RESUMEN

In Streptococcus pneumoniae, a fermentative aerotolerant and catalase-deficient human pathogen, oxidases with molecular oxygen as substrate are important for virulence and for competence. The signal-transducing two-component systems CiaRH and ComDE mediate the response to oxygen, culminating in competence. In this work we show that the two-component MicAB system, whose MicB kinase carries a PAS domain, is also involved in competence repression under oxygen limitation. Autophosphorylation of recombinant MicB and phosphotransfer to recombinant MicA have been demonstrated. Mutational analysis and in vitro assays showed that the C-terminal part of the protein and residue L100 in the N-terminal cap of its PAS domain are both crucial for autokinase activity in vitro. Although no insertion mutation in micA was obtained, expression of the mutated allele micA59DA did not change bacterial growth and overcame competence repression under microaerobiosis. This was related to a strong instability of MicA59DA-PO(4) in vitro. Thus, mutations which either reduced the stability of MicA-PO(4) or abolished kinase activity in MicB were related to competence derepression under microaerobiosis, suggesting that MicA-PO(4) is involved in competence repression when oxygen becomes limiting. The micAB genes are flanked by mutY and orfC. MutY is an adenine glycosylase involved in the repair of oxidized pyrimidines. OrfC shows the features of a metal binding protein. We did not obtain insertion mutation in orfC, suggesting its requirement for growth. It is proposed that MicAB, with its PAS motif, may belong to a set of functions important in the protection of the cell against oxidative stress, including the control of competence.

Asunto(s)

ADN Glicosilasas , N-Glicosil Hidrolasas/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal/fisiología , Streptococcus pneumoniae/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Datos de Secuencia Molecular , Mutagénesis , N-Glicosil Hidrolasas/genética , Sistemas de Lectura Abierta , Oxígeno , Fenotipo , Fosforilación , Proteínas Quinasas/genética , Análisis de Secuencia de ADN , Streptococcus pneumoniae/genética

RESUMEN

In Streptococcus pneumoniae oxygen availability is a major determinant for competence development in exponentially growing cultures. NADH oxidase activity is required for optimal competence in cultures grown aerobically. The implication of oxidative metabolism and more specifically of Nox on central metabolism has been examined. Glycolytic flux throughout exponential growth revealed homolactic fermentation with a lactate production/glucose utilization ratio close to 2, whatever the aerobiosis level of the culture. Loss-of-function mutations in nox, which encodes NADH oxidase, did not change this trait. Consistently, mRNA levels of glyceraldehyde-3-phosphate dehydrogenase, L-lactate dehydrogenase, pyruvate oxidase, and NADH oxidase remained comparable to wild-type levels, as did the specific activities of key enzymes which control central metabolism. Competence regulation by oxygen involving the NADH oxidase activity is not due to significant modification of carbon flux through glycolysis. Failure to obtain loss-of-function mutation in L-ldh, which encodes the L-lactate dehydrogenase, indicates its essential role in pneumococci whatever their growth status.

Asunto(s)

Complejos Multienzimáticos/genética , NADH NADPH Oxidorreductasas/genética , Oxígeno/metabolismo , Streptococcus pneumoniae/metabolismo , Northern Blotting , Medios de Cultivo , Glucosa/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/análisis , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , L-Lactato Deshidrogenasa/análisis , L-Lactato Deshidrogenasa/genética , Ácido Láctico/análisis , Ácido Láctico/metabolismo , Mutagénesis Insercional , Piruvato Oxidasa/análisis , Piruvato Oxidasa/genética , ARN Bacteriano/genética , ARN Mensajero/análisis , Streptococcus pneumoniae/enzimología , Streptococcus pneumoniae/genética

RESUMEN

Oxygen controls competence development in Streptococcus pneumoniae. Oxygen signaling involves the two-component signal transduction systems CiaRH and ComDE and the competence-stimulating peptide encoded by comC and processed by ComAB. We found that NADH oxidase (Nox) was required for optimal competence. Transcriptional analysis and genetic dissection showed that Nox was involved in post-transcriptional activation of the response regulator ComE and in the transcriptional control of ciaRH and comCDE. Thus, in S. pneumoniae, Nox, with O(2) as its secondary substrate, is part of the O(2)-signaling pathway.

