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WO2001092471A1 - Bacteries pouvant unir des metaux lourds et leur utilisation dans la detoxication de milieux contamines par des metaux lourds - Google Patents

Bacteries pouvant unir des metaux lourds et leur utilisation dans la detoxication de milieux contamines par des metaux lourds Download PDF

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WO2001092471A1
WO2001092471A1 PCT/ES2001/000214 ES0100214W WO0192471A1 WO 2001092471 A1 WO2001092471 A1 WO 2001092471A1 ES 0100214 W ES0100214 W ES 0100214W WO 0192471 A1 WO0192471 A1 WO 0192471A1
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Prior art keywords
heavy metals
bacterium
soil
heavy
protein
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English (en)
Spanish (es)
Inventor
Victor De Lorenzo Prieto
Marc Valls Matheu
Silvia Atrian Ventura
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Consejo Superior de Investigaciones Cientificas CSIC
Universitat de Barcelona UB
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Consejo Superior de Investigaciones Cientificas CSIC
Universitat de Barcelona UB
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P3/00—Preparation of elements or inorganic compounds except carbon dioxide
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/825—Metallothioneins
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/10—Inorganic compounds
    • C02F2101/20—Heavy metals or heavy metal compounds

Definitions

  • This invention relates to the development of bacteria with a high capacity to bind heavy metals on their surface and their use in the recovery of soils and / or liquid media contaminated with heavy metals.
  • Bacteria, adapted to the soil and resistant to heavy metals, either naturally or by genetic manipulation, have been modified to produce a protein, or a peptide sequence, on their surface, capable of binding heavy metals.
  • the invention faces the problem of recovering a medium, solid or liquid, contaminated with one or more heavy metals through the use of biological agents.
  • the solution provided by this invention is based on the fact that the inventors have observed that by anchoring proteins or peptide sequences with the ability to bind heavy metals on the surface of bacteria adapted to the soil and resistant to heavy metals, a medium can be efficiently recovered Contaminated with heavy metals.
  • Example 2 illustrates the adsorption of cadmium from a liquid medium by the Ralstonia eutropha MTB bacteria provided by this invention, while Example 3 illustrates the ability of said bacterium to counteract the toxic effect of cadmium in soil.
  • an object of this invention is bacteria adapted to the ground and resistant to heavy metals that contain on their surface a protein, or a peptide sequence, with the ability to bind one or more heavy metals
  • the use of said bacteria in the recovery of a medium, solid or liquid, contaminated with heavy metals constitutes an additional object of this invention.
  • Another additional object of this invention is a method for recovering a soil contaminated by heavy metals comprising contacting said bacteria with the soil to be recovered.
  • Another additional object of this invention is a method for treating an effluent loaded with heavy metals, in order to reduce the heavy metal content in said effluent, which comprises contacting the effluent to be treated with said bacteria or, alternatively, with non-viable biomass from said dead bacteria, said biomass being attached to a solid support.
  • Figure 1 shows the genetic structure of pMT ⁇ -1 and the production of MT ⁇ in E. coli.
  • Figure 1A shows the scheme of plasmid pMT ⁇ -1 containing the fusion of the mtb gene. The Jad gene and the lac (plac) promoter are indicated. The domain structure and basic topology of the MT ⁇ protein are also outlined. The locations of the pelB signal peptide (ss, not present in the mature protein), the MT domain, the E-tag peptide and the ⁇ domain of the Nei sseria gonorrhoeae IgA protease are shown.
  • Figure IB shows the results of the immunodetection of proteins from the soluble, inner membrane (MI) and outer membrane (ME) fractions of E.
  • MI inner membrane
  • ME outer membrane
  • E. coli JCB570 (wt) and E. coli JCB571 (dsbA) cells that carry the pMT ⁇ -1 and induced with IPTG.
  • the anti-E-tag monoclonal antibody was used to test for the presence of the MT ⁇ -1 hybrid (57 kDa) in protein extracts transferred after SDS-PAGE electrophoresis.
  • the Figure 1C shows the presentation of MT ⁇ on the surface of E. coli.
  • the accessibility of the MT ⁇ protein in intact cells (I), subjected to osmotic shock (S) and Used (L) was tested using an anti-E-tag monoclonal antibody in an ELISA [see Example 1.5].
  • the negative control included isogenic E. coli cells devoid of plasmid pMT ⁇ -1.
  • Figure 2 shows the accumulation of Cd 2+ by E. coli and R. eutropha.
