RESUMO
Membrane proteins responsible for the active efflux of structurally and functionally unrelated drugs were first characterized in higher eukaryotes. To date, a vast number of transporters contributing to multidrug resistance (MDR transporters) have been reported for a large variety of organisms. Predictions about the functions of genes in the growing number of sequenced genomes indicate that MDR transporters are ubiquitous in nature. The majority of described MDR transporters in bacteria use ion motive force, while only a few systems have been shown to rely on ATP hydrolysis. However, recent reports on MDR proteins from gram-positive organisms, as well as genome analysis, indicate that the role of ABC-type MDR transporters in bacterial drug resistance might be underestimated. Detailed structural and mechanistic analyses of these proteins can help to understand their molecular mode of action and may eventually lead to the development of new strategies to counteract their actions, thereby increasing the effectiveness of drug-based therapies. This review focuses on recent advances in the analysis of ABC-type MDR transporters in bacteria.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Farmacorresistência Bacteriana Múltipla , Transportadores de Cassetes de Ligação de ATP/química , Animais , Proteínas de Bactérias/metabolismo , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Humanos , Modelos Biológicos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
The cytoplasmic membrane of bacteria is the matrix for metabolic energy transducing processes such as proton motive force generation and solute transport. Passive permeation of protons across the cytoplasmic membrane is a crucial determinant in the proton motive generating capacity of the organisms. Adaptations of the membrane composition are needed to restrict the proton permeation rates especially at higher temperatures. Thermophilic bacteria cannot sufficiently restrict this proton permeation at their growth temperature and have to rely on the much lower permeation of Na + to generate a sodium motive force for driving metabolic energy-dependent membrane processes. Specific transport systems mediate passage across the membrane at physiological rates of all compounds needed for growth and metabolism and of all end products of metabolism. Some of transport systems, the secondary transporters, transduce one form of electrochemical energy into another form. These transporters can play crucial roles in the generation of metabolic energy. This is especially so in anaerobes such as Lactic Acid Bacteria which live under energy-limited conditions. Several transport systems are specifically aimed at the generation of metabolic energy during periods of energy-limitation. In their natural environment bacteria are also often exposed to cytotoxic compounds, including antibiotics. Many bacteria can respond to this live-threatening condition by overexpressing powerful drug-extruding multidrug resistance systems.
Assuntos
Archaea/metabolismo , Bactérias/metabolismo , Membrana Celular/metabolismo , Adaptação Fisiológica , Anti-Infecciosos/farmacologia , Archaea/química , Archaea/citologia , Archaea/efeitos dos fármacos , Bactérias/química , Bactérias/citologia , Bactérias/efeitos dos fármacos , Transporte Biológico , Membrana Celular/química , Resistência Microbiana a Medicamentos , Metabolismo Energético , Temperatura Alta , Transporte de Íons , Ácido Láctico/metabolismo , Lipídeos/análise , Proteínas de Membrana Transportadoras/metabolismoRESUMO
LmrCD is a lactococcal, heterodimeric multidrug transporter, which belongs to the ABC superfamily. It consists of two half-transporters, LmrC and LmrD, that are necessary and sufficient for drug extrusion and ATP hydrolysis. LmrCD is asymmetric in terms of the conservation of the functional motifs of the nucleotide-binding domains (NBDs). Important residues of the nucleotide-binding site of LmrC and the C loop of LmrD are not conserved. To investigate the functional importance of the LmrC and LmrD subunits, the putative catalytic base residue adjacent to the Walker B motif of both NBDs were substituted for the respective carboxamides. Our data demonstrate that Glu587 of LmrD is essential for both drug transport and ATPase activity of the LmrCD heterodimer, whereas mutation of Asp495 of LmrC has a less severe effect on the activity of the complex. Structural and/or functional asymmetry is further demonstrated by differential labeling of both subunits by 8-azido-[alpha-32P]ATP, which, at 4 degrees C, occurs predominantly at LmrC, while aluminiumfluoride (AlF(x))-induced trapping of the hydrolyzed nucleotide at 30 degrees C results in an almost exclusive labeling of LmrD. It is concluded that the LmrCD heterodimer contains two structurally and functionally distinct NBDs.
