Добірка наукової літератури з теми "CzcCBA transporter"

ABSTRACT According to in silico analysis, the genome of Pseudomonas putida KT2440 encodes at least four Zn/Cd/Pb efflux transporters—two P-type ATPases (CadA1 and CadA2) and two czc chemiosmotic transporters (CzcCBA1 and CzcCBA2). In this study we showed that all these transporters are functional, but under laboratory conditions only two of them were involved in the mediation of heavy metal resistance in P. putida KT2440. CadA2 conferred Cd2+ and Pb2+ resistance, whereas CzcCBA1 was involved in export of Zn2+, Cd2+, and possibly Pb2+. CadA1, although nonfunctional in P. putida, improved Zn2+ resistance and slightly improved Cd2+ resistance when it was expressed in Escherichia coli. CzcCBA2 contributed to Zn resistance of a czcA1-defective P. putida strain or when the CzcA2 subunit was overexpressed in a transporter-deficient strain. It seemed that CzcA2 could complex with CzcC1 and CzcB1 subunits and therefore complement the loss of CzcA1. The CzcCBA2 transporter itself, however, did not function. Expression of cadA1, cadA2, and czcCBA1 was induced by heavy metals, and the expression levels were dependent on the growth medium and growth phase. Expression of cadA2 and czcCBA1 was nonspecific; both genes were induced by Zn2+, Cd2+, Pb2+, Ni2+, Co2+, and Hg2+. On the other hand, remarkably, expression of cadA1 was induced only by Zn2+. Possible roles of distinct but simultaneously functioning transporters are discussed.

ABSTRACT The Czc system of Ralstonia sp. strain CH34 mediates resistance to cobalt, zinc, and cadmium through ion efflux catalyzed by the CzcCB2A cation-proton antiporter. The CzcD protein is involved in the regulation of the Czc system. It is a membrane-bound protein with at least four transmembrane α-helices and is a member of a subfamily of the cation diffusion facilitator (CDF) protein family, which occurs in all three domains of life. The deletion ofczcD in a Ralstonia sp. led to partially constitutive expression of the Czc system due to an increased transcription of the structural czcCBA genes, both in the absence and presence of inducers. The czcD deletion could be fully complemented in trans by CzcD and two other CDF proteins from Saccharomyces cerevisiae, ZRC1p and COT1p. All three proteins mediated a small but significant resistance to cobalt, zinc, and cadmium in Ralstonia, and this resistance was based on a reduced accumulation of the cations. Thus, CzcD appeared to repress the Czc system by an export of the inducing cations.

Resistance to the last-line antibiotics against invasive Gram-negative bacterial infection is a rising concern in public health. Multidrug resistant (MDR) Acinetobacter baumannii Aci46 can resist colistin and carbapenems with a minimum inhibitory concentration of 512 µg/mL as determined by microdilution method and shows no zone of inhibition by disk diffusion method. These phenotypic characteristics prompted us to further investigate the genotypic characteristics of Aci46. Next generation sequencing was applied in this study to obtain whole genome data. We determined that Aci46 belongs to Pasture ST2 and is phylogenetically clustered with international clone (IC) II as the predominant strain in Thailand. Interestingly, Aci46 is identical to Oxford ST1962 that previously has never been isolated in Thailand. Two plasmids were identified (pAci46a and pAci46b), neither of which harbors any antibiotic resistance genes but pAci46a carries a conjugational system (type 4 secretion system or T4SS). Comparative genomics with other polymyxin and carbapenem-resistant A. baumannii strains (AC30 and R14) identified shared features such as CzcCBA, encoding a cobalt/zinc/cadmium efflux RND transporter, as well as a drug transporter with a possible role in colistin and/or carbapenem resistance in A. baumannii. Single nucleotide polymorphism (SNP) analyses against MDR ACICU strain showed three novel mutations i.e., Glu229Asp, Pro200Leu, and Ala138Thr, in the polymyxin resistance component, PmrB. Overall, this study focused on Aci46 whole genome data analysis, its correlation with antibiotic resistance phenotypes, and the presence of potential virulence associated factors.