Asunto(s)

Complejos Multienzimáticos/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Transducción de Señal , Streptococcus pneumoniae/genética , Transformación Bacteriana , Proteínas Bacterianas/metabolismo , Modelos Biológicos , Proteínas Quinasas/metabolismo

RESUMEN

In the extracellular pathogen Streptococcus pneumoniae, transformable by soluble DNA, calcium transport is shown to play a key role for vegetative growth, developement of competence for genetic transformation and experimental virulence. To get a more precise localisation of Ca2+ in the cell, we cloned the cDNA of apoaequorine in the chromosome of Streptococcus pneumoniae. This allowed the reconstitution of the acquorine system and chemoluminescence measurements of the cytoplasmic free calcium concentration in the bacteria. Intracellular free Ca2+ is 2 microM at the steady state and can reach 14 microM when calcium is added to the bacterial suspension. Increase in free Ca2+ in response to an imposed Ca2+ gradient depends on the initial velocity (Vi) of the DMB-sensitive Ca2+ transport, showing that changes in cytoplasmic Ca2+ involve active transport.

Asunto(s)

Aequorina , Calcio/análisis , Mediciones Luminiscentes , Streptococcus pneumoniae/química , Aequorina/genética , Transporte Biológico Activo , Calcio/metabolismo , Citoplasma/química , Indicadores y Reactivos , Proteínas Recombinantes , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/ultraestructura

RESUMEN

Anaerobic aerotolerant Streptococcus pneumoniae modulates its genetic transformability and its virulence in response to the oxygen concentration. The activity of a single protein encoded by nox and showing NADH oxidase activity is involved in these adaptive responses to O2. Northern blot analysis of wild-type cultures grown under aerobic and microaerobic conditions indicated transcriptional control of comCDE by O2. An O2-independent mutant strain carrying the gain-of-function mutation comE38KE was isolated and its analysis showed that ComE is a key point in competence stimulation by O2. Plasmid insertion mutations in ciaRH revealed that this two component signal-transducing system negatively regulates comCDE transcription. The level of comCDE transcripts appears as a major control point in competence regulation by O2 and also by growth phase and cell density.

Asunto(s)

Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Complejos Multienzimáticos , Streptococcus pneumoniae/fisiología , Anaerobiosis , Proteínas Bacterianas/metabolismo , Genotipo , Histidina Quinasa , Mutagénesis , Oxígeno/farmacología , Fenotipo , Mutación Puntual , Proteínas Quinasas/genética , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/genética , Transcripción Genética , Transformación Bacteriana , Virulencia

RESUMEN

Mutational analysis of a 5.5 kb fragment of the genome Streptococcus pneumoniae led to the identification of a putative new virulence gene, designated orfD. Insertion mutagenesis of flanking genes on the fragment suggested that the corresponding gene products were required for in vitro growth. In contrast, insertion mutation of orfD did not alter in vitro growth or the transformability pattern of the mutated strain. However, it did reduce bacterial growth in mice and attenuated virulence in an intraperitoneal model of infection. orfD is flanked by orfC (63 codons) and ftsL (105 codons) and all three genes are upstream of pbpx. orfC showed no similarity with other known proteins. ftsL of S. pneumoniae exhibits minimal sequence similarity with ftsL of E. coli, but shares 16% identical residues with the ftsL homologue encoded by ylld of B. subtilis. Also, ftsL of S. pneumoniae has a predicted topology similar to that described for ftsL of E. coli. Putative promoters with an extended -10 box could be identified upstream of both orfC or orfD. The four open reading frames (including pbpx) are orientated in the same direction, and polycistronic transcription could theoretically start at either promoter. Interestingly, this region shows organizational and sequence homologies with genes controlling division and cell wall biosynthesis (DCW) in other bacteria. The attenuation of virulence in the orfD insertion mutant might be due to the loss of function of the orfD gene product or to an altered level of expression of downstream genes.

