Hop resistance in the beer spoilage bacterium Lactobacillus brevis is mediated by the ATP-binding cassette multidrug transporter HorA

Lactobacillus brevis is a major contaminant of spoiled beer. The organism can grow in beer in spite of the presence of antibacterial hop compounds that give the beer a bitter taste. The hop resistance in L. brevis is, at least in part, dependent on the expression of the horA gene. The deduced amino acid sequence of HorA is 53% identical to that of LmrA, an ATP-binding cassette multidrug transporter in Lactococcus lactis. To study the role of HorA in hop resistance, HorA was functionally expressed in L. lactis as a hexa-histidine-tagged protein using the nisin-controlled gene expression system. HorA expression increased the resistance of L. lactis to hop compounds and cytotoxic drugs. Drug transport studies with L. lactis cells and membrane vesicles and with proteoliposomes containing purified HorA protein identified HorA as a new member of the ABC family of multidrug transporters.     

Characterization of a highly hop-resistant Lactobacillus brevis strain lacking hop transport

Resistance to hops is a prerequisite for lactic acid bacteria to spoil beer. In this study we analyzed mechanisms of hop resistance of Lactobacillus brevis at the metabolism, membrane physiology, and cell wall composition levels. The beer-spoiling organism L. brevis TMW 1.465 was adapted to high concentrations of hop compounds and compared to a nonadapted strain. Upon adaptation to hops the metabolism changed to minimize ethanol stress. Fructose was used predominantly as a carbon source by the nonadapted strain but served as an electron acceptor upon adaptation to hops, with concomitant formation of acetate instead of ethanol. Furthermore, hop adaptation resulted in higher levels of lipoteichoic acids (LTA) incorporated into the cell wall and altered composition and fluidity of the cytoplasmic membrane. The putative transport protein HitA and enzymes of the arginine deiminase pathway were overexpressed upon hop adaptation. HorA was not expressed, and the transport of hop compounds from the membrane to the extracellular space did not account for increased resistance to hops upon adaptation. Accordingly, hop resistance is a multifactorial dynamic property, which can develop during adaptation. During hop adaptation, arginine catabolism contributes to energy and generation of the proton motive force until a small fraction of the population has established structural improvements. This acquired hop resistance is energy independent and involves an altered cell wall composition. LTA shields the organism from accompanying stresses and provides a reservoir of divalent cations, which are otherwise scarce as a result of their complexation by hop acids. Some of the mechanisms involved in hop resistance overlap with mechanisms of pH resistance and ethanol tolerance and as a result enable beer spoilage by L. brevis.     

Proteomic approach for characterization of hop-inducible proteins in Lactobacillus brevis

Resistance to hops is a prerequisite for the capability of lactic acid bacteria to grow in beer and thus cause beer spoilage. Bactericidal hop compounds, mainly iso-alpha-acids, are described as ionophores which exchange H+ for cellular divalent cations, e.g., Mn2+, and thus dissipate ion gradients across the cytoplasmic membrane. The acid stress response of Lactobacillus brevis TMW 1.465 and hop adaptation in its variant L. brevis TMW 1.465A caused changes at the level of metabolism, membrane physiology, and cell wall composition. To identify the basis for these changes, a proteomic approach was taken. The experimental design allowed the discrimination of acid stress and hop stress. A strategy for improved protein identification enabled the identification of 84% of the proteins investigated despite the lack of genome sequence data for this strain. Hop resistance in L. brevis TMW 1.465A implies mechanisms to cope with intracellular acidification, mechanisms for energy generation and economy, genetic information fidelity, and enzyme functionality. Interestingly, the majority of hop-regulated enzymes are described as manganese or divalent cation dependent. Regulation of the manganese level allows fine-tuning of the metabolism, which enables a rapid response to environmental (stress) conditions. The hop stress response indicates adaptations shifting the metabolism into an energy-saving mode by effective substrate conversion and prevention of exhaustive protein de novo synthesis. The findings further demonstrate that hop stress in bacteria not only is associated with proton motive force depletion but obviously implies divalent cation limitation.     

