RESUMEN
Lactobacillus sakei is one of the most important lactic acid bacteria of meat and fermented meat products. It is able to degrade arginine with ammonia and ATP production by the arginine deiminase pathway (ADI). This pathway is composed of three enzymes: arginine deiminase, ornithine transcarbamoylase and carbamate kinase, and an arginine transport system. The transcription of the ADI pathway is induced by arginine and subjected to catabolite repression. In order to understand the physiological role of the degradation of this amino acid we investigated the growth of bacteria under various conditions. We show that arginine degradation is responsible for an enhanced viability during the stationary phase when cells are grown under anaerobiosis. Arginine is necessary for the induction of the ADI pathway but in association with another environmental signal. Using a mutant of the L-lactate dehydrogenase unable to lower the pH we could clearly demonstrate that (i) low pH is not responsible for cell death during the stationary phase, so survival is due to another factor than elevated pH, (ii) neither low pH nor oxygen limitation is responsible for the induction of the ADI pathway together with arginine since the ldhL mutant is able to degrade arginine under aerobiosis.
Asunto(s)
Arginina/metabolismo , Lactobacillus/crecimiento & desarrollo , Lactobacillus/metabolismo , Medios de Cultivo , Glucosa/metabolismo , Concentración de Iones de Hidrógeno , Hidrolasas/genética , Hidrolasas/metabolismo , Carne/microbiología , Operón , Ribosa/metabolismoRESUMEN
The ptsH and ptsI genes of Lactobacillus sake, encoding the general enzymes of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS), were cloned and sequenced. HPr (88 amino acids), encoded by ptsH, and enzyme I (574 amino acids), encoded by ptsI, are homologous to the corresponding known enzymes of other bacteria. Nucleotide sequence and mRNA analysis showed that the two genes are cotranscribed in a large transcript encoding both HPr and enzyme I. The transcription of ptsHI was shown to be independent of the carbon source. Four ptsI mutants were constructed by single-crossover recombination. For all mutants, growth on PTS carbohydrates was abolished. Surprisingly, the growth rates of mutants on ribose and arabinose, two carbohydrates which are not transported by the PTS, were accelerated. This unexpected phenotype suggests that the PTS negatively controls ribose and arabinose utilization in L. sake by a mechanism different from the regulation involving HPr described for other gram-positive bacteria.
Asunto(s)
Genes Bacterianos , Lactobacillus/enzimología , Lactobacillus/genética , Operón , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Secuencia de Aminoácidos , Arabinosa/metabolismo , Secuencia de Bases , Metabolismo de los Hidratos de Carbono , Clonación Molecular , Cartilla de ADN/genética , Lactobacillus/metabolismo , Datos de Secuencia Molecular , Mutación , Fenotipo , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Ribosa/metabolismo , Transcripción GenéticaRESUMEN
Single-crossover homologous integration in Lactobacillus sake was studied. Integration was conducted with nonreplicative delivery vector pRV300. This vector is composed of a pBluescript SK- replicon for propagation in Escherichia coli and an erythromycin resistance marker. Random chromosomal DNA fragments of L. sake 23K ranging between 0.3 and 3.4 kb were inserted into pRV300. The resulting plasmids were able to integrate into the chromosome by homologous recombination as single copies and were maintained stably. The single cross-over integration frequency was logarithmically proportional to the extent of homology between 0.3 and 1.2 kb and reached a maximum value of 1.4 x 10(3) integrants/micrograms of DNA. We used this integration strategy to inactivate the ptsI gene, encoding enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system, and the lacL gene, which is one of the two genes required for the synthesis of a functional beta-galactosidase. The results indicated that our method facilitates genetic analysis of L. sake.