Asunto(s)

Proteínas de Ciclo Celular , Proteínas de Escherichia coli , Genes Bacterianos , Mutagénesis Insercional , Sistemas de Lectura Abierta/genética , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/patogenicidad , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , División Celular/genética , Pared Celular/genética , Pared Celular/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Metiltransferasas/genética , Metiltransferasas/metabolismo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Infecciones Neumocócicas/microbiología , Alineación de Secuencia , Análisis de Secuencia de ADN , Streptococcus pneumoniae/crecimiento & desarrollo , Virulencia/genética

RESUMEN

A soluble flavoprotein that reoxidizes NADH and reduces molecular oxygen to water was purified from the facultative anaerobic human pathogen Streptococcus pneumoniae. The nucleotide sequence of nox, the gene which encodes it, has been determined and was characterized at the functional and physiological level. Several nox mutants were obtained by insertion, nonsense or missense mutation. In extracts from these strains, no NADH oxidase activity could be measured, suggesting that a single enzyme encoded by nox, having a C44 in its active site, was utilizing O2 to oxidize NADH in S. pneumoniae. The growth rate and yield of the NADH oxidase-deficient strains were not changed under aerobic or anaerobic conditions, but the efficiency of development of competence for genetic transformation during growth was markedly altered. Conditions that triggered competence induction did not affect the amount of Nox, as measured using Western blotting, indicating that nox does not belong to the competence-regulated genetic network. The decrease in competence efficiency due to the nox mutations was similar to that due to the absence of oxygen in the nox+ strain, suggesting that input of oxygen into the metabolism via NADH oxidase was important for controlling competence development throughout growth. This was not related to regulation of nox expression by O2. Interestingly, the virulence and persistence in mice of a blood isolate was attenuated by a nox insertion mutation. Global cellular responses of S. pneumoniae, such as competence for genetic exchange or virulence in a mammalian host, could thus be modulated by oxygen via the NADH oxidase activity of the bacteria, although the bacterial energetic metabolism is essentially anaerobic. The enzymatic activity of the NADH oxidase coded by nox was probably involved in transducing the external signal, corresponding to O2 availability, to the cell metabolism and physiology; thus, this enzyme may function as an oxygen sensor. This work establishes, for the first time, the role of O2 in the regulation of pneumococcal transformability and virulence.

Asunto(s)

Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , Streptococcus pneumoniae/enzimología , Streptococcus pneumoniae/patogenicidad , Transformación Bacteriana , Aerobiosis , Secuencia de Aminoácidos , Anaerobiosis , Animales , Western Blotting , Clonación Molecular , Regulación Bacteriana de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Complejos Multienzimáticos/aislamiento & purificación , Mutación , NADH NADPH Oxidorreductasas/aislamiento & purificación , Infecciones Neumocócicas/microbiología , Conejos , Análisis de Secuencia de ADN , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/crecimiento & desarrollo , Virulencia

RESUMEN

Genetic construction of virulence deficient mutant is a strategy to analyse virulence genes of Streptococcus pneumoniae and was used to virulence factors as capsule, pneumolysin, autolysin and PspA. We perform a model allowing the in vivo positive selection of virulent S. pneumoniae mutants. Mice which are the most susceptible animals to pneumococcal infection, offer the best model for screening virulent S. pneumoniae. Indeed, after intraperitoneal injection of bacterial mix which was composed to a lot of avirulent bacteria (6 log10 CFU per mouse) (V1015 strain, DL50 = 7.05) and few virulent pneumococci (1 to 2 log10 CFU per mouse) (P4241 strain, DL50 < 1), mice cleared all avirulent bacteria but not virulent pneumococci. Thus, mice dead in 3 to 4 days with septicaemia and positive hemoculture contained only virulent strain. This model was validated by in vivo selection of a virulent mutant (V1042, DL50 = 4.1) which was obtained after transformation of avirulent strain V1015 with the genomic fragment of virulent strain P4241. Our model of screening was the only one allowing detection of virulent S. pneumoniae mutants. This new genetic strategy which consisted in gene addition and used mouse as selection agent, could be used to discover new virulence genes required to in vivo bacterial development.