Membrane-bound ATPase contributes to hop resistance of Lactobacillus brevis

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.     

Random amplified polymorphic DNA-PCR based cloning of markers to identify the beer-spoilage strains of Lactobacillus brevis, Pediococcus damnosus, Lactobacillus collinoides and Lactobacillus coryniformis

AIMS: Beer-spoilage ability of lactic acid bacteria such as Lactobacillus brevis is a strain-dependent phenomenon in which the mechanism has not yet been completely clarified. In order to systematically identify genes that contribute to beer-spoilage, large-scale random amplified polymorphic DNA (RAPD)-based cloning methods was carried out. METHODS AND RESULTS: A systematic RAPD polymerase chain reaction (PCR) analysis using 600 primers was performed on beer-spoilage and on nonspoilage strains of L. brevis. Among 600 primers, three were found to amplify a single locus highly specific to beer-spoilage strains. DNA sequencing of this locus revealed a three-part operon encoding a putative glycosyl transferase, membrane protein and teichoic acid glycosylation protein. PCR analysis of typical beer-spoilage lactic acid bacteria suggested that this locus is highly specific to beer-spoilage strains. CONCLUSION: The cloned markers are highly specific to identify the beer-spoilage strains not only in L. brevis but also in Pediococcus damnosus, Lactobacillus collinoides and Lactobacillus coryniformis. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper proves that RAPD-PCR is an efficient method for cloning the strain-specific genes from bacteria. The markers described here is one of the most useful tools to identify the beer-spoilage strains of lactic acid bacteria.     

A bacterial glutathione transporter (Escherichia coli CydDC) exports reductant to the periplasm

Glutathione (GSH), a major biological antioxidant, maintains redox balance in prokaryotes and eukaryotic cells and forms exportable conjugates with compounds of pharmacological and agronomic importance. However, no GSH transporter has been characterized in a prokaryote. We show here that a heterodimeric ATP-binding cassette-type transporter, CydDC, mediates GSH transport across the Escherichia coli cytoplasmic membrane. In everted membrane vesicles, GSH is imported via an ATP-driven, protonophore-insensitive, orthovanadate-sensitive mechanism, equating with export to the periplasm in intact cells. GSH transport and cytochrome bd quinol oxidase assembly are abolished in the cydD1 mutant. Glutathione disulfide (GSSG) was not transported in either Cyd(+) or Cyd(-) strains. Exogenous GSH restores defective swarming motility and benzylpenicillin sensitivity in a cydD mutant and also benzylpenicillin sensitivity in a gshA mutant defective in GSH synthesis. Overexpression of the cydDC operon in dsbD mutants defective in disulfide bond formation restores dithiothreitol tolerance and periplasmic cytochrome b assembly, revealing redundant pathways for reductant export to the periplasm. These results identify the first prokaryotic GSH transporter and indicate a key role for GSH in periplasmic redox homeostasis.     

Bacterial multidrug resistance mediated by a homologue of the human multidrug transporter P-glycoprotein

Most ATP-binding cassette (ABC) multidrug transporters known to date are of eukaryotic origin, such as the P-glycoproteins (Pgps) and multidrug resistance-associated proteins (MRPs). Only one well-characterized ABC multidrug transporter, LmrA, is of bacterial origin. On the basis of its structural and functional characteristics, this bacterial protein is classified as a member of the P-glycoprotein cluster of the ABC transporter superfamily. LmrA can even substitute for P-glycoprotein in human lung fibroblast cells, suggesting that this type of transporter is conserved from bacteria to man. The functional similarity between bacterial LmrA and human P-glycoprotein is further exemplified by their currently known spectrum of substrates, consisting mainly of hydrophobic cationic compounds. In addition, LmrA was found to confer resistance to eight classes of broad-spectrum antibiotics, and homologs of LmrA have been found in pathogenic bacteria, supporting the clinical and academic value of studying this bacterial protein. Current studies are focused on unraveling the mechanism by which ABC multidrug transporters, such as LmrA, couple the hydrolysis of ATP to the translocation of drugs across the membrane. Recent evidence indicates that LmrA mediates drug transport by an alternating two-site transport mechanism.     

