansucrase (SacB) leader region in a degU32(Hy) mutant, it was observed that the production yields of the various extracellular proteins were quite different. This is mainly due to differences in the stabilities of their corresponding mRNAs which lead to discrepancies between the steady-state level of mRNA of sacB and csn on the one hand and amyE and sacC on the other. In contrast to levansucrase mRNA, the decay curves of alpha-amylase and levanase mRNAs obtained by Northern blotting analysis did not match the decay curves of their functional mRNA. This suggested that only a part of the population of the amyE and sacC transcripts was fully translated, while the others were possibly poorly bound to ribosomes and thus were only partially translated or not at all and consequently submitted to rapid endonuclease degradation. This hypothesis was substantiated by the finding that the introduction of a Shine-Dalgarno sequence upstream from the ribosome-binding site in the sacC transcript resulted in a fourfold increase in both the half-life of this transcript and the production of levanase. An additional cause of low-level levanase production is the premature release of mRNA by the polymerase. It was attempted to correlate this event with internal secondary structures of sacC mRNA.
Asunto(s)
Regiones no Traducidas 5' , Bacillus subtilis/genética , Proteínas Bacterianas , Glicósido Hidrolasas/genética , Hexosiltransferasas/genética , alfa-Amilasas/genética , Bacillus subtilis/metabolismo , Northern Blotting , Glicósido Hidrolasas/biosíntesis , Hexosiltransferasas/biosíntesis , Cinética , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Transcripción Genética , alfa-Amilasas/biosíntesisRESUMEN
The Bacillus subtilis levanase structural gene sacC was expressed under the regulated control of sacR, the inducible levansucrase leader region, in a degU32(Hy) strain. In this genetic context, exocellular levanase is overproduced (0.5% of total protein) during the exponential phase of growth upon induction by sucrose at 37 degrees C and pH 7. No precursor form that comprised a signal peptide was detected in pulse-chase experiments. The subsequent release of the cell-associated processed protein is a slow event (t(1,2) = 80+/-10 s). The unfolding-folding transition of pure levanase monitored in vitro by the resistance to proteolysis was achieved within the same time range (t(1/2) = 50 s) under the same conditions of pH and temperature. Calcium ions, which modulate the rate and the yield of refolding, have a low affinity for the protein. Comparison of these results with those obtained previously with levansucrase and alpha-amylase overproduced in the same genetic and physiological context suggests that the precursor processing is more efficient in levanase and alpha-amylase than in levansucrase. This discrepancy could lie in information borne by the signal peptide sequence of these exoproteins. However, the rate of the ultimate stage of release of these three proteins, which includes the passage through the cell wall, is correlated with the rate of folding and appears to be independent of their molecular size.
Asunto(s)
Bacillus subtilis/crecimiento & desarrollo , Proteínas Bacterianas , Glicósido Hidrolasas/metabolismo , Bacillus subtilis/enzimología , Calcio/farmacología , Glicósido Hidrolasas/genética , Cinética , Pliegue de Proteína , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/metabolismoRESUMEN
The Bacillus subtilis alpha-amylase gene, amyE, was expressed under the regulated control of sacR, the levansucrase leader region. The gene fusion including the complete amyE coding sequence with the signal peptide sequence was integrated into the chromosome of a degU32(Hy) strain deleted of the sacB DNA fragment. In this genetic contex, alpha-amylase is produced in the culture supernatant at a high level (2% of total protein) during the exponential phase of growth upon induction by sucrose. Pulse-chase experiments showed that the rate-limiting step (t1/2 = 120 s) of the secretion process is the release of a cell-associated precursor form whose signal peptide has been cleaved. The efficiency of this ultimate step of secretion decreased dramatically in the presence of a metal chelator (EDTA) or when the cells were converted to protoplasts. The hypothesis that this step is tightly coupled with the folding process of alpha-amylase occurring within the cell wall environment was substantiated by in vitro folding studies. The unfolding-folding transition, monitored by the resistance to proteolysis, was achieved within the same time range (t1/2 = 60 s) and required the presence of calcium. This metal requirement could possibly be satisfied in vivo by the integrity of the cell wall. The t1/2 of the alpha-amylase release step is double that of levansucrase, although their folding rates are similar. This perhaps indicates that the passage through the cell wall may depend on parietal properties (e.g. metal ion binding and porosity) and on certain intrinsic properties of the protein (molecular mass and folding properties).
Asunto(s)
Bacillus subtilis/enzimología , alfa-Amilasas/metabolismo , Secuencia de Aminoácidos , Fusión Artificial Génica , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Secuencia de Bases , Pared Celular/enzimología , Pared Celular/metabolismo , Quelantes/farmacología , Cartilla de ADN/genética , Ácido Edético/farmacología , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Cinética , Plásmidos/genética , Pliegue de Proteína , Señales de Clasificación de Proteína/genética , Protoplastos/enzimología , Sacarosa/farmacología , alfa-Amilasas/química , alfa-Amilasas/genéticaRESUMEN
The Acetobacter diazotrophicus SRT4 gene encoding levansucrase (EC 2.4.1.10) (IsdA) was isolated from a genomic library. The nucleotide sequence of a 2.3 kb DNA fragment sufficient for complementation of a levansucrase-deficient mutant (obtained by EMS treatment) was determined. The IsdA gene (1751 bp) coded for a polypeptide of molecular mass 64.9 kDa with an isoelectric point of 5.2. The N-terminal amino acid sequence of the extracellular levansucrase indicated the presence of a precursor protein with a putative signal sequence of 51 residues which is possibly cleaved in two successive steps. Expression of the IsdA gene from the lac promoter in Escherichia coli resulted in the production of a protein with levansucrase activity. The deduced amino acid sequence of the IsdA gene was 48% and 46% identical with the levansucrases from the Gram-negative bacteria Zymomonas mobilis and Erwinia amylovora, respectively, but only 28-31% identical with levansucrases from Gram-positive bacteria. Multiple alignments of published levansucrase sequences from Gram-negative and Gram-positive bacteria revealed eight conserved motifs. A comparison of the catalytic properties and the sequence of the A. diazotrophicus levansucrase with those of the Bacillus subtilis levansucrase suggested that one of these motifs may be involved in the specificity of the synthetized product. Disruption of the IsdA gene in the genome of A. diazotrophicus resulted in a mutant lacking both levansucrase activity and the ability to utilize sucrose as a carbon source, suggesting that levansucrase is the key enzyme in sucrose metabolism of A. diazotrophicus.