  • the amount of Cd 2+ bound by bacterial cells was determined by atomic absorption spectroscopy and is indicated in nmoles of Cd 2+ per g of dry cell mass. The values shown are the average of two independent experiments using duplicate samples in each Cd determination.
  • Figure 2A shows the results of the E. coli JCB570 (wt) and E. coli JCB571 (dsbA) cells transformed with pMT ⁇ -1 ( +), or not transformed (-), induced with IPTG in MJS medium containing 30 ⁇ M CdCl 2 .
  • Figure 2B shows the results of the accumulation of Cd 2+ by the strains of R. eutropha.
  • the cell cultures of R. eutropha MTB and its parental strain R. eutropha CH34 were induced with 3-methylbenzoate (3-MB) in MJS medium containing 30 ⁇ M CdCl 2 (left) or 300 ⁇ M (right).
  • 3-methylbenzoate (3-MB) 3-methylbenzoate
  • Figure 3 shows the structure of the InMT ⁇ -1 transposon and the production of MT ⁇ in R. eutropha.
  • Figure 3A shows the genetic elements within ⁇ MT ⁇ -1: the mtJb gene, the kanamycin resistance gene (Km), the xylS gene
  • FIG. 3B shows the results of immunoblotting of whole-cell protein extracts of R. eutropha MTB and its parental control strain (CH34) tested with the anti-E-tag monoclonal antibody to detect the presence of the MT ⁇ protein.
  • the Pm promoter inducer, 3-methyl-benzoate (3-MB) was added (+) or not (-) to the bacterial culture.
  • Figure 3C shows the results of protein immunodetection, after SDS-PAGE electrophoresis and transfer, from the soluble fractions, of the inner membrane (MI) and outer membrane (ME) of R.
  • Figure 4 shows the results of Cd 2+ toxicity repair on plant growth with R. eutropha MTB. Seedlings of 15 days of N. bentamiana were grown for 40 days in sterile control soil (1), or in soil inoculated with 10 8 cells of R. eutropha CH34 / g (2), or 10 8 cells of R. eutropha MTB / g (3), previously grown in MS medium containing citrate and 3-MB; the control soil was also mixed with the same medium [Figure 4A].
  • Figure 4B The same procedure as described for Figure 4A was performed but on land containing 150 ⁇ moles of Cd 2+ / kg. The toxicity of Cd (compare Figures 4A and 4B) and the protective effect of R. eutropha MTB on plant growth can be observed (compare pots 1 to 3).
  • the invention provides a bacterium capable of binding heavy metals, hereinafter bacteria of the invention, characterized in that it is a bacterium adapted to the soil and resistant to heavy metals and containing on its surface a protein, or a peptide sequence, with the ability to join heavy metals.
  • bacteria adapted to the soil and resistant to heavy metals means that the bacteria can live in the soil and that its growth is not affected by the presence of heavy metals in toxic concentrations for most organisms and other bacteria, and includes both soil-adapted bacteria and naturally resistant to heavy metals, that is, whose habitat Soil is natural, such as bacteria adapted to the soil that have acquired resistance to heavy metals by genetic engineering techniques, for example, by transforming a bacterium that naturally is not resistant to heavy metals with a system capable of inducing resistance to metals heavy
  • the resistance to heavy metals is mainly due to the presence of heavy metal pumping systems from the cytoplasm to the external environment. Thanks to these mechanisms, heavy metal does not accumulate inside the bacteria.
  • Bacteria adapted to the soil and naturally resistant to heavy metals include bacteria belonging to the gram-negative genera Ralstonia and Pseudomonas, and Bacill us in gram-positive.
  • the bacterium of the invention comes from Ral stonia eutropha (previously called Al caligenes eutrophus) CH34, a bacterial strain that is adapted to develop in soils highly contaminated with metal ions.
  • heavy metals as used in this description includes a series of metals and metalloids from the Periodic Table of elements with metallic and potentially toxic properties. Heavy toxic metals include Ag, As, Bi, Cd, Co, Cr, Cu, Hg, Ni, Pb, Pd, Pt, Te, TI, Sb, Se, Sn and Zn. In general, the most significant heavy metals are Cd, Co, Cu, Hg, Ni, Pb and Zn.
  • heavy Ametal includes any chemical form of said metal that may be present in a solid or liquid medium.