啤酒花抗性机制的研究进展

酒花苦味酸是构成啤酒风味的重要组分,也是啤酒生产过程中的天然抑菌剂,酒花苦味酸通过降低pH梯度而抑制啤酒花敏感菌生长。研究发现,啤酒花抗性菌是通过膜上转运蛋白将酒花苦味酸泵出细胞外,以降低膜上的质子流速,维持了细胞内的pH梯度。结合近年来酒花抗性相关研究结果,讨论了细胞膜上酒花抗性相关组分与酒花抗性间的关系,提出了酒花抗性机制的模型。     

Emerging themes in manganese transport,biochemistry and pathogenesis in bacteria

Though an essential trace element, manganese is generally accorded little importance in biology other than as a cofactor for some free radical detoxifying enzymes and in the photosynthetic photosystem II. Only a handful of other Mn2+-dependent enzymes are known. Recent data, primarily in bacteria, suggest that Mn2+-dependent processes may have significantly greater physiological importance. Two major classes of prokaryotic Mn2+ uptake systems have now been described, one homologous to eukaryotic Nramp transporters and one a member of the ABC-type ATPase superfamily. Each is highly selective for Mn2+ over Fe2+ or other transition metal divalent cations, and each can accumulate millimolar amounts of intracellular Mn2+ even when environmental Mn2+ is scarce. In Salmonella enterica serovar Typhimurium, simultaneous mutation of both types of transporter results in avirulence, implying that one or more Mn2+-dependent enzymes is essential for pathogenesis. This review summarizes current literature on Mn2+ transport, primarily in the Bacteria but with relevant comparisons to the Archaea and Eukaryota. Mn2+-dependent enzymes are then discussed along with some speculations as to their role(s) in cellular physiology, again primarily in Bacteria. It is of particular interest that most of the enzymes which interconvert phosphoglycerate, pyruvate, and oxaloacetate intermediates are either strictly Mn2+-dependent or highly stimulated by Mn2+. This suggests that Mn2+ may play an important role in central carbon metabolism. Further studies will be required, however, to determine whether these or other actions of Mn2+ within the cell are the relevant factors in pathogenesis.     

The Role of Flexible Loops in Folding,Trafficking and Activity of Equilibrative Nucleoside Transporters

Equilibrative nucleoside transporters (ENTs) are integral membrane proteins, which reside in plasma membranes of all eukaryotic cells and mediate thermodynamically downhill transport of nucleosides. This process is essential for nucleoside recycling, and also plays a key role in terminating adenosine-mediated cellular signaling. Furthermore, ENTs mediate the uptake of many drugs, including anticancer and antiviral nucleoside analogues. The structure and mechanism, by which ENTs catalyze trans-membrane transport of their substrates, remain unknown. To identify the core of the transporter needed for stability, activity, and for its correct trafficking to the plasma membrane, we have expressed human ENT deletion mutants in Xenopus laevis oocytes and determined their localization, transport properties and susceptibility to inhibition. We found that the carboxyl terminal trans-membrane segments are essential for correct protein folding and trafficking. In contrast, the soluble extracellular and intracellular loops appear to be dispensable, and must be involved in the fine-tuning of transport regulation.     

Molecular mechanisms of sugar transport across mammalian and microbial cell membranes

Recent DNA cloning studies have revealed the existence of a large family of homologous sugar transporters in both prokaryotic and eukaryotic organisms. The family includes passive transporters typical of mammalian tissues and active, H(+)-linked sugar transporters from bacteria. Each of these transporters characteristically contains two groups of six putative membrane-spanning alpha-helices separated by a large, hydrophilic, cytoplasmic region. Both the N-terminal and C-terminal regions of the sequence are also predicted to be cytoplasmic. Biophysical and other studies on the human erythrocyte glucose transporter, the only member of the family so far isolated in functional form, suggest that the membrane-spanning alpha-helices associate to form a hydrophilic channel or a substrate-binding cleft extending across the membrane. It is likely that the mechanism of substrate translocation involves alternate exposure of the substrate-binding site to each face of the membrane via a conformational change. Studies in progress on the erythrocyte transporter are beginning to identify regions of the protein involved in substrate binding and the conformational change, and should throw light on the mechanism of sugar translocation in the sugar transporter family as a whole.     