Assuntos
Proteínas de Bactérias/metabolismo , Lactococcus lactis/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Nucleotídeos/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Compostos de Alumínio/metabolismo , Sequência de Aminoácidos , Azidas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Transporte Biológico Ativo , Domínio Catalítico , Dimerização , Resistência a Múltiplos Medicamentos/genética , Fluoretos/metabolismo , Lactococcus lactis/enzimologia , Lactococcus lactis/genética , Dados de Sequência Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutagênese Sítio-Dirigida , Nucleotídeos/química , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
Binding-protein-dependent secondary transporters make up a unique transport protein family. They use a solute-binding protein in proton-motive-force-driven transport. Only a few systems have been functionally analysed. The yiaMNO genes of Escherichia coli K-12 encode one family member that transports the rare pentose l-xylulose. Its physiological role is unknown, since wild-type E. coli K-12 does not utilize l-xylulose as sole carbon source. Deletion of the yiaMNO genes in E. coli K-12 strain MC4100 resulted in remarkable changes in the transition from exponential growth to the stationary phase, high-salt survival and biofilm formation.
Assuntos
Proteínas de Transporte/metabolismo , Escherichia coli K12/crescimento & desenvolvimento , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismo , Biofilmes/crescimento & desenvolvimento , Transporte Biológico , Proteínas de Transporte/genética , Meios de Cultura , Escherichia coli K12/efeitos dos fármacos , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/genética , Óperon , Transdução de Sinais , Cloreto de Sódio/farmacologia , Xilulose/metabolismoRESUMO
ATP-binding cassette (ABC) transporters are vital to any living system and are involved in the translocation of a wide variety of substances, from ions and nutrients to high molecular weight proteins. This chapter describes methods used to purify and membrane reconstitute ABC transporters in a fully functional state. The procedures are largely based on our experience with substrate-binding protein-dependent ABC uptake systems from bacteria, but the approaches should be applicable to multisubunit membrane complexes in general. Also, we present simple methods, based on substrate binding or translocation, to follow the activity of the protein complexes in detergent-solubilized and/or membrane-reconstituted state(s).
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Estruturas da Membrana Celular/química , Detergentes/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Cromatografia , Vesículas Citoplasmáticas/química , Fluorescência , Modelos Moleculares , Solubilidade , Análise EspectralRESUMO
The extensive progress of genome sequencing projects in recent years has demonstrated that multidrug resistance (MDR) transporters are widely spread among all domains of life. This indicates that they play crucial roles in the survival of organisms. Moreover, antibiotic and chemotherapeutic treatments have revealed that microorganisms and cancer cells may use MDR transporters to fight the cytotoxic action of drugs. Currently, several MDR extrusion systems are being investigated in detail. It is expected that understanding of the molecular basis of multidrug recognition and the transport mechanisms will allow a more rational design of new drugs which either will not be recognized and expelled by or will efficiently inhibit the activity of the MDR transporters. MDR transporters either utilize ATP hydrolysis or an ion motive force as an energy source to drive drugs out of the cell. This review summarizes the recent progress in the field of bacterial proton motive force driven MDR transporters.
Assuntos
Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Força Próton-Motriz/fisiologia , Animais , Sítios de Ligação , Membrana Celular/fisiologia , Humanos , Família Multigênica/fisiologiaRESUMO
In previous studies, it has been shown that inactivation of opp or even oppA abolishes the capacity of Lactococcus lactis to utilize oligopeptides. We now show that the opp operon has been duplicated in L. lactis MG1363. The nucleotide sequence of the oppA and oppC homologues (appA and appC) and most of the oppB homologue (appB) indicate that the corresponding protein sequences are 83%, 92% and 91% identical, respectively. Inactivation of appA, via homologous recombination, as well as complementation studies were carried out to determine the possible function of appA in peptide utilization. As anticipated from studies with an oppA knock-out, peptide utilization was not impaired in an appA disruption mutant. Importantly, AppA expressed from a plasmid could restore the ability of oppA deletion mutants to utilize Leu-enkephalin, albeit with a lower efficiency than OppA. The differences in the ability to utilize this pentapeptide were not due to differences in expression levels but most likely reflect a different catalytic efficiency in oligopeptide utilization when AppA is used as ligand receptor.