  • the protein or peptide sequence must bind at least one heavy metal, including, but not limited to, Ag, As, Bi, Cd, Co, Cr, Cu, Hg, Ni, Pb, Pd, Pt, Te, TI, Sb, Se, Sn and Zn, and their chemical forms corresponding.
  • Illustrative examples of proteins capable of binding one or more heavy metals include metallothioneins and plant phytokelatins.
  • Metallothioneins (MT) are a group of small proteins (approximately 60 amino acids) rich in cysteine, which are specialized in the binding of heavy metals, for example, Zn 2+ , Cd 2+ , Hg 2+ [Nordberg, M ., (1998)]. Genes encoding MT can be found in different animal species (eg, in mice and humans), plants, and fungi.
  • the nucleotide sequence encoding said protein or peptide sequence capable of binding one or more heavy metals can be obtained from any available source, both natural and artificial.
  • the nucleotide sequence encoding said proteins, or peptide sequence can be isolated from its natural source by using the appropriate restriction enzymes or can be obtained by recombinant DNA techniques or can be obtained by synthetic techniques.
  • It can also be a polymeric form of a DNA sequence that codes for a protein or peptide sequence with the ability to bind one or more heavy metals, as well as a variant in which some codons have been replaced by others in order to encode for amino acids that increase the binding affinity of heavy metals and / or to obtain proteins or peptide sequences that have affinity for heavy metals other than heavy metals for which the native peptide protein or sequence has affinity.
  • peptide protein or sequence capable of binding one or more heavy metals includes not only native and synthetic forms but also derivatives thereof, for example, fusion products of said protein or peptide sequence. and another protein or peptide that can provide different functions, for example, improve the stability and / or activity of the heavy metal bioadsorbent agent.
  • US patents US 5,824,512 and US 5,965,796 describe natural, synthetic and derivative MTs, as well as metal binding sequences, respectively, which could be used in the present invention.
  • Other proteins and metal binding sequences that can be used in the present invention have been described by Klemba et al. (1995), Brown, (1997), and Bae & Mehra, (1997).
  • the protein capable of binding one or more heavy metals present on the surface of the bacterium is a mouse MT, specifically the mouse MT-I.
  • the soil-adapted and heavy metal resistant bacteria that serves as the basis for the development of the bacterium of the invention can be Gram positive or Gram negative.
  • the protein or peptide sequence capable of binding one or more heavy metals is linked directly to the peptidoglycan or by suitable coupling elements, such as peptidoglycan binding domains such as those obtained from protein A of Staphyl ococcus aureus [Stáhl et al., (1997)].
  • the anchoring of said protein or peptide sequence capable of joining one or more heavy metals on the outer membrane can be carried out by means of a self-transporting system or by any other external membrane anchoring system, such as maltoporin or the lpp-ompA hybrid [Valls et al., (1998)].
  • Anchoring systems must allow the protein or peptide sequence with binding capacity of one or more heavy metals to manifest on the surface of the bacteria.
  • Motor carriers are a very extended family of secreted proteins found in Gram negative bacteria and that are capable of translocating through the outer membrane without any auxiliary protein machinery
  • Neisseria gonorrhoeae involves the production of a large protein precursor that contains an N-terminal signal peptide that directs transport through the inner membrane (MI) in the periplasm. This is followed by the insertion in the outer membrane (ME) of the C-terminal domain of the protein (the collaborator or ⁇ domain) which, forming an amphipathic ⁇ barrel, allows the accompanying passenger N domain to cross the ME [Klauser et al. , (1993); Veiga et al., (1999)].
  • the bacteria provided by this invention can be obtained by using conventional recombinant DNA techniques.
  • the sequence encoding the protein or peptide sequence capable of binding one or more heavy metals is cloned into an expression vector such as a plasmid.
  • said coding sequence is cloned for the protein or peptide sequence capable of binding one or more heavy metals in the expression vector so that they are expressed as a fusion product with a cell membrane protein or with an anchoring system, such as an auto transporter.
  • the recombinant expression vector thus obtained is introduced, by conventional methods, into a bacterium adapted to the soil and resistant to metals.
  • heavy whose resistance to heavy metals has been acquired either naturally or by genetic manipulation, capable of expressing the product of the DNA sequence coding for the protein or peptide sequence capable of binding one or more heavy metals.
  • multiple copies of different expression vectors provided by this invention can be inserted into a single bacterial cell.
  • the transformed bacteria are subjected to an induction process by using the appropriate inductors so as to express said protein or peptide sequence with the ability to bind one or more heavy metals on the surface of the bacteria.