Asunto(s)

Bacteriemia/fisiopatología , Infecciones Neumocócicas/fisiopatología , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/patogenicidad , Animales , Modelos Animales de Enfermedad , Femenino , Dosificación Letal Mediana , Ratones , Ratones Endogámicos , Mutagénesis , Streptococcus pneumoniae/aislamiento & purificación , Virulencia/genética

RESUMEN

The kinetics and pharmacological characterization of a Na+/Ca2+ exchange system, essential for the growth of the extracellular pathogen Streptococcus pneumoniae in high-calcium media, demonstrated that calcium transport, in addition to its role in calcium homeostasis, is involved in the induction of autolysis and of competence for genetic transformation. These responses are expressed respectively in cultures entering the stationary phase and growing with exponential rates. Experimental virulence also appears to be modulated by the kinetics of calcium transport. Calcium transport in S. pneumoniae is electrogenic and shows sigmoidicity, indicating a cooperative mechanism with an inflexion point at 1 mM Ca2+. Mutant strains with Hill number values of 4 and 1, compared to 2 in the wild-type strain, were isolated. These changes were associated with altered regulation of competence and autolysis, and also with reduced experimental virulence. By contrast, they could not be related to a specific calcium requirement for growth. This indicates that the cooperativity of Ca2+ transport is not involved in vegetative growth, but rather regulates competence and autolysis. Competence and autolysis represent two growth-phase-dependent responses to an oligopeptide-activator exported to the medium, the competence-stimulating peptide. Addition of this activator to noncompetent cells, triggers net and transient 45Ca2+ influx. One effect of the activator might be to activate a calcium transporter by enhancing its cooperativity. In addition to an increase in intracellular calcium, a transient membrane depolarization induced by electrogenic calcium influx may be part of the signaling mechanism. The competence activator is a quorum-sensing molecule whose synthesis is autoregulated. This regulation might involve calcium-mediated signaling. As an extracellular pathogen, S. pneumoniae probably develops in niches with variable calcium concentration. Interestingly, virulence depends strongly upon the kinetics of Ca2+ transport. Regulation of calcium influx may represent a common mechanism of sensing the environment, if the Na+/Ca2+ exchanger is the target for external mediators including the competence activator.

Asunto(s)

Señalización del Calcio , Calcio/metabolismo , Streptococcus pneumoniae/metabolismo , Transporte Iónico , Cinética , Sodio/metabolismo , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/patogenicidad , Virulencia

RESUMEN

Streptococcus pneumoniae is a pathogen in which the extracellular calcium concentration plays a major physiological role, in growth as well as in the induction of competence for genetic transformation and activation of autolysis. Both responses are under the control of a protein activator exported in the medium. We have checked the impact of mutations which alter the regulation of competence and autolysis on experimental virulence. Isogenic encapsulated derivatives carrying the relevant mutations were serotype 3 smooth clones, obtained by transformation of the relevant rough strains with DNA from a serotype 3 smooth isolate. Survival kinetics and bacterial clearance from the blood were followed after intraperitoneal infection of Swiss mice with the different bacterial cultures. In this model, mutants showing an attenuation of virulence relative to the wild type fell into two classes. In the first, represented by the lytA::ery mutant V1095 defective for calcium-induced autolysis, attenuated virulence could be correlated with rapid bacterial clearance from the blood. In the second, represented by the dmb mutants V2200 and V3300, attenuation was associated with delayed bacterial clearance from the blood, and correlated with altered kinetics of calcium transport and of regulation of competence and autolysis. It appeared unlikely that attenuation of virulence for strains V2200 and V3300 was a direct consequence of their competence phenotype, since the com::ery mutants V1008 and V1019, defective for the production of the competence activator, were as virulent as the wild-type strain. Autolysis involving an N-acetyl-muramyl-alanine amidase encoded by lytA was also regulated by calcium. The inserted allele lytA0::ery further reduced virulence in the dmb1 background (V2200). This additive effect of lytA- to dmb1 points to different routes of virulence regulation by LYT and DMB1 and suggests that the kinetics of calcium traffic controls several pathways involved in the virulence of pneumococcus.