ABSTRACT Cadmium and zinc are removed from cells of Ralstonia metallidurans by the CzcCBA efflux pump and by two soft-metal-transporting P-type ATPases, CadA and ZntA. The czcCBA genes are located on plasmid pMOL30, and the cadA and zntA genes are on the bacterial chromosome. Expression of zntA from R. metallidurans in Escherichia coli predominantly mediated resistance to zinc, and expression of cadA predominantly mediated resistance to cadmium. Both transporters decreased the cellular content of zinc or cadmium in this host. In the plasmid-free R. metallidurans strain AE104, single gene deletions of cadA or zntA had only a moderate effect on cadmium and zinc resistance, but zinc resistance decreased 6-fold and cadmium resistance decreased 350-fold in double deletion strains. Neither single nor double gene deletions affected zinc resistance in the presence of czcCBA. In contrast, cadmium resistance of the cadA zntA double mutant could be elevated only partially by the presence of CzcCBA. lacZ reporter gene fusions indicated that expression of cadA was induced by cadmium but not by zinc in R. metallidurans strain AE104. In the absence of the zntA gene, expression of cadA occurred at lower cadmium concentrations and zinc now served as an inducer. In contrast, expression of zntA was induced by both zinc and cadmium, and the induction pattern did not change in the presence or absence of CadA. However, expression of both genes, zntA and cadA, was diminished in the presence of CzcCBA. This indicated that CzcCBA efficiently decreased cytoplasmic cadmium and zinc concentrations. It is discussed whether these data favor a model in which the cations are removed either from the cytoplasm or the periplasm by CzcCBA.

ABSTRACTMetal ion transport systems have been studied extensively, but the specificity of a given transporter is often unclear from amino acid sequence data alone. In this study, predicted Cu2+and Zn2+resistance systems inPseudomonas stutzeristrain RCH2 are compared with those experimentally implicated in Cu2+and Zn2+resistance, as determined by using a DNA-barcoded transposon mutant library. Mutant fitness data obtained under denitrifying conditions are combined with regulon predictions to yield a much more comprehensive picture of Cu2+and Zn2+resistance in strain RCH2. The results not only considerably expand what is known about well-established metal ion exporters (CzcCBA, CzcD, and CusCBA) and their accessory proteins (CzcI and CusF), they also reveal that isolates with mutations in some predicted Cu2+resistance systems do not show decreased fitness relative to the wild type when exposed to Cu2+. In addition, new genes are identified that have no known connection to Zn2+(corB,corC, Psest_3226, Psest_3322, and Psest_0618) or Cu2+resistance (Mrp antiporter subunit gene, Psest_2850, and Psest_0584) but are crucial for resistance to these metal cations. Growth of individual deletion mutants lackingcorB,corC, Psest_3226, or Psest_3322 confirmed the observed Zn-dependent phenotypes. Notably, to our knowledge, this is the first time a bacterial homolog of TMEM165, a human gene responsible for a congenital glycosylation disorder, has been deleted and the resulting strain characterized. Finally, the fitness values indicate Cu2+- and Zn2+-based inhibition of nitrite reductase and interference with molybdenum cofactor biosynthesis for nitrate reductase. These results extend the current understanding of Cu2+and Zn2+efflux and resistance and their effects on denitrifying metabolism.IMPORTANCEIn this study, genome-wide mutant fitness data inP. stutzeriRCH2 combined with regulon predictions identify several proteins of unknown function that are involved in resisting zinc and copper toxicity. For zinc, these include a member of the UPF0016 protein family that was previously implicated in Ca2+/H+antiport and a human congenital glycosylation disorder, CorB and CorC, which were previously linked to Mg2+transport, and Psest_3322 and Psest_0618, two proteins with no characterized homologs. Experiments using mutants lacking Psest_3226, Psest_3322,corB,corC, orczcIverified their proposed functions, which will enable future studies of these little-characterized zinc resistance determinants. Likewise, Psest_2850, annotated as an ion antiporter subunit, and the conserved hypothetical protein Psest_0584 are implicated in copper resistance. Physiological connections between previous studies and phenotypes presented here are discussed. Functional and mechanistic understanding of transport proteins improves the understanding of systems in which members of the same protein family, including those in humans, can have different functions.