  • the bacteria of the invention can be used in the recovery of a medium, solid or liquid, contaminated with heavy metals.
  • the invention therefore provides a method for recovering a soil contaminated by heavy metals comprising contacting an amount effective to recover said contaminated soil of at least one culture of bacteria provided by this invention with the soil to be recovered.
  • an amount effective to recover said contaminated soil of at least one culture of bacteria provided by this invention with the soil to be recovered.
  • between 10 5 and 10 11 bacteria / g of soil, preferably 10 8 bacteria / g soil are applied.
  • the bacteria of the invention are applied on the soil to be treated by any conventional method.
  • the bacteria can be in contact with the soil to be treated either in solid or liquid phase, for example, by introducing or injecting a suspension of bacteria of the invention onto the soil to be treated, or by watering the soil to be treated with a formulation containing a suspension of the bacteria of the invention; alternatively, the soil to be treated can be physically mixed with the bacteria of the invention.
  • the method provided by this invention is suitable for recovering soils contaminated with heavy metals containing between 0.1 ⁇ moles of heavy metal / kg of soil and 100 millimoles of heavy metal / kg of soil.
  • the invention also provides a method for treating an effluent charged with heavy metals, in order to reduce the heavy metal content in said effluent, which comprises contacting an effective amount of at least one culture of bacteria provided by this invention. With the effluent to be treated.
  • the reduction of the heavy metal content in said effluent is intended to reach a level of contaminating heavy metals in the effluent that is not toxic to the environment, whereby said effluent, substantially discharged from contaminating heavy metals, can be discharged so controlled.
  • the effluent Prior to contacting the bacteria of the invention with the effluent to be treated, the effluent is analyzed and the pH and the existence of adequate nutrients for the growth of the bacteria of the invention are determined, so that, in case If the pH does not have the necessary nutrients in adequate amounts to guarantee the viability of the bacteria, appropriate measures are taken, for example, in the correction of the pH and in supplementing the effluent with the necessary nutrients.
  • the removal of heavy metals from the effluent comprises (i) killing, by conventional methods, for example, modifying the viability conditions of the Bacteria in question, bacteria provided by this invention, which express on their surface a protein or peptide sequence capable of joining one or more heavy metals, to obtain a non-viable biomass capable of joining one or more heavy metals, (ii) joining said biomass to the surface of a solid support, (iii) contacting said surface that has biomass attached, with an effluent that contains at least one heavy metal such that said heavy metal binds to said protein or peptide sequence capable of joining said heavy metal, and (iv) removing from said effluent the support containing said biomass.
  • the biomass can be attached to the solid support either by a covalent or non-covalent bond, for example, by adsorption, etc.
  • US Patent 5,824,512 describes a method for removing heavy metals from an aqueous medium by using non-viable biomass obtained from dead bacteria capable of attaching at least one heavy metal, said biomass being attached to a solid support. The teachings of said US patent are included in this description as a reference.
  • the invention is illustrated below by an example showing the expression of mouse MT-I on the cell surface of R. eutropha CH34, Gram negative bacterial strain adapted to develop in soils highly contaminated with heavy metal ions.
  • the DNA sequence encoding the mouse MT-I was fused to the ⁇ domain of the autotransporter of the IgA protease of N. gonorrhoeae, which places the hybrid protein (Mt ⁇ ) towards the external bacterial membrane.
  • the translocation, presentation on the bacterial surface, and functionality of the chimeric protein MT ⁇ was initially demonstrated in E. coli before the transfer of its coding gene (mtb) to R. eutropha, observing that the resulting bacterial strain, called R.
  • Example 3 an inducer (3-MB) is used for the expression of mouse MT-I in R.
  • the promoter used in the construction is an inducible promoter, for use in the treatment It is more convenient to use a construct in which the promoter of the gene encoding the protein or peptide sequence capable of binding one or more heavy metals is a constitutive promoter so that the expression of said protein or peptide sequence is carried out in continuously and do not depend on the availability of an inductor. Any constitutive promoter could be used in such constructions.
  • E. coli JCB570 (MC1000 phoR zihl:: Tnl O) [Bardwell et al., (1991)]
  • E. coli JCB571 (MC1000 phoR zihl2:: Tnl O dsbA:: kanl) [Bardwell et al., (1991)]
  • the bacteria were grown at 301c in LB medium [Miller et al., (1992)] liquid or in 1.5% w / v LB agar plates supplemented with appropriate antibiotics [Miller et al. , (1992)].