Asunto(s)

Calcio/metabolismo , Mutación , N-Acetil Muramoil-L-Alanina Amidasa , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/patogenicidad , Animales , Autólisis , Bacteriólisis , Transporte Biológico , Enzimas/genética , Ratones , Infecciones Neumocócicas/sangre , Transformación Bacteriana , Virulencia/genética

RESUMEN

In Streptococcus pneumoniae, Ca2+ induces a stress response which is regulated by a proteic activator known as competence factor (CF). This stress response is expressed as the induction of competence for DNA uptake and genetic transformation in exponentially growing cultures and by autolysis in late exponential phase. DNA transport during competence can be described as a homeostatic response that prevents autolysis of the cultures. Electrogenic and cooperative calcium transport with a Hill number (nH) of 2 appears to mediate this Ca2+ response. Mutant strains altered in their kinetics for Ca2+ transport, with nHs of 1 and 4, were isolated and characterized in order to address the role of the kinetics of Ca2+ transport in the Ca2+ response. The reduced cooperativity of Ca2+ uptake in mutant strain Cp2200 was associated with an absolute requirement for added CF to develop competence and with resistance to autolysis. The enhanced cooperativity of Ca2+ uptake in mutant strain Cp3300 was associated with facilitated competence and hypersensitivity to autolysis. Moreover, the mutation carried by strain Cp3300 increases the CF response of previously described competence-defective mutants. The pleiotropic mutants Cp2200 and Cp3300 allowed us to demonstrate that cooperativity of transport determines the Ca2+ response in S. pneumoniae.

Asunto(s)

Calcio/metabolismo , Streptococcus pneumoniae/metabolismo , Transformación Genética/fisiología , Regulación Alostérica , Amilorida/análogos & derivados , Amilorida/farmacología , Transporte Biológico , División Celular , Farmacorresistencia Microbiana/genética , Mutación , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/crecimiento & desarrollo , Equilibrio Hidroelectrolítico/genética

Asunto(s)

Streptococcus pneumoniae/efectos de los fármacos , Transporte Biológico , Farmacorresistencia Microbiana/genética , Transporte de Electrón , Modelos Biológicos , Streptococcus pneumoniae/genética

RESUMEN

It is shown that Streptococcus pneumoniae possesses a Ca2+ transporter, sensitive to the amiloride derivative 2',4'-dimethylbenzamil (DMB), which is essential for grown at high Ca(2+)-concentrations, and which mediates the triggering by Ca2+ of competence for genetic transformation in the exponential phase and autolysis in the late exponential phase. DMB inhibited both Ca2+ transport and the Ca2+ response. Kinetic analysis of 45Ca2+ transport in ATP-depleted S. pneumoniae revealed an electrogenic influx sensitive to DMB. This transport was cooperative with respect to Ca2+ concentration, and exhibited a Hill coefficient (nH) of 2. In bacteria pre-loaded with 45Ca2+, a DMB-sensitive efflux could be triggered by an imposed Na+ gradient. The efflux kinetics showed the same cooperativity profile as Ca2+ concentration and a similar nH value to that of influx, suggesting a possible Na+/Ca2+ antiport. Cooperativity of transport was lowered (nH = 1) by a mutation that confers resistance to DMB and abolishes the Ca2+ response. These results demonstrate that DMB-sensitive Ca2+ transport is essential for growth and competence regulation. The role of the DMB-sensitive porter involved in Ca2+ circulation and in Ca2+ homeostasis and its possible regulation by competence factor are discussed.