Assuntos
Proteínas de Transporte/genética , Genes Bacterianos , Lactococcus lactis/genética , Lipoproteínas/genética , Oligopeptídeos/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas de Transporte/metabolismo , Lipoproteínas/metabolismo , Leite/metabolismo , Dados de Sequência Molecular , Óperon , Plasmídeos/genética , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
Lactic acid and 3-hydroxypropanoic acid are industrially relevant microbial products. This paper reviews the current knowledge on export of these compounds from microbial cells and presents a theoretical analysis of the bioenergetics of different export mechanisms. It is concluded that export can be a key constraint in industrial production, especially under the conditions of high product concentration and low extracellular pH that are optimal for recovery of the undissociated acids. Under these conditions, the metabolic energy requirement for product export may equal or exceed the metabolic energy yield from product formation. Consequently, prolonged product formation at low pH and at high product concentrations requires the involvement of alternative, ATP-yielding pathways to sustain growth and maintenance processes, thereby reducing the product yield on substrate. Research on export mechanisms and energetics should therefore be an integral part of the development of microbial production processes for these and other weak acids.
Assuntos
Bactérias/crescimento & desenvolvimento , Microbiologia Industrial , Ácido Láctico/análogos & derivados , Ácido Láctico/metabolismo , Transporte Biológico/fisiologia , Reatores Biológicos/microbiologia , Fermentação/fisiologiaRESUMO
LmrP, a proton/multidrug antiporter of Lactococcus lactis, transports a variety of cationic substrates. Previously, two membrane-embedded acidic residues, Asp142 and Glu327, have been reported to be important for multidrug transport activity of LmrP. Here we show that neither Glu327 nor Asp142 is essential for ethidium binding but that Glu327 is a critical residue for the high affinity binding of Hoechst 33342. Substitution of these two residues, however, negatively influences the transport activity. The energetics of transport was studied of two closely related cationic substrates ethidium and propidium that carry one and two positive charges, respectively. Extrusion of monovalent ethidium is dependent on both the electrical membrane potential (Deltapsi) and transmembrane proton gradient (DeltapH), while extrusion of propidium predominantly depends on the DeltapH only. The LmrP mutants D142C and E327C, however, mediate electroneutral ethidium extrusion, but are unable to mediate DeltapH-dependent extrusion of propidium. These data indicate that Asp142 and Glu327 are involved in proton translocation.
Assuntos
Proteínas de Bactérias/metabolismo , Resistência Microbiana a Medicamentos , Resistência a Múltiplos Medicamentos , Lactococcus lactis/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Bactérias/genética , Benzimidazóis/metabolismo , Metabolismo Energético , Etídio/metabolismo , Proteínas de Membrana Transportadoras/genética , MutaçãoRESUMO
Multidrug resistance presents a major obstacle to the treatment of infectious diseases and cancer. LmrA, a bacterial ATP-dependent multidrug transporter, mediates efflux of hydrophobic cationic substrates, including antibiotics. The substrate-binding domain of LmrA was identified by using photo-affinity ligands, proteolytic degradation of LmrA, and identification of ligand-modified peptide fragments with matrix-assisted laser desorption ionization/time of flight mass spectrometry. In the nonenergized state, labeling occurred in the alpha-helical transmembrane segments (TM) 3, 5 and 6 of the membrane-spanning domain. Upon nucleotide binding, the accessibility of TM5 for substrates increased, whereas that of TM6 decreased. Inverse changes were observed upon ATP-hydrolysis. An atomic-detail model of dimeric LmrA was generated based on the template structure of the homologous transporter MsbA from Vibrio cholerae, allowing a three-dimensional visualization of the substrate-binding domain. Labeling of TM3 of one monomer occurred in a predicted area of contact with TM5 or TM6 of the opposite monomer, indicating substrate-binding at the monomer/monomer interface. Inverse changes in the reactivity of TM segments 5 and 6 suggest that substrate binding and release involves a repositioning of these helices during the catalytic cycle.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Lactococcus lactis/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Propafenona/farmacocinética , Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Sítios de Ligação , Transporte Biológico , Modelos Moleculares , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Marcadores de Fotoafinidade , Propafenona/análogos & derivados , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