Asunto(s)
Genes Bacterianos , Lactobacillus/genética , Intercambio Genético , Farmacorresistencia Microbiana/genética , Eritromicina , Escherichia coli/genética , Vectores Genéticos , Lactobacillus/enzimología , Carne/microbiología , Fenotipo , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Plásmidos , Recombinación Genética , Replicón , beta-Galactosidasa/genéticaRESUMEN
The ability of Lactobacillus sake to use various carbon sources was investigated. For this purpose we developed a chemically defined medium allowing growth of L. sake and some related lactobacilli. This medium was used to determine growth rates on various carbohydrates and some nutritional requirements of L. sake. Mutants resistant to 2-deoxy-d-glucose (a nonmetabolizable glucose analog) were isolated. One mutant unable to grow on mannose and one mutant deficient in growth on mannose, fructose, and sucrose were studied by determining growth characteristics and carbohydrate uptake and phosphorylation rates. We show here that sucrose, fructose, mannose, N-acetylglucosamine, and glucose are transported and phosphorylated by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The PTS permease specific for mannose, enzyme II(supMan), was shown to be responsible for mannose, glucose, and N-acetylglucosamine transport. A second, non-PTS system, which was responsible for glucose transport, was demonstrated. Subsequent glucose metabolism involved an ATP-dependent phosphorylation. Ribose and gluconate were transported by PTS-independent permeases.
RESUMEN
To test the effects of theta-type replication on homologous DNA recombination, we integrated in the chromosome of Bacillus subtilis a structure comprising a conditional replication region and direct repeats of approximately 4 kb. The replicon was derived from a broad-host-range plasmid, pAM beta 1, which replicates by a unidirectional theta mechanism and its thermosensitive. The direct repeats were derived from plasmid pBR322 and flanked the chloramphenicol-resistance gene of plasmid pC194. Recombination between the repeats could therefore lead to a loss of the resistance gene or the appearance of additional repeats. The integrated replicon was active at the permissive temperature, and approximately 25% of the integrated plasmids could be isolated as Y-shaped molecules after restriction, having a branch at the replication origin. Replicon activity stimulated recombination four- to fivefold, as estimated from the proportion of chloramphenicol-sensitive cells at the restrictive and permissive temperature, and also led to the appearance of additional direct repeats. We conclude that theta-type replication stimulates homologous recombination and suggest that many or even most recombination events between long homologous sequences present in a bacterial genome may be the consequence of DNA replication.
Asunto(s)
Bacillus subtilis/genética , Replicación del ADN , ADN Bacteriano/biosíntesis , Recombinación Genética , Bacillus subtilis/metabolismo , Resistencia al Cloranfenicol/genética , Cromosomas Bacterianos/genética , Electroforesis en Gel de Campo Pulsado , Electroforesis en Gel Bidimensional , Enterococcus faecalis/genética , Genotipo , Microscopía Electrónica , Plásmidos/genética , Plásmidos/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos/genética , Replicón/genética , Eliminación de Secuencia , TemperaturaRESUMEN
The gene infB codes for two forms of translational initiation factor IF2; IF2 alpha (97,300 Da) and IF2 beta (79,700 Da). IF2 beta arises from an independent translational event on a GUG codon located 471 bases downstream from IF2 alpha start codon. By site-directed mutagenesis we constructed six different mutations of this GUG codon. In all cases, IF2 beta synthesis was variably affected by the mutations but not abolished. We show that the residual expression of IF2 beta results from translational initiation on an AUG codon located 21 bases downstream from the mutated GUG. Furthermore, two forms of IF2 beta have been separated by fast protein liquid chromatography and the determination of their N-terminal sequences indicated that they resulted from two internal initiation events, one occurring on the previously identified GUG start codon, the other on the AUG codon immediately downstream. We conclude that two forms of IF2 beta exist in the cell, which differ by seven aminoacid residues at their N terminus. Only by mutating both IF2 beta start codons could we construct plasmids that express only IF2 alpha. A plasmid expressing only IF2 beta was obtained by deletion of the proximal region of the infB gene. Using a strain that carries a null mutation in the chromosomal copy of infB and a functional copy of the same gene on a thermosensitive lysogenic lambda phage, we could cure the lambda phage when the plasmids expressing only one form of IF2 were supplied in trans. We found that each one of the two forms of IF2, at near physiological levels, can support growth of Escherichia coli, but that growth is retarded at 37 degrees C. This result shows that both forms of IF2 are required for maximal growth of the cell and suggests that they have acquired some specialized but not essential function.