Asunto(s)

Calcio/metabolismo , Streptococcus pneumoniae/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacología , Autólisis , Electroquímica , Transporte Iónico/efectos de los fármacos , Transporte Iónico/genética , Cinética , Mutación , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/crecimiento & desarrollo

RESUMEN

Streptococcus pneumoniae requires 0.15 mM-Ca2+ in the medium for optimal growth. Increasing the Ca2+ concentration to 1 mM triggers either a differentiative state, competence for genetic transformation during exponential growth, or partial lysis as soon as the cultures enter stationary phase. Genetic and physiological data both suggest that these responses are under the control of activator(s), excreted in the presence of high Ca2+ concentrations. 45Ca2+ transport is also stimulated by the activator(s). The amiloride derivative 2',4'-dimethylbenzamil (DMB) inhibits 45Ca2+ transport and prevents lysis and competence development. This provides evidence in favour of the involvement of Ca2+ transport in competence and culture lysis. On the other hand, addition of DNA to a competent culture prevents lysis of wild-type bacteria while a mutant, defective for DNA uptake, is not protected from lysis by exogenous DNA. An hypothesis is proposed for competence induction as a global metabolic response to Ca2+, under the control of competence factor.

Asunto(s)

Calcio/metabolismo , Streptococcus pneumoniae/crecimiento & desarrollo , Transformación Genética , Amilorida/análogos & derivados , Amilorida/farmacología , Bacteriólisis , Transporte Biológico/efectos de los fármacos , Calcimicina/farmacología , Medios de Cultivo , ADN/farmacología , Mutación , Potasio/metabolismo , Sodio/metabolismo , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Transformación Genética/efectos de los fármacos

RESUMEN

The complete nucleotide sequence of the ami locus of Streptococcus pneumoniae revealed the presence of six open reading frames, amiABCDEF. The predicted Ami proteins are probably involved in a transport system. The AmiA, C, D, E, and F proteins exhibit homology with components of the oligopeptide permeases (opp) of Salmonella typhimurium and Escherichia coli. Intriguingly, the AmiB protein is homologous to ArsC, a cytosolic modifier subunit of the anion pump encoded by the arsenical resistance operon of the R-factor R773 from E. coli. Data are presented which indicate that Ami is indeed a transport system.

Asunto(s)

Proteínas Portadoras/genética , Operón , Streptococcus pneumoniae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico , Datos de Secuencia Molecular , Oligopéptidos/metabolismo , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Streptococcus pneumoniae/crecimiento & desarrollo

RESUMEN

DNA uptake in competent Streptococcus pneumoniae was strongly dependent on intracellular pH. Ionophore treatments that either acidified or alkalinized the cytoplasm reduced DNA transport. This indicates that the optimum pH for DNA uptake corresponds to the intracellular pH of competent bacteria which is 8.3 +/- 0.2. In addition, the ATP pool of the bacteria appeared to be a critical parameter in the process. The pattern of inhibition by arsenate, when the culture was treated at different steps of the competence cycle, suggested firstly, that a threshold ATP level was required to trigger transport and secondly, an ATP requirement for the process itself. This may indicate an ATP involvement in the activation of an uptake machinery functioning at the expense of ATP.

