Removal of copper by Pseudomonas putida strain S4 isolated from copper mines

A bacterial strain, Pseudomonas putida S4, was isolated from smelter drainage of copper mines. The strain exhibited resistance to several heavy metals, like aluminium (Al), zinc (Zn), nickel (Ni), cobalt (Co) besides copper (Cu). Strain S4 could accumulate Cu from the Cu-supplemented growth medium. In the present study, we have demonstrated the Cu2+ removal capacity of this strain from various samples such as mine effluent, low-grade ore and ore-tailings, collected from the mining site. Moreover, approximately 80% of the accumulated Cu2+ could be recovered from the loaded biomass by a simple desorption procedure.     

A bacterial view of the periodic table: genes and proteins for toxic inorganic ions

Essentially all bacteria have genes for toxic metal ion resistances and these include those for Ag+, AsO2-, AsO4(3-), Cd2+ Co2+, CrO4(2-), Cu2+, Hg2+, Ni2+, Pb2+, TeO3(2-), Tl+ and Zn2+. The largest group of resistance systems functions by energy-dependent efflux of toxic ions. Fewer involve enzymatic transformations (oxidation, reduction, methylation, and demethylation) or metal-binding proteins (for example, metallothionein SmtA, chaperone CopZ and periplasmic silver binding protein SilE). Some of the efflux resistance systems are ATPases and others are chemiosmotic ion/proton exchangers. For example, Cd2+-efflux pumps of bacteria are either inner membrane P-type ATPases or three polypeptide RND chemiosmotic complexes consisting of an inner membrane pump, a periplasmic-bridging protein and an outer membrane channel. In addition to the best studied three-polypeptide chemiosmotic system, Czc (Cd2+, Zn2+, and Co2), others are known that efflux Ag+, Cu+, Ni2+, and Zn2+. Resistance to inorganic mercury, Hg2+ (and to organomercurials, such as CH3Hg+ and phenylmercury) involve a series of metal-binding and membrane transport proteins as well as the enzymes mercuric reductase and organomercurial lyase, which overall convert more toxic to less toxic forms. Arsenic resistance and metabolizing systems occur in three patterns, the widely-found ars operon that is present in most bacterial genomes and many plasmids, the more recently recognized arr genes for the periplasmic arsenate reductase that functions in anaerobic respiration as a terminal electron acceptor, and the aso genes for the periplasmic arsenite oxidase that functions as an initial electron donor in aerobic resistance to arsenite.     

Increasing the copper sensitivity of microorganisms by restricting iron supply,a strategy for bio-management practices

Pollution by copper (Cu²⁺) extensively used as antimicrobial in agriculture and farming represents a threat to the environment and human health. Finding ways to make microorganisms sensitive to lower metal concentrations could help decreasing the use of Cu²⁺ in agriculture. In this respect, we showed that limiting iron (Fe) uptake makes bacteria much more susceptible to Cu²⁺ or Cd²⁺ poisoning. Using efflux mutants of the purple bacterium Rubrivivax gelatinosus, we showed that Cu⁺ and Cd²⁺ resistance relies on the expression of the Fur-regulated FbpABC and Ftr iron transporters. To support this conclusion, inactivation of these Fe-importers in the Cu⁺ or Cd²⁺-ATPase efflux mutants gave rise to hypersensitivity towards these ions. Moreover, in metal overloaded cells the expression of FbpA, the periplasmic iron-binding component of the ferric ion transport FbpABC system was induced, suggesting that cells perceived an ‘iron-starvation’ situation and responded to it by inducing Fe-importers. In this context, the Fe-Sod activity increased in response to Fe homoeostasis dysregulation. Similar results were obtained for Vibrio cholerae and Escherichia coli, suggesting that perturbation of Fe-homoeostasis by metal excess appeared as an adaptive response commonly used by a variety of bacteria. The presented data support a model in which metal excess induces Fe-uptake to support [4Fe-4S] synthesis and thereby induce ROS detoxification system.     

