RESUMEN
A 7 kb DNA fragment was cloned from Lactobacillus sakei which contains the IdhL gene encoding the L(+)-lactate dehydrogenase (L-LDH). Analysis of the DNA sequence, Northern experiments and primer extension experiments showed that IdhL is transcribed from a single promoter, leading to a monocistronic 1.15 kb mRNA which yields the L-LDH. A stable mutant was constructed by chromosomal integration of a chloramphenicol cassette into IdhL by a double-crossover event. Both L- and D-lactate were produced by the wild-type strain whereas only residual amounts of both isomers were produced by the mutant. This demonstrates that L. sakei possesses an L-LDH producing L-lactate and a lactate racemase able to transform it to D-lactate, but is devoid of D-LDH activity. Moreover the ability to degrade L-lactate present in the medium that was observed with the mutant strain grown aerobically suggests that an L-lactate oxidase activity is also present in L. sakei.
Asunto(s)
L-Lactato Deshidrogenasa/genética , Lactatos/metabolismo , Lactobacillus/genética , Clonación Molecular , ADN Bacteriano/análisis , Escherichia coli , Regulación Bacteriana de la Expresión Génica , L-Lactato Deshidrogenasa (Citocromo) , Lactobacillus/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis , Análisis de Secuencia de ADN , Estereoisomerismo , Transcripción GenéticaRESUMEN
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
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
The first anaesthetic record was introduced into medical practice in 1940. Since then few changes have been made to it and it remains a rudimentary memorandum. However, since the beginning of the 1980s, interest in automatic recording of the anaesthetic file has been increasing and numerous arguments can be put forward in its favour. Apart from theoretical and experimental arguments, in practice one has to master the automatic collection of data, management of alarms and the technology of the networks involved in order to manage the flow of information by channelling it and organizing it into a hierarchy. Four other objectives can be added to the clinical recording and its medico-legal applications: anaesthetic cost evaluation, quality of care, research and clinical teaching which will provide the basis of anaesthetic epidemiological research.