  • Glucose (2% w / v) was included in the growth medium for total repression of the lac promoter of pMT ⁇ -1 in E. coli. The expression of
  • Smtl 5'-CGGGCCCAGCCGGCCATGGCGGACCCCAACTGCTCCTGC-3 '
  • Smt2 5' -GCGGCCCCCGACGCCGCGGCACAGACAGTGCACTTGTC-3 '
  • the 200 bp amplified DNA fragment containing the MT-I gene flanked by the Sfi l sites was digested with Sfil and ligated to the 5.2 kb fragment obtained with the Sfi l digestion of pPvH ⁇ [Veiga et al., (1999)].
  • the Xjal and HindI II sites it contained flanking the mtb gene were converted into ATOt ⁇ sites in pMT ⁇ -1 [see Figure 1] by ligation of the Xbal-Notl and ⁇ findlII-Notl couplers.
  • the Xbal-Notl coupler was constructed by hybridization of the oligonucleotides: 5'-CTAGGCGGCCGC-3 '(SEQ. ID. Ni: 3) and 5'-CTAGGCGGCCGC-3' (SEQ. ID. Ni: 4).
  • the HindlII-Notl coupler was constructed by hybridization of the oligonucleotides: 5'- AGCTGCGGCCGC-3 '(SEQ. ID. Ni: 5) and 5'-AGCTGCGGCCGC-3' (SEQ. ID. Ni: 6) .
  • pTnMT ⁇ -1 For the construction of pTnMT ⁇ -1, the 1.7 kb DNA fragment containing the mtb gene was isolated by Noti digestion of pMT ⁇ -1 and ligated into the single Notl site of pCNBl [by Lorenzo et al., (1993 )] in the orientation that places mtb under the control of the Pm promoter [see Figure 3].
  • TnMT ⁇ -1 Transfer of TnMT ⁇ -1 to Ralstonia eutropha CH34
  • the mini-Tn5 element TnMT ⁇ -1 was transferred to the chromosome of R. eutropha CH34 by conjugation with E. coli S17-1 ⁇ pir transformed with plasmid p7nMT ⁇ -l using the protocol previously described [ de Lorenzo et al., (1994)].
  • the selection of the transconjugants of R. eutropha CH34 was carried out in plates with minimal medium M9 [Miller et al., (1992)] containing 0.2% w / v citrate and supplemented with kanamycin (1 mg / ml).
  • PVDF polyvinylidene difluoride membrane
  • an anti-mouse peroxidase IgG conjugate (0.03 U / ml; Boehringer Mannheim) was used.
  • the membrane was revealed by a chemiluminescence reaction [Veiga et al., (1999)] and was exposed to an X-ray film (X-OMAT; Kodak).
  • ELISA Enzyme-linked immunosorbent assay
  • MT ⁇ modular protein
  • MT ⁇ modular protein
  • E-tag short peptide epitope
  • E. coli cells were collected and the soluble fraction proteins, MI and ME were analyzed by SDS-PAGE, transferred and tested with the anti-E-tag monoclonal antibody.
  • a major protein of the expected size for MT ⁇ (approximately 57 kDa) was located in the ME fraction of wild-type E. coli and dsbA cells (pMT ⁇ -1). It is interesting to note that the presence or absence of DbsA did not make a significant difference in MT ⁇ stability or in its direction towards ME.
  • an ELISA was performed with intact E. coli cells, using the anti-E-tag monoclonal antibody to detect the presence of the hybrid.
  • the mtb gene was placed in a mini-Tn5 transposon (Km r ) in the direction of the Pm promoter of the plasmid p WO of Pseudomonas putida, together with the gene coding for its transcriptional activator xyls [Ramos et al., (1997 )].
  • plnMT ⁇ -1 enables the stable integration of the TnMT ⁇ -1 mini-transposon into the chromosome of a wide variety of Gram-negative bacteria and allows the induction of the expression of j ⁇ t £> by the addition of 3-methyl-benzoate (3- MB) in the growth medium ( Figure 3A).
  • the ⁇ nMT ⁇ -1 mini-transposon was inserted into the chromosome of
  • R. eutropha CH34 and MT ⁇ production was assayed by immunoblot after induction with 3-MB.
  • the production of MT ⁇ was strictly dependent on the presence of 3-MB in the growth medium, which demonstrates the strict control of the Pm promoter in R. eutropha CH34.