Characterization of a Highly Hop-Resistant Lactobacillus brevis Strain Lacking Hop Transport

Resistance to hops is a prerequisite for lactic acid bacteria to spoil beer. In this study we analyzed mechanisms of hop resistance of Lactobacillus brevis at the metabolism, membrane physiology, and cell wall composition levels. The beer-spoiling organism L. brevis TMW 1.465 was adapted to high concentrations of hop compounds and compared to a nonadapted strain. Upon adaptation to hops the metabolism changed to minimize ethanol stress. Fructose was used predominantly as a carbon source by the nonadapted strain but served as an electron acceptor upon adaptation to hops, with concomitant formation of acetate instead of ethanol. Furthermore, hop adaptation resulted in higher levels of lipoteichoic acids (LTA) incorporated into the cell wall and altered composition and fluidity of the cytoplasmic membrane. The putative transport protein HitA and enzymes of the arginine deiminase pathway were overexpressed upon hop adaptation. HorA was not expressed, and the transport of hop compounds from the membrane to the extracellular space did not account for increased resistance to hops upon adaptation. Accordingly, hop resistance is a multifactorial dynamic property, which can develop during adaptation. During hop adaptation, arginine catabolism contributes to energy and generation of the proton motive force until a small fraction of the population has established structural improvements. This acquired hop resistance is energy independent and involves an altered cell wall composition. LTA shields the organism from accompanying stresses and provides a reservoir of divalent cations, which are otherwise scarce as a result of their complexation by hop acids. Some of the mechanisms involved in hop resistance overlap with mechanisms of pH resistance and ethanol tolerance and as a result enable beer spoilage by L. brevis.     

The mgtB Mg2+ transport locus of Salmonella typhimurium encodes a P-type ATPase.

The mgtB locus codes for one of three distinct Mg2+ transport systems of Salmonella typhimurium. The system encoded by the mgtB locus mediates Mg2+ influx only. The nucleotide sequence of a 4.6-kilobase fragment of DNA carrying mgtB has been determined. Two open reading frames were apparent. The most 5' (mgtC) could encode a hydrophobic protein of up to 25 kDa depending on which translation starts are used. A plasmid carrying this region downstream from a phage T7 promoter expresses a 22.5-kDa protein. The second open reading frame encoded a 101-kDa polypeptide (MgtB) consistent with our previous observation that a plasmid carrying the mgtB locus expresses a 102-kDa protein in maxicells. Insertions into either open reading frame abolished the ability of the plasmid to relieve the requirement for added Mg2+ and to restore Mg2+ uptake to a Mg2+ transport-deficient strain of S. typhimurium. The predicted amino acid sequence of MgtC showed no similarity to any other known protein. In contrast, the predicted sequence of MgtB indicated that it is a member of the family of cation transport P-type ATPases. Strikingly, however, MgtB was significantly more similar to eukaryotic Ca2(+)-ATPases than to prokaryotic P-type ATPases or other classes of eukaryotic P-type ATPases such as the Na+,K(+)-ATPase. MgtB is most closely related to Ca2(+)-ATPases of mammalian sarcoplasmic reticulum and yeast. A number of features of the Ca2(+)-ATPases thought to be important for cation transduction across the membrane are present in MgtB but not in other prokaryotic members of this enzyme family. Unlike the Ca2(+)-ATPases, however, which mediate efflux of cation from the cytosol, MgtB mediates influx of cation into the cytosol.     

Comparative analyses of fundamental differences in membrane transport capabilities in prokaryotes and eukaryotes

Whole-genome transporter analyses have been conducted on 141 organisms whose complete genome sequences are available. For each organism, the complete set of membrane transport systems was identified with predicted functions, and classified into protein families based on the transporter classification system. Organisms with larger genome sizes generally possessed a relatively greater number of transport systems. In prokaryotes and unicellular eukaryotes, the significant factor in the increase in transporter content with genome size was a greater diversity of transporter types. In contrast, in multicellular eukaryotes, greater number of paralogs in specific transporter families was the more important factor in the increase in transporter content with genome size. Both eukaryotic and prokaryotic intracellular pathogens and endosymbionts exhibited markedly limited transport capabilities. Hierarchical clustering of phylogenetic profiles of transporter families, derived from the presence or absence of a certain transporter family, showed that clustering patterns of organisms were correlated to both their evolutionary history and their overall physiology and lifestyles.     