ABSTRACTA novel microcalorimetric approach was used to analyze the responses of a metal-tolerant soil bacterium (Pseudomonas putidastrain KT2440) to metal resistance gene deletions in cadmium-amended media. As hypothesized, under cadmium stress, the wild-type strain benefited from the resistance genes by entering the exponential growth phase earlier than two knockout strains. In the absence of cadmium, strain KT1, carrying a deletion in the main component (czcA1) of a Cd/Zn chemiosmotic efflux transporter (CzcCBA1), grew more efficiently than the wild type and released ∼700 kJ (per mole of biomass carbon) less heat than the wild-type strain, showing the energetic cost of maintaining CzcCBA1 in the absence of cadmium. A second mutant strain (KT4) carrying a different gene deletion, ΔcadA2, which encodes the main Cd/Pb efflux transporter (a P-type ATPase), did not survive beyond moderate cadmium concentrations and exhibited a decreased growth yield in the absence of cadmium. Therefore, CadA2 plays an essential role in cadmium resistance and perhaps serves an additional function. The results of this study provide direct evidence that heavy metal cation efflux mechanisms facilitate shorter lag phases in the presence of metals and that the maintenance and expression of tolerance genes carry quantifiable energetic costs and benefits.

ABSTRACT Genomic sequencing of the β-proteobacterium Wautersia (previously Ralstonia) metallidurans CH34 revealed the presence of three genes encoding proteins of the cation diffusion facilitator (CDF) family. One, CzcD, was previously found to be part of the high-level metal resistance system Czc that mediates the efflux of Co(II), Zn(II), and Cd(II) ions catalyzed by the CzcCBA cation-proton antiporter. The second CDF protein, FieF, is probably mainly a ferrous iron detoxifying protein but also mediated some resistance against other divalent metal cations such as Zn(II), Co(II), Cd(II), and Ni(II) in W. metallidurans or Escherichia coli. The third CDF protein, DmeF, showed the same substrate spectrum as FieF, but with different preferences. DmeF plays the central role in cobalt homeostasis in W. metallidurans, and a disruption of dmeF rendered the high-level metal cation resistance systems Czc and Cnr ineffective against Co(II). This is evidence for the periplasmic detoxification of substrates by RND transporters of the heavy metal efflux family subgroup.

Acinetobacter species are ubiquitous Gram-negative bacteria that can be found in water, soil, and as commensals of the human skin. The successful inhabitation of Acinetobacter species in diverse environments is primarily attributable to the expression of an arsenal of stress resistance determinants, which includes an extensive repertoire of metal ion efflux systems. Metal ion homeostasis in the hospital pathogen Acinetobacter baumannii contributes to pathogenesis, however, insights into its metal ion transporters for environmental persistence are lacking. Here, we studied the impact of cadmium stress on A. baumannii . Our functional genomics and independent mutant analyses revealed a primary role for CzcE, a member of the cation diffusion facilitator (CDF) superfamily, in resisting cadmium stress. We also show that the CzcCBA heavy metal efflux system contributes to cadmium efflux. Collectively, these systems provide A. baumannii with a comprehensive cadmium translocation pathway from the cytoplasm to the periplasm and subsequently the extracellular space. Furthermore, analysis of the A. baumannii metallome under cadmium stress showed zinc depletion, as well as copper enrichment, which are likely to influence cellular fitness. Overall, this work provides new knowledge on the role of a broad arsenal of membrane transporters in A. baumannii metal ion homeostasis. IMPORTANCE Cadmium toxicity is a widespread problem, yet the interaction of this heavy metal with biological systems is poorly understood. Some microbes have evolved traits to proactively counteract cadmium toxicity, including Acinetobacter baumannii , which is notorious for persisting in harsh environments. Here we show that A. baumannii utilises a dedicated cadmium efflux protein in concert with a system that is primarily attuned to zinc efflux to efficiently overcome cadmium stress. The molecular characterization of A. baumannii under cadmium stress revealed how active cadmium efflux plays a key role in preventing the dysregulation of bacterial metal ion homeostasis, which appeared to be a primary means by which cadmium exerts toxicity upon the bacterium.