Mechanism of copper resistance in a copper mine isolate Pseudomonas putida strain S4

The mechanism of copper resistance in a multiple-metal-resistant natural isolate Pseudomonas putida strain S4 is based on inducible efflux. Active extrusion of copper ions occurs from the cytoplasm during the exponential phase of growth. Involvement of ATPase in the efflux of copper ions has been demonstrated by employing specific inhibitors. The effluxed copper is not thrown out of the cell, but remains in a bound form (to a protein) in the periplasm. Thus, a balance between the intracellular level, to fulfill the metabolic requirements, and the periplasmic sequestration, to evade toxicity, is maintained by this isolate.     

The cobalt, zinc, and cadmium efflux system CzcABC from Alcaligenes eutrophus functions as a cation-proton antiporter in Escherichia coli.

The function of the CzcABC protein complex, which mediates resistance to Co2+, Zn2+, and Cd2+ in Alcaligenes eutrophus by cation efflux, was investigated by using everted membrane vesicles of Escherichia coli and an acridine orange fluorescence quenching assay. Since metal cation uptake could not be measured with inside-out membrane vesicles prepared from A. eutrophus and since available E. coli strains did not express the Czc-mediated resistance to cobalt, zinc, and cadmium salts, mutants of E. coli which exhibited a Czc-dependent increase in heavy metal resistance were isolated. E. coli mutant strain EC351 constitutively accumulated Co2+, Zn2+, and Cd2+. In the presence of Czc, net uptake of these heavy metal cations was reduced to the wild-type level. Inside-out vesicles prepared from E. coli EC351 cells displayed a Czc-dependent uptake of Co2+, Zn2+, and Cd2+ and a cation-triggered acridine orange fluorescence increase. The czc-encoded protein complex CzcABC was shown to be a zinc-proton antiporter.     

Subcellular distribution of accumulated heavy metals in Saccharomyces cerevisiae and Kluyveromyces marxianus

Summary Kluyveromyces spp. have been found to be more efficient than a CUP1^R strain of S. cerevisiae in heavy metal resistance and accumulation. The present study describes the subcellular distribution of the accumulated metals (Ag, Cd, Cu) in S. cerevisiae and K. marxianus. Absorption by insoluble cellular material of the metals appears as the main mechanism of metal accumulation in both organisms.     

Functional Properties of Pro Region of Escherichia coli Heat-Stable Enterotoxin

Escherichia coli heat-stable enterotoxin Ip (STp) is synthesized as the 72-amino-acid residue precursor consisting of three regions: pre region (amino acid residues 1 to 19), pro region (amino acid residues 20 to 54), and mature ST (mST) region (amino acid residues 55 to 72). We examined the role of the pro sequence of STp in enterotoxigenicity of a strain by deleting the gene fragment encoding amino acids 22 to 57. This deletion caused a remarkable reduction of its enterotoxic activity of culture supernatant. In order to analyze the sequence responsible for the function of the pro region, two additional deletion mutants were made. The deletion of the sequence covering amino acids 29 to 38, which is conserved in all sequences of ST reported, brought about a significant reduction of enterotoxic activity but the deletion of the non-conserved sequence (amino acids 40 to 53) did not. This result shows that conserved sequence is mainly responsible for the function. Subsequently, to examine the mechanism of action of the pro region, plasmids carrying DNA sequences of hybrid proteins consisting of pre-pro-nuclease, pre-mST-nuclease, pre-pro-mST-nuclease and pre-pro-nuclease-mST were constructed. Amino acid sequence determination and SDS-polyacrylamide gel analysis revealed that these fusion proteins were cleaved between pre sequence and pro sequence during secretion and the cleaved fusion proteins were accumulated in periplasmic space. But the amount of hybrid protein accumulated in the periplasmic space varied among the strains. That is, the amount of the pre-pro-nuclease gene product that accumulated in the periplasmic space was the highest of all fusion gene products. These results indicate that the existence of the mST region strongly interferes with the translocation of the gene product into the periplasmic space and that the pro region functions to guide the mST region into the periplasmic space.     