Asunto(s)
Codón , Escherichia coli/crecimiento & desarrollo , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Clonación Molecular , ADN Bacteriano , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/fisiología , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutagénesis , ARN BacterianoRESUMEN
A system allowing the induction of DNA amplification in Bacillus subtilis was developed, based on a thermosensitive plasmid, pE194, stably integrated in the bacterial chromosome. An amplification unit, comprising an antibiotic resistance marker flanked by directly repeated sequences, was placed next to the integrated plasmid. Activation of pE194 replication led to DNA amplification. Two different amplification processes appeared to take place: one increased the copy number of all sequences in the vicinity of the integrated plasmid and was possibly of the onion skin type, while the other increased the copy number of the amplification unit only and generated long arrays of amplification units. These arrays were purified and shown to consist mainly of directly repeated amplification units but to also contain non-linear regions, such as replication forks and recombination intermediates. They were attached to the chromosome at one end only, and were, in general, not stably inherited, which suggests that they are early amplification intermediates. Longer arrays were detected before the shorter ones during amplification. When the parental amplification unit contained repeats which differed by a restriction site the arrays which derived thereof contained in a majority of cases only a single type of repeat. We propose that the amplified DNA is generated by rolling circle replication, and that such a process might underlie a number of amplification events.
Asunto(s)
Bacillus subtilis/genética , Cromosomas Bacterianos , ADN Bacteriano/genética , Amplificación de Genes , Intercambio Genético , Electroforesis , Modelos Genéticos , Conformación de Ácido Nucleico , Plásmidos , Mapeo RestrictivoRESUMEN
The gene for initiation factor IF2, infB, represents one of the few examples in Escherichia coli of genes encoding two protein products in vivo. In a previous work, our group showed that both forms of IF2 (alpha and beta) are closely related and may arise from two independent translational events on infB mRNA. Unambiguous mapping and rigorous determination of the nature of the initiation triplet for IF2 beta, the smaller form of IF2, is critical for future mutagenesis of this codon, required for investigating the biological importance of both IF2 alpha and IF2 beta. Three types of experiments were carried out. First, a 77-bp deletion was created at the beginning of the structural gene leading to premature termination of IF2 alpha synthesis. Under these conditions, IF2 beta is still formed. Second, various Bal31 digests of infB containing the 77-bp deletion were fused to lacZ. Any synthesis of a fused protein with beta-galactosidase activity should reflect the occurrence of an initiation event on the messenger corresponding to this DNA segment. It was consequently possible to locate the IF2 beta initiation site within an 18-base region containing an in-phase GUG codon. Third, to avoid any artefactual reinitiation event possibly occurring under our experimental conditions, we fused to lacZ an infB fragment devoid of IF2 alpha start sequences but containing genetic information for this 18-base region. A hybrid protein with beta-galactosidase activity was synthesized. Moreover, its NH2-terminal amino acid sequence coincided with that of IF2 beta, demonstrating that GUG, located 471 bases downstream from the IF2 alpha external start codon, is the internal start codon for the shorter form of IF2.
Asunto(s)
ADN Bacteriano/análisis , Escherichia coli/genética , Factor 2 Eucariótico de Iniciación/genética , Galactosidasas/biosíntesis , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Biosíntesis de Proteínas/fisiología , ARN Mensajero/análisis , Proteínas Recombinantes de Fusión/biosíntesis , beta-Galactosidasa/biosíntesis , Secuencia de Bases , Sitios de Unión , Deleción Cromosómica , Codón/análisis , Escherichia coli/metabolismo , Factor 2 Eucariótico de Iniciación/biosíntesis , Datos de Secuencia Molecular , Mapeo Peptídico , PlásmidosRESUMEN
The assembly of ribosomes in bacterial cells is a complex process that remains poorly characterized. The in vitro assembly of active ribosomal subunits from purified RNA and protein components indicates that all of the information for proper assembly resides in the primary sequences of these macromolecules. On the other hand, the in vitro requirement of unphysiological heating steps suggests that this pathway may not accurately reflect the in vivo pathway, and that other proteins may be required. One approach to identify any additional proteins is to isolate second-site revertants of mutants defective in ribosome assembly. Ribosomal protein L24 is essential in the assembly of 50S subunits. We have identified an Escherichia coli gene, srmB, that, when expressed at high copy number, can suppress the effect of a temperature-sensitive lethal mutation in L24. The SrmB amino-acid sequence has sequence identity with mouse translation initiation factor eIF-4A and with the human nuclear protein, p68. The purified SrmB protein is a nucleic acid-dependent ATPase, like eIF-4A, but can also bind RNA in the absence of ATP and other auxiliary protein factors. The RNA dependent ATPase activity of SrmB suggests that like, eIF-4A, it could be involved in specific alterations of RNA secondary structure.