Le gène copH est l'un des 19 gènes constituant le cluster cop, impliqué dans la détoxication du cuivre dans le cytoplasme et le périplasme, chez Cupriavidus meta//idurans CH34, souche modèle pour l'étude de la résistance aux métaux lourds chez les bactéries. La protéine CopH, localisée dans le périplasme, possède une structure et une fonction inconnues mais son expression est stimulée par la présence de cuivre. Nous avons analysé les propriétés de liaison CopH-Cu: les données montrent la présence d'un centre Cu(II) de type 2 dans un champ de ligands azotés. Les 2 seuls résidus histidine, a priori ligands du cuivre, n'interagiraient pas directement avec les ions Cu(II). Le site cuivre serait constitué d'atomes d'azote appartenant à la chaîne principale de la protéine. L'étude des transporteurs CusA et CzcA a nécessité une purification optimisée en détergents et l'analyse de leur comportement en solution
The copH gene is one of the 19 genes found in the cop c1uster, involved in detoxification of copper from the cytoplasme as weil as from the periplasm, in Cupriavidus meta//idurans CH34, used as a model to study heavy metals resistance in bacteria. The function of CopH protein, located in the periplasm, is not clear yet, but its expression is induced by copper. We analysed the copper binding properties of CopH. The features are consistent with the presence of Cu(II) type 2 centers in a nitrogen ligand field. The only two histidine residus, ligands of copper as we thought, would not interact directly avec Cu(II) ions. The copper site wou Id consist with 45 nitrogen atoms from the backbone of CopH. The study of membrane transporters CusA and CzcA has needed the optimised purification in detergents and the analysis oftheir behaviour in solution

Negli ultimi decenni aree sempre maggiori sono state sfruttate, degradate ed inquinate dalle attività antropiche. Tra gli inquinanti più pericolosi rilasciati nell’ambiente vi sono i metalli pesanti. Questi vengono dispersi nell’ambiente attraverso processi industriali, rilascio di fanghi di depurazione, attività minerarie e pratiche agricole (Hüttermann et al., 1999; Shull, 1998). Queste attività causano un accumulo nell’ambiente di metalli pesanti che sono già naturalmente presenti nei suoli (Godbold and Hüttermann, 1985; Breckle, 1991; Nies, 1999). I metalli pesanti grazie alle loro caratteristiche chimiche–fisiche sono stabili e persistenti nell’ambiente e spesso non sono sensibili a degradazioni chimiche o microbiologiche (Gisbert et al., 2003). Alte concentrazioni di metalli pesanti nel suolo causano una forte riduzione della crescita delle piante e quelli più solubili in acqua, diffondono nell’ambiente ed entrano facilmente nella catena alimentare, diventando un serio problema per la salute umana. Negli organismi viventi, la tossicità dei metalli pesanti è legata ai danni che provocano a livello cellulare, quali l’induzione di stress ossidativo, gli effetti negativi sulla permeabilità ed integrità di membrana, l’inibizione delle attività enzimatiche e l’interferenza con il folding proteico (Schützendübel and Polle, 2002). Piante e microorganismi per sopravvivere in ambienti inquinati da metalli pesanti hanno evoluto meccanismi di controllo dell’omeostasi dei metalli, evolvendo tolleranza o resistenza (Ramos et al., 2001; Moore and Helmann, 2005; Clemens 2001). I meccanismi generalmente sfruttati dai batteri sono la produzione di molecole chelanti (peptidi, proteine, polisaccaridi), l’attivazione di enzimi di detossificazione e di meccanismi di efflusso, intesi come il trasporto attivo dei metalli al di fuori della cellula. Quest’ultimo meccanismo è uno dei più utilizzati dai batteri per sfuggire alla tossicità dei metalli pesanti (Nies, 2003). Al fine di ottenere produzioni agricole più sicure è possibile trasferire meccanismi batterici di efflusso dei metalli pesanti in piante di interesse agrario. L’obiettivo è quello di coltivare, su terreni leggermente contaminati, piante caratterizzate da un ridotto assorbimento di metalli pesanti. Lo scopo di questo progetto di dottorato è quello di trasferire in pianta il trasportatore di efflusso di Pseudomonas putida CzcCBA, per cercare di modulare il contenuto dei metalli pesanti nella pianta trasformata. Il complesso di efflusso CzcCBA utilizzato in questo lavoro è stato isolato da un ceppo di P. putida individuato nella rizosfera autoctona di un ecotipo di A. halleri cresciuto su un suolo contaminato da Cd, Zn e Pb (Farinati et al., 2011), questo ceppo è stato denominato P. putida Cd001. Il trasportatore di efflusso è risultato sovra espresso in P. putida Cd001 in condizioni di stress da metalli pesanti (CdSO4 250 μM) (Manara et al., 2012). CzcCBA è una pompa di efflusso tripartita appartenente ad una famiglia di trasportatori che basano il loro funzionamento sulla proteina trans-membrana RND (Nies et