Genetic basis of multidrug resistance of tumor cells

Multidrug resistance in animal cells is defined as the simultaneous resistance to a variety of compounds which appear to be structurally and mechanistically unrelated. One type of multidrug resistance is characterized by the decreased accumulation of hydrophobic natural product drugs, a phenotype which is mediated by an ATP-dependent integral membrane multidrug transporter termed P-glycoprotein or P170. The gene coding for P170 is calledMDR. The nucleotide-binding domain of P-glycoprotein shares sequence homology with a family of bacterial permease ATP-binding components. In addition, P170 as a whole is structurally very similar to a number of prokaryotic and eukaryotic proteins believed to be involved in transport activities. This review summarizes our current knowledge of the molecular biology and clinical significance ofMDR expression and P-glycoprotein transport activity, as well as some theories about the function of this protein in normal cells.     

Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles

Gram-negative bacteria shed outer membrane vesicles composed of outer membrane and periplasmic components. Since vesicles from pathogenic bacteria contain virulence factors and have been shown to interact with eukaryotic cells, it has been proposed that vesicles behave as delivery vehicles. We wanted to determine whether heterologously expressed proteins would be incorporated into the membrane and lumen of vesicles and whether these altered vesicles would associate with host cells. Ail, an outer membrane adhesin/invasin from Yersinia enterocolitica, was detected in purified outer membrane and in vesicles from Escherichia coli strains DH5alpha, HB101, and MC4100 transformed with plasmid-encoded Ail. In vesicle-host cell co-incubation assays we found that vesicles containing Ail were internalized by eukaryotic cells, unlike vesicles without Ail. To determine whether lumenal vesicle contents could be modified and delivered to host cells, we used periplasmically expressed green fluorescent protein (GFP). GFP fused with the Tat signal sequence was secreted into the periplasm via the twin arginine transporter (Tat) in both the laboratory E. coli strain DH5alpha and the pathogenic enterotoxigenic E. coli ATCC strain 43886. Pronase-resistant fluorescence was detectable in vesicles from Tat-GFP-transformed strains, demonstrating that GFP was inside intact vesicles. Inclusion of GFP cargo increased vesicle density but did not result in morphological changes in vesicles. These studies are the first to demonstrate the incorporation of heterologously expressed outer membrane and periplasmic proteins into bacterial vesicles.     

Common patterns and unique features of P-type ATPases: a comparative view on the KdpFABC complex from Escherichia coli (Review)

P-type ATPases are ubiquitously abundant primary ion pumps, which are capable of transporting cations across the cell membrane at the expense of ATP. Since these ions comprise a large variety of vital biochemical functions, nature has developed rather sophisticated transport machineries in all kingdoms of life. Due to the importance of these enzymes, representatives of both eu- and prokaryotic as well as archaeal P-type ATPases have been studied intensively, resulting in detailed structural and functional information on their mode of action. During catalysis, P-type ATPases cycle between the so-called E1 and E2 states, each of which comprising different structural properties together with different binding affinities for both ATP and the transport substrate. Crucial for catalysis is the reversible phosphorylation of a conserved aspartate, which is the main trigger for the conformational changes within the protein. In contrast to the well-studied and closely related eukaryotic P-type ATPases, much less is known about their homologues in bacteria. Whereas in Eukarya there is predominantly only one subunit, which builds up the transport system, in bacteria there are multiple polypeptides involved in the formation of the active enzyme. Such a rather unusual prokaryotic P-type ATPase is the KdpFABC complex of the enterobacterium Escherichia coli, which serves as a highly specific K(+) transporter. A unique feature of this member of P-type ATPases is that catalytic activity and substrate transport are located on two different polypeptides. This review compares generic features of P-type ATPases with the rather unique KdpFABC complex and gives a comprehensive overview of common principles of catalysis as well as of special aspects connected to distinct enzyme functions.     