Multidrug resistance (MDR)-type transporters mediate the active extrusion of structurally and functionally dissimilar compounds from the cells, thereby rendering cells resistant to a range of drugs. The ydaG and ydbA genes of Lactococcus lactis encode two ATP-binding cassette half-transporters, which both share homology with MDR proteins such as LmrA from L. lactis or the mammalian P-glycoprotein. The ydaG/ydbA genes were cloned and expressed separately and jointly in L. lactis using the nisin-inducible system. When both proteins are co-expressed, several structurally dissimilar drugs such as ethidium, daunomycin, and BCECF-AM are extruded from the cell. YdaG and YdbA could be co-purified as a stable heterodimer. ATPase activity was found to be associated with the YdaG/YdbA heterodimer only and not with the individual subunits. Both the ydaG and ydbA genes are up-regulated in multidrug-resistant L. lactis strains selected for growth in the presence of a variety of toxic compounds. This is the first demonstration of a functional heterodimeric ATP-binding cassette-type MDR transporter.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/metabolismo , Resistência a Múltiplos Medicamentos , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Transportadores de Cassetes de Ligação de ATP/fisiologia , Adenosina Trifosfatases/metabolismo , Antibióticos Antineoplásicos/farmacologia , Transporte Biológico , Membrana Celular/metabolismo , DNA/química , Daunorrubicina/farmacologia , Dimerização , Eletroforese em Gel de Poliacrilamida , Etídio/farmacologia , Fluoresceínas/farmacologia , Corantes Fluorescentes/farmacologia , Ligação Proteica , RNA/metabolismo , Fatores de Tempo , Regulação para CimaRESUMO
In archaea, ATP-binding cassette (ABC) transporters play a crucial role in substrate uptake, export, and osmoregulation. Archael substrate-binding-protein-dependent ABC transporters are equipped with a very high affinity for their cognate substrates which provide these organisms with the ability to efficiently scavenge substrates from their environment even when present only at low concentration. Further adaptations to the archaeal way of life are especially found in the domain organization and anchoring of the substrate-binding proteins to the membrane. Examination of the signal peptides of binding proteins of 14 archael genomes showed clear differences between euryarchaeotes and crenarchaeotes. Furthermore, a profiling and comparison of ABC transporters in the three sequenced pyrococcal strains was performed.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Archaea/fisiologia , Membrana Celular/fisiologia , Regulação da Expressão Gênica em Archaea/fisiologia , Sinais Direcionadores de Proteínas/fisiologia , Transdução de Sinais/fisiologia , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Transporte Biológico Ativo/fisiologia , Dados de Sequência Molecular , Ligação Proteica , Pyrococcus/fisiologia , Homologia de Sequência de AminoácidosRESUMO
The majority of bacterial multidrug resistance transporters belong to the class of secondary transporters. LmrP is a proton/drug antiporter of Lactococcus lactis that extrudes positively charged lipophilic substrates from the inner leaflet of the membrane to the external medium. This study shows that LmrP is a true secondary transporter. In the absence of a proton motive force, LmrP facilitates downhill fluxes of ethidium in both directions. These fluxes are inhibited by other substrates of LmrP. The cysteine-reactive agent p-chloromercuri-benzene sulfonate inhibits these fluxes in wild type LmrP but not in the cysteine-less LmrP C270A mutant. Cysteine mutagenesis of LmrP resulted in three mutants, D68C/C270A, D128C/C270A, and E327C/C270A, with an energy-uncoupled phenotype. Asp68 is located in the conserved motif GXXX(D/E)(R/K)XGRK for the major facilitator superfamily of secondary transporters and was found to play an important role in energy coupling, whereas the negatively charged residues Asp128 and Glu327 have indirect effects on the transport process. L. lactis strains expressing these uncoupled mutants of LmrP show an increased rate of ethidium influx and an increased drug susceptibility compared with cells harboring an empty vector. The rate of influx in these mutants is enhanced by a transmembrane electrical potential, inside negative. These observations suggest a new strategy for eliminating drug-resistant microbial pathogens, i.e. the design and use of modulators of secondary multidrug resistance transporters that uncouple drug efflux from proton influx, thereby allowing transmembrane electrical potential-driven influx of cationic drugs.