al., 1989). Esso è in grado di trasportare Zn2+, Co2+ e Cd2, al di fuori della cellula, sfruttando un meccanismo di efflusso in antiporto di tipo protone/catione (Goldberg et al., 1999; Nies, 1995). Il complesso è formato da tre componenti CzcA, CzcB and CzcC, rispettivamente la proteina RND, la proteina di fusione di membrana e il fattore della membrana esterna. Il primo attraversa la membrana interna e trasporta i cationi metallici dal citosol allo spazio periplasmico, il componente CzcB funziona da connettore e direziona gli ioni verso il terzo componente CzcC, che apre un canale nella membrana esterna permettendo l’estrusione dei metalli al di fuori della cellula (Rensing et al., 1997). I geni codificanti per questi tre trasportatori sono stati amplificati a partire dal DNA genomico di P. putida Cd001 e clonati in vettori di espressione adatti all’espressione in pianta, sotto il controllo del promotore costitutivo 35SCaMV. Sono state scelte tre specie modello per le trasformazioni, Arabidopsis, tabacco e pomodoro. Per ciascuna specie sono state ottenute diverse linee transgeniche stabilmente trasformate con ciascuno dei tre trasportatori. Incrociando successivamente i singoli trasformati sono state ottenute piante che esprimono parzialmente (CzcA/CzcB) o per intero il complesso (CzcCBA). Le analisi di Real Time PCR sui transgeni hanno evidenziato diversi livelli di espressione tra piante trasformate appartenenti allo stesso genotipo. Si è evidenziato inoltre un livello di espressione maggiore per il gene CzcB quando risulta espresso simultaneamente a CzcA, o a CzcA e CzcC. La modulazione del contenuto di metalli pesanti nelle piante transgeniche è stata verificata attraverso esperimenti di accumulo. Le piante trasformate sono state trasferite da condizioni in vitro e cresciute in soluzione idroponica in condizioni controllate, utilizzando piante wild-type come controllo. L’aggiunta del Cd (CdSO4) è stata diversificata nelle tre specie analizzate Arabidopsis, tabacco e pomodoro che sono state trattate rispettivamente con concentrazioni di 0,45, 0,7 e 5 µM. Dopo 22 giorni di crescita la parte aerea è stata campionata ed il contenuto di Cd analizzato. Le piante transgeniche di tabacco mostrano una riduzione del contenuto di Cd in confronto alle piante wild-type. La riduzione aumenta quando nella pianta vengono sovra espressi più componenti del sistema di efflusso CzcCBA. L’espressione singola di CzcA determina una riduzione media del contenuto di Cd (considerando tutte le linee testate) pari al 30% rispetto al wild-type, la contemporanea presenza di CzcA e CzcB determina una riduzione media del 39%. In presenza dell’intero sistema di efflusso i tabacchi riducono il contenuto di Cd nella parte aerea mediamente del 55%. In Arabidopsis la sola presenza di CzcA o CzcB non determina un chiaro effetto sul contenuto di Cd mentre la loro contemporanea espressione causa una riduzione media dei livelli di Cd nelle parte aerea delle piante pari al 23% rispetto al wild-type. Né in tabacco né in Arabidopsis la sola espressione di CzcC è in gradi di modulare il contenuto di Cd rispetto alle piante di controllo. Poiché in P. putida il sistema CzcCBA trasporta oltre al Cd anche lo Zn, piante di tabacco sovra esprimenti l’intero complesso CzcCBA sono state testate per il contenuto di quest’ultimo metallo. Differenze molto basse nel contenuto di Zn sono state rilevate tra piante trasformate e di controllo. Al fine di caratterizzare il meccanismo di efflusso del sistema CzcCBA in pianta, la localizzazione sub-cellulare dei tre componenti è stata studiata. Sono stati creati tre costrutti di fusione sotto il controllo del promotore 35SCaMV: CzcA::eGFP, CzcB::dsRED, CzcC::eYFP e sono stati utilizzati per la trasformazione stabile di tabacco e successivamente per la trasformazione transiente di protoplasti. In entrambi i casi i campioni sono stati osservati al microscopio confocale. La trasformazione dei protoplasti con i tre costrutti di fusione è stata combinata con la co-espressione di marcatori di comparti sub-cellulari per approfondire il grado di specificità della localizzazione. Le analisi microscopiche hanno indicato una localizzazione a livello del reticolo endoplasmico per CzcA; la proteina non sembra tuttavia essere indirizzata verso il vacuolo litico. I risultati preliminari suggeriscono che CzcB tende a formare aggregati a livello citosolico. CzcC appare localizzato in maniera diffusa nel citosol. Ulteriori analisi sono necessarie per stabilire l’eventuale co-localizzazione dei tre componenti del complesso CzcCBA. In conclusione, i risultati presentati dimostrano che l’espressione dei singoli componenti ed ancor più dell’intero sistema di efflusso batterico CzcCBA determinano una riduzione del contenuto di Cd a livello della porzione aerea delle piante trasformate in confronto alle piante wild-type.