Multidrug resistance mechanisms: drug efflux across two membranes

A set of multidrug efflux systems enables Gram-negative bacteria to survive in a hostile environment. This review focuses on the structural features and the mechanism of major efflux pumps of Gram-negative bacteria, which expel from the cells a remarkably broad range of antimicrobial compounds and produce the characteristic intrinsic resistance of these bacteria to antibiotics, detergents, dyes and organic solvents. Each efflux pump consists of three components: the inner membrane transporter, the outer membrane channel and the periplasmic lipoprotein. Similar to the multidrug transporters from eukaryotic cells and Gram-positive bacteria, the inner membrane transporters from Gram-negative bacteria recognize and expel their substrates often from within the phospholipid bilayer. This efflux occurs without drug accumulation in the periplasm, implying that substrates are pumped out across the two membranes directly into the medium. Recent data suggest that the molecular mechanism of the drug extrusion across a two-membrane envelope of Gram-negative bacteria may involve the formation of the membrane adhesion sites between the inner and the outer membranes. The periplasmic components of these pumps are proposed to cause a close membrane apposition as the complexes are assembled for the transport.     

Divalent metal transport in the green microalga <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> is mediated by a protein similar to prokaryotic Nramp homologues

Information about the molecular mechanisms of metal transport in algae is scarce, despite the significant status these organisms have in aquatic ecosystems. In the present study, we describe the cloning and functional characterization of a divalent metal transporter (named DMT1) in the green microalga Chlamydomonas reinhardtii Dangeard. The longest open reading frame of the cloned DMT1 cDNA encodes a protein of 513 amino acids with 11 putative transmembrane domains. The protein belongs to the Nramp family of divalent metal transporters and shows surprisingly higher similarity to some prokaryotic than to eukaryotic polypeptides. Especially the N-terminus, which is longer than of every other homologue considered in this study, displays – uniquely among selected eukaryotic Nramps – exclusively prokaryotic characteristics. Functional complementation experiments in yeast strains with impaired metal transport systems, revealed that C. reinhardtii DMT1 has a broad specificity, acting in the transport of several divalent metals (manganese, iron, cadmium, copper), but excluding zinc. Published online December 2004     

Recent progress in structure-function analyses of Nramp proton-dependent metal-ion transporters.

The natural resistance-associated macrophage protein (Nramp) homologs form a family of proton-coupled transporters that facilitate the cellular absorption of divalent metal ions (Me2+, including Mn2+, Fe2+, Co2+, and Cd2+). The Nramp, or solute carrier 11 (SLC11), family is conserved in eukaryotes and bacteria. Humans and rodents express 2 parologous genes that are associated with iron disorders and immune diseases. The NRAMP1 (SLC11A1) protein is specific to professional phagocytes and extrudes Me2+ from the phagosome to defend against ingested microbes; polymorphisms in the NRAMP1 gene are associated with various immune diseases. Several isoforms of NRAMP2 (SLC11A2, DMT1, DCT1) are expressed ubiquitously in recycling endosomes or specifically at the apical membrane of epithelial cells in intestine and kidneys, and can contribute to iron overload, whereas mutations impairing NRAMP2 function cause a form of congenital microcytic hypochromic anemia. Structure-function studies, using various experimental models, and mutagenesis approaches have begun to reveal the overall transmembrane organization of Nramp, some of the transmembrane segments (TMS) that are functionally important, and an unusual mechanism coupling Me2+ and proton H+ transport. The approaches used include functional complementation of yeast knockout strains, electrophysiology analyses in Xenopus oocytes, and transport assays that use mammalian and bacterial cells and direct and indirect measurements of SLC11 transporter properties. These complementary studies enabled the identification of TMS1 and 6 as crucial structural segments for Me2+ and H+ symport, and will help develop a deeper understanding of the Nramp transport mechanism and its contribution to Me2+ homeostasis in human health and diseases.