Assuntos
4-Cloromercuriobenzenossulfonato/farmacologia , Proteínas de Bactérias/metabolismo , Lactococcus lactis/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Transporte Biológico/efeitos dos fármacos , Sequência Conservada , Etídio/metabolismo , Cinética , Lactococcus lactis/efeitos dos fármacos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Mutagênese Sítio-Dirigida , Plasmídeos , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
The yiaMNO genes of Escherichia coli K-12 encode a binding protein-dependent secondary, or tri-partite ATP-independent periplasmic (TRAP), transporter. Since only a few members of this family have been functionally characterized to date, we aimed to identify the substrate for this transporter. Cells that constitutively express the yiaK-S gene cluster metabolized the rare pentose L-xylulose, while deletion of the yiaMNO transporter genes reduced L-xylulose metabolism. The periplasmic substrate-binding protein YiaO was found to bind L-xylulose, and stimulated L-xylulose uptake by spheroplasts. These date indicate that the yiaMNO transporter mediates uptake of this rare pentose.
Assuntos
Proteínas de Transporte/genética , Proteínas de Escherichia coli/genética , Proteínas de Membrana Transportadoras/genética , Xilulose/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Bases , Transporte Biológico/genética , Proteínas de Transporte/metabolismo , Cromossomos Bacterianos , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Óperon/genética , Mapeamento por Restrição , Esferoplastos/metabolismoRESUMO
The Lactococcus lactis multidrug resistance ABC transporter protein LmrA has been shown to confer resistance to structurally and functionally diverse antibiotics and anti-cancer drugs. Using a previously characterized photoreactive drug analogue of Rhodamine 123 (iodo-aryl azido-Rhodamine 123 or IAARh123), direct and specific photoaffinity labeling of LmrA in enriched membrane vesicles could be achieved under non-energized conditions. This photoaffinity labeling of LmrA occurs at a physiologically relevant site as it was inhibited by molar excess of ethidium bromide>Rhodamine 6G>vinblastine>doxorubicin>MK571 (a quinoline-based drug) while colchicine had no effect. The MDR-reversing agents PSC 833 and cyclosporin A were similarly effective in inhibiting IAARh123 photolabeling of LmrA and P-glycoprotein. In-gel digestion with Staphyloccocus aureus V8 protease of IAARh123-photolabeled LmrA revealed several IAARh123 labeled polypeptides, in addition to a 6.8kDa polypeptide that comprises the last two transmembrane domains of LmrA.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Lactococcus lactis/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Vimblastina , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Membrana Celular/metabolismo , Colchicina/farmacologia , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos , Etídio/farmacologia , Corantes Fluorescentes/farmacologia , Substâncias Intercalantes/farmacologia , Luz , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Peptídeos/química , Testes de Precipitina , Propionatos/farmacologia , Estrutura Terciária de Proteína , Quinolinas/farmacologia , Rodaminas/farmacologia , Vimblastina/farmacologiaRESUMO
For brewing industry, beer spoilage bacteria have been problematic for centuries. They include some lactic acid bacteria such as Lactobacillus brevis, Lactobacillus lindneri and Pediococcus damnosus, and some Gram-negative bacteria such as Pectinatus cerevisiiphilus, Pectinatus frisingensis and Megasphaera cerevisiae. They can spoil beer by turbidity, acidity and the production of unfavorable smell such as diacetyl or hydrogen sulfide. For the microbiological control, many advanced biotechnological techniques such as immunoassay and polymerase chain reaction (PCR) have been applied in place of the conventional and time-consuming method of incubation on culture media. Subsequently, a method is needed to determine whether the detected bacterium is capable of growing in beer or not. In lactic acid bacteria, hop resistance is crucial for their ability to grow in beer. Hop compounds, mainly iso-alpha-acids in beer, have antibacterial activity against Gram-positive bacteria. They act as ionophores which dissipate the pH gradient across the cytoplasmic membrane and reduce the proton motive force (pmf). Consequently, the pmf-dependent nutrient uptake is hampered, resulting in cell death. The hop-resistance mechanisms in lactic acid bacteria have been investigated. HorA was found to excrete hop compounds in an ATP-dependent manner from the cell membrane to outer