In the last years, more and more areas were exploited, degraded and polluted by antropic activities. Among the different pollutants released into the environment, heavy metals are one of the most life-threatening, being diffused into the environment through a great variety of activities and industrial processes, such as release of sewage sludge, mining, agricultural practices, and usage of contaminated fertilizers (Hüttermann et al., 1999; Shull, 1998). As, Hg, Ag, Cd e Pb, are examples of heavy metals with no biological functions and toxic to all living organisms even at low concentrations, they can be considered very dangerous to human health (Godbold and Hüttermann, 1985; Breckle, 1991; Nies, 1999). Due to their chemical and physical properties, heavy metals are highly stable and persistent for long periods in the environment. The majority is poorly sensitive to microbial or chemical degradation and usually cannot be biologically destroyed but only modified from one oxidation state or organic complex to another (Garbisu and Alkorta, 2001; Gisbert et al., 2003). Excess concentrations of heavy metals in soil cause strong declines in plant growth and these elements (due to their solubility in water) can easily enter the food-chain, becoming a serious problem to human health. Heavy metal toxicity shows its effect by causing damages at cellular level, leading, for instance, to oxidative stress, perturbation of membrane integrity and permeability, inhibition of enzymatic activities, and interference with protein folding (Schützendübel and Polle, 2002). Plants and microorganisms developed several strategies to survive in dynamic environments and to face different unfavourable conditions, being able to continuously adapt to rapid changes in the environment. During evolution same bacterial and plant species have developed metal homeostasis mechanisms allowing the achievement of tolerance and resistance conditions in order to survive in heavy metals polluted environments (Ramos et al., 2001; Moore and Helmann, 2005; Clemens, 2001). Bacterial resistance mechanisms include the synthesis of metal-chelating molecules (peptides, proteins or polysaccharides), active metal efflux outside the cell and induction of detoxification enzymes that modify a toxic ion into a less toxic or less available form. One of the most important mechanisms of metal resistance is the translocation of toxic ions towards the outside of the cell. Efflux transporters participate in the reduction of heavy metals content into the cell (Nies, 2003). An interesting biotechnological application aimed at improving safe crop production, is the transfer of bacterial mechanisms deputed to extrude heavy metals into plants, that can be evaluated for metal accumulation when grown on slightly polluted soils. In this project, the Pseudomonas putida CzcCBA efflux system (membrane exporter of Cd2+, Zn2+, Co2+), was transferred into plant, in order to modulate heavy metals content. The CzcCBA complex was isolated from P. putida Cd001, a strain of Pseudomonas inhabiting the rhizosphere of an ecotype of A. halleri growing in soils contaminated by high concentrations of Cd, Zn and Pb (Farinati et al., 2009). The CzcCBA complex was chosen among several membrane transporter specifically induced by Cd stress (Manara et al., 2012). Such system is a trans-envelope efflux pump, belonging to the heavy metal efflux family of the Gram-negative bacteria RND-driven tripartite protein complexes (Nies et al., 1989 ). This transporter utilizes the proton motive force, pumping the substrate across the membranes, through a proton/cation antiporter (Goldberg et al., 1999; Nies, 1995). The complex is made up of three proteins defined CzcA, CzcB and CzcC, respectively the RND protein, the Membrane Fusion Protein, and the Outer Membrane Factor. The first is localized at the inner membrane of the cell and allows the movement of metal cations from the cytosol to the periplasmic space, whereas the B component is located in the periplasmic space, directing ions towards the third element, located in the outer membrane, that opens a membrane channel in order to extrude ions from the bacterium (Rensing et al., 1997). The sequences encoding for CzcA, CzcB and CzcC were amplified from the genomic DNA of P. putida Cd001 and cloned into constructs enabling their overexpression in plants under the control of a plant-specific