An essential role for UshA in processing of extracellular flavin electron shuttles by Shewanella oneidensis

The facultative anaerobe Shewanella oneidensis can reduce a number of insoluble extracellular metals. Direct adsorption of cells to the metal surface is not necessary, and it has been shown that S. oneidensis releases low concentrations flavins, including riboflavin and flavin mononucleotide (FMN), into the surrounding medium to act as extracellular electron shuttles. However, the mechanism of flavin release by Shewanella remains unknown. We have conducted a transposon mutagenesis screen to identify mutants deficient in extracellular flavin accumulation. Mutations in ushA, encoding a predicted 5′‐nucleotidase, resulted in accumulation of flavin adenine dinucleotide (FAD) in culture supernatants, with a corresponding decrease in FMN and riboflavin. Cellular extracts of S. oneidensis convert FAD to FMN, whereas extracts of ushA mutants do not, and fractionation experiments show that UshA activity is periplasmic. We hypothesize that S. oneidensis secretes FAD into the periplasmic space, where it is hydrolysed by UshA to FMN and adenosine monophosphate (AMP). FMN diffuses through outer membrane porins where it accelerates extracellular electron transfer, and AMP is dephosphorylated by UshA and reassimilated by the cell. We predict that transport of FAD into the periplasm also satisfies the cofactor requirement of the unusual periplasmic fumarate reductase found in Shewanella.     

Escherichia coli gamma-glutamyltranspeptidase mutants deficient in processing to subunits.

Arginyl residues 513 and 571 of Escherichia coli K-12 gamma-glutamyl-transpeptidase (EC 2.3.2.2) were substituted with alanyl and glycyl residues, respectively, by oligonucleotide-directed in vitro mutagenesis. Both mutants were devoid of the enzymatic activity. On Western blot analysis, we found that both mutants accumulated a gamma-glutamyltranspeptidase precursor which was not processed into large and small subunits in the periplasmic space of Escherichia coli.     

基因工程菌大肠杆菌JM109富集废水中镍离子的研究

利用通过基因工程技术所构建的在细胞内同时表达出高特异性镍转运蛋白和金属硫蛋白的基因工程菌富集水体中的镍离子。菌体细胞对Ni²⁺的富集速率很快,富集过程满足Langmuir等温线模型。与原始宿主菌相比,经基因改造的基因工程菌不仅最大镍富集容量增加了5倍多,而且对pH值、离子强度的变化及其它共存重金属离子的影响都呈现出更强的适应性。相比而言,Na⁺、Ca²⁺、Cd²⁺、Pb²⁺的影响较小,但Mg²⁺、Hg²⁺和Cu²⁺所引起的负面效应较大。进一步的实验表明基因工程菌对Ni2+的富集行为不需要外加营养物质。     

Defective export in Escherichia coli caused by DsbA'-PhoA hybrid proteins whose DsbA' domain cannot fold into a conformation resistant to periplasmic proteases.

The disulfide bond-forming factor DsbA and the alkaline phosphatase are stable in the Escherichia coli periplasmic space and can be overproduced without significant perturbation of the cell's physiology. By contrast, DsbA'-PhoA hybrid proteins resulting from TnphoA insertions into different regions of a plasmid-borne dsbA gene could become toxic (lethal) to bacteria. Toxicity was concomitant with an impairment of some step of the export mechanism and depended on at least three parameters, i.e., (i) the rate of expression of the hybrid protein, (ii) the ability of the amino-terminal DsbA' domain of the hybrid protein to fold into a protease-resistant conformation in the periplasmic space, and (iii) the activity of the DegP periplasmic protease. Even under viable conditions of low expression, DsbA' folding-deficient hybrid proteins accumulated more than the folding-proficient ones in the insoluble material and this was aggravated in a strain lacking the DegP protease. When production was more elevated, the folding-deficient hybrid proteins became lethal, but only in strains lacking the DegP activity, while the folding-proficient ones were not. Under conditions of very high production by degP+ or degP strains, both types of hybrid proteins accumulated as insoluble preproteins. Meanwhile, the export machinery was dramatically handicapped and the cells lost viability. However, the folding-deficient hybrid proteins had a higher killing efficiency than the folding-proficient ones. Free DsbA'-truncated polypeptides, although not toxic, were processed more slowly when they could not fold into a protease-resistant form in the periplasmic space. This provides indications in E. coli for a direct or indirect influence of the folding of a protein in the periplasmic environment on export efficiency.     