medium. Additionally, increased proton pumping by the membrane bound H(+)-ATPase contributes to hop resistance. To energize such ATP-dependent transporters hop-resistant cells contain larger ATP pools than hop-sensitive cells. Furthermore, a pmf-dependent hop transporter was recently presented. Understanding the hop-resistance mechanisms has enabled the development of rapid methods to discriminate beer spoilage strains from nonspoilers. The horA-PCR method has been applied for bacterial control in breweries. Also, a discrimination method was developed based on ATP pool measurement in lactobacillus cells. However, some potential hop-resistant strains cannot grow in beer unless they have first been exposed to subinhibitory concentration of hop compounds. The beer spoilage ability of Pectinatus spp. and M. cerevisiae has been poorly studied. Since all the strains have been reported to be capable of beer spoiling, species identification is sufficient for the breweries. However, with the current trend of beer flavor (lower alcohol and bitterness), there is the potential risk that not yet reported bacteria will contribute to beer spoilage. Investigation of the beer spoilage ability of especially Gram-negative bacteria may be useful to reduce this risk.
Assuntos
Bactérias/crescimento & desenvolvimento , Cerveja/microbiologia , Microbiologia de Alimentos , Humulus , Transportadores de Cassetes de Ligação de ATP , Ácidos/farmacologia , Bactérias/genética , Meios de Cultura , Farmacorresistência Bacteriana/genética , Contaminação de Alimentos , Genes Bacterianos , Lactobacillus/crescimento & desenvolvimentoRESUMO
Di- and tricarboxylates found as intermediates in the tricarboxylic acid cycle can be utilized by many bacteria and serve as carbon and energy source under aerobic and anaerobic conditions. A prerequisite for metabolism is that the carboxylates are transported into the cells across the cytoplasmic membrane. Bacillus subtilis is able to metabolize many di- and tricarboxylates and in this overview the available data on all known and putative di- and tricarboxylate transporters in B. subtilis is summarized. The B. subtilis transporters, that are of the secondary type, are discussed in the context of the protein families to which they belong. Available data on biochemical characterization, regulation of gene expression and the physiological function is summarized. It is concluded that in B. subtilis multiple transporters are present for tricarboxylic acid cycle intermediates.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Ácidos Dicarboxílicos/metabolismo , Ácidos Tricarboxílicos/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Sequência de Bases , Transporte Biológico , Proteínas de Transporte/genética , Transportadores de Ácidos Dicarboxílicos/genética , Transportadores de Ácidos Dicarboxílicos/metabolismo , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Família Multigênica , Alinhamento de Sequência , SimportadoresRESUMO
The cytoplasmic membrane of bacteria and archaea determine to a large extent the composition of the cytoplasm. Since the ion and in particular the proton and/or the sodium ion electrochemical gradients across the membranes are crucial for the bioenergetic conditions of these microorganisms, strategies are needed to restrict the permeation of these ions across their cytoplasmic membrane. The proton and sodium permeabilities of all biological membranes increase with the temperature. Psychrophilic and mesophilic bacteria, and mesophilic, (hyper)thermophilic and halophilic archaea are capable of adjusting the lipid composition of their membranes in such a way that the proton permeability at the respective growth temperature remains low and constant (homeo-proton permeability). Thermophilic bacteria, however, have more difficulties to restrict the proton permeation across their membrane at high temperatures and these organisms have to rely on the less permeable sodium ions for maintaining a high sodium-motive force for driving their energy requiring membrane-bound processes. Transport of solutes across the bacterial and archaeal membrane is mainly catalyzed by primary ATP driven transport systems or by proton or sodium motive force driven secondary transport systems. Unlike most bacteria, hyperthermophilic bacteria and archaea prefer primary ATP-driven uptake systems for their carbon and energy sources. Several high-affinity ABC transporters for sugars from hyperthermophiles have been identified and characterized. The activities of these ABC transporters allow these organisms to thrive in their nutrient-poor environments.