Asunto(s)

Adenosina Trifosfato/metabolismo , ADN Bacteriano/metabolismo , Streptococcus pneumoniae/genética , Transformación Bacteriana , Arseniatos/farmacología , Transporte Biológico Activo , Concentración de Iones de Hidrógeno , Monensina/farmacología , Nigericina/farmacología , Potasio/metabolismo , Sodio/metabolismo , Streptococcus pneumoniae/metabolismo

RESUMEN

Addition of competence factor extracts to trigger competence in a culture of Streptococcus pneumoniae induced an increase in the intracellular pH and the Na+ content of the bacteria without any change in the K+ pool or in the membrane potential. These ionic shifts were concomitant with a stimulation of glycolysis that resulted in an enhanced ATP pool. Thus, in transforming conditions, at extracellular pH 7.8, competent bacteria presented a particularly high energetic state resulting from an increase in delta pH and in the ATP pool, associated with an enhanced Na+ content. These features are discussed in the context of homeostasis regulation in response to an environmental stimulus.

Asunto(s)

Streptococcus pneumoniae/metabolismo , Adenosina Trifosfato/metabolismo , Benzoatos/metabolismo , Ácido Benzoico , Glucosa/metabolismo , Concentración de Iones de Hidrógeno , Lactatos/metabolismo , Espectroscopía de Resonancia Magnética , Potasio/metabolismo , Sodio/metabolismo , Streptococcus pneumoniae/genética , Transformación Genética

RESUMEN

Mutations in the amiA locus of the Gram-positive bacterium Streptococcus pneumoniae confer a complex phenotype including resistance to various antineoplastic drugs. As a first step towards the understanding of the molecular organization and the function(s) of this locus, we have cloned DNA fragments carrying its 5'- and 3'-extremities. We have isolated and characterized a down-promoter mutation and have located the functional limits of the locus. The amiA locus is between 5.8 and 7.5 kb long strongly suggesting that it encodes several proteins.

Asunto(s)

Clonación Molecular , Genes Bacterianos , Streptococcus pneumoniae/genética , Aminopterina , Antineoplásicos , ADN Bacteriano/genética , Farmacorresistencia Microbiana/genética , Mutación , Plásmidos , Regiones Promotoras Genéticas , Mapeo Restrictivo , Streptococcus pneumoniae/efectos de los fármacos

RESUMEN

Celiptium, a cationic and amphiphilic drug currently employed in cancer chemotherapy, was found to be accumulated against its concentration gradient by the bacterium Streptococcus pneumoniae. Accumulation was reduced in Celiptium resistant amiA mutants which were also observed to have reduced electric transmembrane potentials delta psi. This suggested a relationship between Celiptium toxicity and accumulation in S. pneumoniae, and indicated a delta psi - driven uptake in a manner reminiscent of that observed for other lipophilic cations such as tetraphenylphosphonium.

Asunto(s)

Alcaloides/metabolismo , Elipticinas/metabolismo , Streptococcus pneumoniae/metabolismo , Farmacorresistencia Microbiana , Potenciales de la Membrana , Metotrexato/metabolismo , Mutación , Compuestos Onio/metabolismo , Compuestos Organofosforados/metabolismo , Streptococcus pneumoniae/genética , Temperatura

RESUMEN

delta psi-reduced amiA mutants of Streptococcus pneumoniae were shown to be resistant to the positively charged antitumoral drugs 2-N-methylellipticinium (NME) and 2-N-methyl-9-hydroxyellipticinium (NMHE). Conversely, mutants selected for their resistance to NMHE were mapped within the amiA locus and exhibited the pleiotropic AmiA- phenotype. This shows that delta psi is a critical parameter in determining resistance to these drugs in S. pneumoniae and suggests that they are accumulated within this bacterium in response to delta psi. As a consequence NME and NMHE appear to be valuable tools for selecting delta psi-reduced mutants in S. pneumoniae.

Asunto(s)

Alcaloides/farmacología , Elipticinas/farmacología , Mutación , Streptococcus pneumoniae/efectos de los fármacos , Fenómenos Químicos , Química , Mapeo Cromosómico , Farmacorresistencia Microbiana , Potenciales de la Membrana , Streptococcus pneumoniae/genética