strong promoter. Arabidopsis thaliana, Nicotiana tabacum and Lycopersicon esculentum were used as test species for transformation. Several stable transgenic lines were obtained for each construct. Singly transformed plants were crossed in order to obtain plants expressing partial (CzcA/CzcB) or whole complex (CzcCBA.). The expression levels of transgenes were analyzed by Real Time PCR. Among transgenic lines of the same genotype, different levels of expression were identified. Worth to note that in both Arabidopsis and tobacco plants overexpressing CzcA/CzcB or CzcA/CzcB/CzcC, the expression level of CzcB is always higher in comparison to the other transgenes. The modulation of metal content in transgenic plants was analyzed carrying out accumulation experiments. Transformed plants were cultured hydroponically and treated with 0.45, 0.7, 5 µM (CdSO4) for Arabidopsis, tobacco and tomato plants respectively. Cd accumulated in shoot was analyzed, after 22 days of treatment by comparing transgenic and wild-type plants. Tobacco transgenic lines showed a reduced Cd content in comparison to wild-type plants. Such reduction in Cd level is enhanced progressively in plants overexpressing simultaneously more components of the CzcCBA system. The single expression of CzcA causes (considering all transgenic lines tested) a Cd reduction on average equal to the 30% in comparison to wild-type, while the concomitant presence of CzcA and CzcB led to 39% reduction. Finally, the overexpression of the whole efflux system, result on average in a 55% reduction in shoot Cd content. In Arabidopsis, the presence of CzcA or CzcB alone does not have a clear effect on Cd content into shoot. Differently, the simultaneous presence of both the components, CzcA and CzcB, results on average in a 23% reduction into shoot in comparison to wild-type. The presence of CzcC does not influence Cd accumulation nor in tobacco neither in Arabidopsis transgenic plants. Since in bacteria the CzcCBA transport system is able to export Cd and Zn, the content of the latter was analyzed in tobacco transgenic lines overexpressing the whole complex CzcCBA. Transgenic lines do not show strong differences in Zn content in comparison to wild-type. In order to better characterize the efflux mechanism of CzcCBA in plant, the sub-cellular localization of the three components was analyzed. The following fusion constructs under the control of the 35SCaMV promoter were obtained: CzcA::eGFP, CzcB::dsRED, CzcC::eYFP. These constructs were used both for stable transformation of tobacco and for protoplast transfection. In both cases samples were observed by confocal microscopy. Tobacco protoplast were transiently transformed with the construct of the three proteins fused to fluorescent reporter, and together with sub-cellular markers, in order to identify the precise location. Microscopy analysis indicates a localization on endoplasmic reticulum for CzcA-GFP; however, the protein seems not to be addressed to the lytic vacuole. Preliminary results suggest that CzcB-RFP forms aggregates with cytosolic localization. CzcC-YFP seems to localize diffusely in the cytosol. Further analyses are necessary to determine the possible co-localization of the three components of the CzcCBA system. In conclusion, results of this study showed that overexpression of single components and even more of the whole efflux system CzcCBA, cause a reduction in Cd content into shoot of transformed plants in comparison to wild-type.

Le gène copH est l'un des 19 gènes constituant le cluster cop, impliqué dans la détoxication du cuivre dans le cytoplasme et le périplasme, chez Cupriavidus metallidurans CH34, souche modèle pour l'étude de la résistance aux métaux lourds chez les bactéries. La protéine CopH, localisée dans le périplasme, possède une structure et une fonction inconnues mais son expression est stimulée par la présence de cuivre. Nous avons analysé les propriétés de liaison CopH-Cu : les données montrent la présence d'un centre Cu(II) de type 2 dans un champ de ligands azotés. Les 2 seuls résidus histidine, a priori ligands du cuivre, n'interagiraient pas directement avec les ions Cu(II). Le site cuivre serait constitué d'atomes d'azote appartenant à la chaîne principale de la protéine.
L'étude des transporteurs CusA et CzcA a nécessité une purification optimisée en détergents et l'analyse de leur comportement en solution.