Assuntos
Archaea/crescimento & desenvolvimento , Bactérias/crescimento & desenvolvimento , Membrana Celular/fisiologia , Temperatura , Transporte Biológico , Permeabilidade da Membrana Celular , Metabolismo EnergéticoRESUMO
The activity of the membrane-bound H+-ATPase of the beer spoilage bacterium Lactobacillus brevis ABBC45 increased upon adaptation to bacteriostatic hop compounds. The ATPase activity was optimal around pH 5.6 and increased up to fourfold when L. brevis was exposed to 666 microM hop compounds. The extent of activation depended on the concentration of hop compounds and was maximal at the highest concentration tested. The ATPase activity was strongly inhibited by N,N'-dicyclohexylcarbodiimide, a known inhibitor of FoF1-ATPase. Western blots of membrane proteins of L. brevis with antisera raised against the alpha- and beta-subunits of FoF1-ATPase from Enterococcus hirae showed that there was increased expression of the ATPase after hop adaptation. The expression levels, as well as the ATPase activity, decreased to the initial nonadapted levels when the hop-adapted cells were cultured further without hop compounds. These observations strongly indicate that proton pumping by the membrane-bound ATPase contributes considerably to the resistance of L. brevis to hop compounds.
Assuntos
Adenosina Trifosfatases/metabolismo , Humulus/química , Lactobacillus/efeitos dos fármacos , Adenosina Trifosfatases/antagonistas & inibidores , Western Blotting , Inibidores Enzimáticos/farmacologia , Lactobacillus/enzimologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Extratos Vegetais/farmacologiaRESUMO
The major life-threatening event for lactic acid bacteria (LAB) in their natural environment is the depletion of their energy sources and LAB can survive such conditions only for a short period of time. During periods of starvation LAB can exploit optimally the potential energy sources in their environment usually by applying proton motive force generating membrane transport systems. These systems include in addition to the proton translocating F0F1-ATPase: a respiratory chain when hemin is present in the medium, electrogenic solute uptake and excretion systems, electrogenic lactate/proton symport and precursor/product exchange systems. Most of these metabolic energy-generating systems offer as additional bonus the prevention of a lethal decrease of the internal and external pH. LAB have limited biosynthetic capacities and rely heavily on the presence of essential components such as sources of amino acids in their environment. The uptake of amino acids requires a major fraction of the available metabolic energy of LAB. The metabolic energy cost of amino acid uptake can be reduced drastically by accumulating oligopeptides instead of the individual amino acids and by proton motive force-generating efflux of excessively accumulated amino acids. Other life-threatening conditions that LAB encounter in their environment are rapid changes in the osmolality and the exposure to cytotoxic compounds, including antibiotics. LAB respond to osmotic upshock or downshock by accumulating or releasing rapidly osmolytes such as glycine-betaine. The life-threatening presence of cytotoxic compounds, including antibiotics, is effectively counteracted by powerful drug extruding multidrug resistance systems. The number and variety of defense mechanisms in LAB is surprisingly high. Most defense mechanisms operate in the cytoplasmic membrane to control the internal environment and the energetic status of LAB. Annotation of the functions of the genes in the genomes of LAB will undoubtedly reveal additional defense mechanisms.