Bacterial resistances to toxic metal ions - a review

Bacterial plasmids encode resistance systems for toxic metal ions, including Ag⁺, AsO₂^-, AsO₄^3-, Cd²⁺, Co²⁺, CrO₄^2-, Cu²⁺ Hg²⁺, Ni²⁺, Pb²⁺, Sb³⁺, TeO₃^2-, Tl⁺ and Zn²⁺. The function of most resistance systems is based on the energy-dependent efflux of toxic ions. Some of the efflux systems are ATPases and others are chemiosmotic cation/proton antiporters. The Cd²⁺-resistance ATPase of Gram-positive bacteria (CadA) is membrane cation pump homologous with other bacterial, animal and plant P-type ATPases. CadA has been labeled with ³²P from [α-³²p]ATP and drives ATP-dependent Cd²⁺ (and Zn²⁺) uptake by inside-out membrane vesicles (equivalent to efflux from whole cells). Recently, isolated genes defective in the human hereditary diseases of copper metabolism, namely Menkes syndrome and Wilson's disease, encode P-type ATPases that are more similar to bacterial CadA than to other ATPases from eukaryotes. The arsenic resistance efflux system transports arsenite [As(III)], alternatively using either a double-polypeptide (ArsA and ArsB) ATPase or a single-polypeptide (ArsB) functioning as a chemiosmotic transporter. The third gene in the arsenic resistance system, arsC, encodes an enzyme that converts intracellular arsenate [As(V)] to arsenite [As(III)], the substrate of the efflux system. The triple-polypeptide Czc (Cd²⁺, Zn²⁺ and Co²⁺) chemiosmotic efflux pump consists of inner membrane (CzcA), outer membrane (CzcC) and membrane-spanning (CzcB) proteins that together transport cations from the cytoplasm across the periplasmic space to the outside of the cell.     

Effect of trichloroethylene on the competitive behavior of toluene-degrading bacteria

The influence of trichloroethylene (TCE) on a mixed culture of four different toluene-degrading bacterial strains (Pseudomonas putida mt-2, P. putida F1, P. putida GJ31, and Burkholderia cepacia G4) was studied with a fed-batch culture. The strains were competing for toluene, which was added at a very low rate (31 nmol mg of cells [dry weight] h). All four strains were maintained in the mixed culture at comparable numbers when TCE was absent. After the start of the addition of TCE, the viabilities of B. cepacia G4 and P. putida F1 and GJ31 decreased 50- to 1,000-fold in 1 month. These bacteria can degrade TCE, although at considerably different rates. P. putida mt-2, which did not degrade TCE, became the dominant organism. Kinetic analysis showed that the presence of TCE caused up to a ninefold reduction in the affinity for toluene of the three disappearing strains, indicating that inhibition of toluene degradation by TCE occurred. While P. putida mt-2 took over the culture, mutants of this strain which could no longer grow on p-xylene arose. Most of them had less or no meta-cleavage activity and were able to grow on toluene with a higher growth rate. The results indicate that cometabolic degradation of TCE has a negative effect on the maintenance and competitive behavior of toluene-utilizing organisms that transform TCE.     

Short Communication: Carbon source-starvation-induced precipitation of copper by Pseudomonas putida strain S4

Pseudomonas putida strain S4, when starved of carbon source, precipitated Cu2+ in the medium. The precipitate, apart from containing copper, consisted of phosphate and hydroxide residues. While high acid phosphatase activity provided the necessary phosphates for Cu2+-precipitation, hydroxyl residues generated by metal efflux pathway may be used for metal hydroxide precipitation. This phenomenon could be exploited in the biorecovery of copper from different sources.     

Biochemical characteristics of new thermosensitive secretory mutants of yeasts

New thermosensitive mutants of the yeast Saccharomyces cerevisiae which block the secretion of periplasmic enzymes at restriction temperature have been obtained. These mutants accumulate active low molecular weight and mature invertase species in the cell; the buoyant density of the cells in a Percoll gradient is higher than that in the wild strain cells. The mutant cells transferred to permissive temperature (25 degrees C) in the absence of protein synthesis can secrete some amount of accumulated invertase. It was found that the secretory defects of conditional mutants do not affect the activity of cytoplasmic enzymes (e.g., alcohol dehydrogenase) or the level of total protein synthesis and glycosylation and do not induce non-specific disturbances in energy metabolism and plasma membrane functions at restriction temperature. Some strains of new secretory mutants revealed uncoupled defective secretion of periplasmic enzymes and intrinsic membrane proteins (proline permease). The possibility of branching of the secretory pathway for periplasmic enzymes and cytoplasmic membrane proteins is discussed.     

Molybdenum requirement for translocation of dimethyl sulfoxide reductase to the periplasmic space in a photodenitrifier, Rhodobacter sphaeroides f. sp. denitrificans.

Translocation of dimethyl sulfoxide (DMSO) reductase to the periplasmic space was studied in vivo with a photodenitrifier, Rhodobacter sphaeroides f. sp. denitrificans, using immunoblotting analysis and radioactive labeling. A polypeptide with an apparent molecular mass about 2,000 Da higher than that of DMSO reductase accumulated during induction of the reductase with DMSO. An uncoupler, carbonyl cyanide-m-chlorophenylhydrazone, inhibited the processing of the polypeptide after cells had been radioactively pulse-labeled with [35S]methionine. These results indicated that the higher-molecular-mass polypeptide was the precursor form of DMSO reductase. The precursor form accumulated in either the cytoplasm or the membrane, whereas the mature form accumulated in the periplasmic space. The membrane-bound precursor was sensitive to proteinase K treatment from both the cytoplasmic and periplasmic sides of the membrane, indicating that the polypeptide binds to the membrane, exposing it to both the outer and inner surfaces of the cytoplasmic membrane. Processing of the precursor was hampered by removal of molybdate from the medium and was restored by its readdition. It was also inhibited by the addition of tungstate in the medium.     

Insight into the selectivity of arsenic trioxide for acute promyelocytic leukemia cells by characterizing Saccharomyces cerevisiae deletion strains that are sensitive or resistant to the metalloid

The genome-wide set of Saccharomyces cerevisiae deletion strains provides the opportunity to analyze how other organisms may respond to toxic agents. Since arsenic trioxide selectively kills human acute promyelocytic leukemia (APL) cells by a poorly understood mechanism we screened the yeast deletion strains for sensitivity or resistance. In addition to confirming mutants previously identified as sensitive to sodium arsenite, a large number of additional genes, and cellular processes, were required for arsenic trioxide tolerance. Of the 4546 mutants, 7.6% were more sensitive to arsenic trioxide than the wild type, while 1.5% was more resistant. IC50 values for all sensitive and resistant mutants were determined. Prominent as sensitive was that missing the MAP kinase, Hog1. The most resistant lacked the plasma-membrane glycerol and arsenite transporter, Fps1. Hog1 and Fps1 control the response to osmotic stress in yeast by regulating glycerol production and plasma membrane flux, respectively. We therefore tested whether APL cells have impaired osmoregulation. The APL cell line NB4 did not produce glycerol in response to osmotic stress and underwent apoptotic cell death. Moreover, the glycerol content of NB4 and differentiated NB4 cells correlated with the level of arsenic trioxide uptake and the sensitivity of the cells. Additionally, NB4 cells accumulated more arsenic trioxide than non-APL cells and were more sensitive. These findings demonstrate the usefulness of the S. cerevisiae deletion set and show that the selectivity of arsenic trioxide for APL cells relates, at least in part, to impaired osmoregulation and control of uptake of the drug.     

Membrane topology of a multidrug efflux transporter, AcrB, in Escherichia coli.

AcrA/B in Escherichia coli is a multicomponent system responsible for intrinsic resistance to a wide range of toxic compounds, and probably cooperates with the outer membrane protein TolC. In this study, acrAB genes were cloned from the E. coli W3104 chromosome. To determine the topology of the inner membrane component AcrB, we employed a chemical labeling approach to analyse mutants of AcrB in which a single cysteine residue had been introduced. The cysteine-free AcrB mutant, in which the two intrinsic Cys residues were replaced by Ala, retained full drug resistance. We constructed 33 cysteine mutants in which a single cysteine was introduced into each putative hydrophilic loop region of the cysteine-free AcrB. The binding of [(14)C]N-ethylmaleimide (NEM) to the Cys residue and the competition of NEM binding with the binding of a membrane-impermeant maleimide, 4-acetamide-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS), in intact cells were investigated. The results revealed that the N- and C-terminals are localized on the cytoplasmic surface of the membrane and the two large loops are localized on the periplasmic surface of the membrane. The results supported the 12-membrane-spanning structure of AcrB. Three of the four short periplasmic loop regions were covered by the two large periplasmic loop domains and were not exposed to the water phase until one of the two large periplasmic loops was removed.