  • the bacterial strain of R. eutropha CH34 that carries the TnMT ⁇ -l mini-transposon stably integrated in its chromosome has been named R. eutropha MTB and has been deposited in the CECT with the deposit number CECT5323.
  • the MT ⁇ hybrid was located entirely in the ME fraction of R. eutropha MTB ( Figure 3C), which demonstrates that the hybrid protein has been correctly directed to the ME by the autotransporter domain.
  • MT ⁇ fusion occurred as an individual polypeptide band, with no indication of proteolytic instability or degradation.
  • E. coli and R. eutropha cells containing the mfcjb gene or its corresponding controls, were grown in LB medium at 301C to an OD600 nm of approximately 0.3.
  • the cells were then collected and resuspended in MJS minimum phosphate medium [12.5 mM HEPES (pH 7.1), with 50 mM NaCl, 20 mM NH 4 C1, 1 mM KC1, 1 mM MgCl 2 , MnCl 2 0.05 mM, casamino acids 0.8% w / v (Gibco BRL), glycerol 4% v / v thiamine 0.005% w / v] supplemented with appropriate concentrations of CdCl 2 and inducer (IPTG or 3-MB) and They were further incubated to an OD600 nm of approximately 1.5.
  • MJS minimum phosphate medium [12.5 mM HEPES (pH 7.1), with 50 mM Na
  • the cells were then collected, washed with 0.8% NaCl in 5 mM HEPES (pH 7.1) and dried for 20 hours at 651C.
  • the dried material was digested overnight with 70% nitric acid and Cd 2+ concentrations of the resulting solution were measured with a Hitachi Z-2800 spectrophotometer [Romeyer et al., (1988)].
  • eutropha MTB was 42 n ol of dry cell Cd 2 Vmg (Figure 2B) when it grew in the presence of 300 ⁇ M CdCl 2 . This phenomenon is produced without any significant variation in the growth rate of Ralstonia cultures (not shown) indicating that neither the production of the MT ⁇ hybrid protein nor the increase in metal adsorption had appreciable effects on the bacteria.
  • Cadmium toxicity was initially tested in N. bentamiana plants grown in soils that showed an increasing increase in CdCl 2 content (not shown). These experiments revealed that 150 ⁇ moles of the Cd salt / kg of soil significantly decreased plant growth and caused severe chlorosis, two typical features of toxicity with Cd 2+ [Ouzounidou et al., (1997)]. This metal content was used for other experiments since it still allowed a slow growth of the plant.
  • Table 1 shows the average values of 4 independent experiments on day 55 after plant germination.
  • the biomass indicates the wet weight of the aerial part of the plant, in grams.
  • the chlorophyll content is indicated as g of pigment per g of plant (wet weight). Where specified, the soil was inoculated with
  • eutropha CH34 a Gram negative strain that resides in the soil and is adapted to environments contaminated with heavy metals [Mergeay, M. (1985); Tibazarwa et al., (2000); Diels et al. (nineteen ninety five)].
  • MT metallothioneins
  • eutropha CH34 involve the active efflux of heavy metal ions, from the cytoplasm to the extracellular environment [Tibazarwa et al., (2000); Diels et al. (nineteen ninety five); Nies et al., (1989); Mergeay, M., (1991)], causes these cells to be excellent detoxifying agents when they are provided with superior adsorption properties of heavy metals on the cell surface.
  • the mtb gene was subsequently inserted into the JR chromosome.
  • Eutropha CH34 and its expression was induced by the addition of 3-MB ( Figure 3). Similar to E. coli, induced R. eutropha MTB cells produced the MT ⁇ protein and directed it towards ME. The production of the MT ⁇ protein produced a 3-fold increase in the intrinsic ability of R. eutropha CH34 cells to bind heavy metals ( Figure 2B). An accumulation of 42 nmoles of Cd / mg atoms of bacterial dry weight was determined, corresponding to a total of approximately 13 ⁇ moles of adsorbed Cd atoms / liter, in R.
  • Plasmid-determined inducible efflux is responsible for resistance to cadmium, zinc, and cobalt in Alcaligenes eutrophus. J. Bacteriol. 171, 896-900.

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Abstract

L'invention concerne une bactérie pouvant unir des métaux lourds. Cette bactérie est adaptée au sol et résistante aux métaux lourds, soit de manière naturelle, soit au moyen d'une manipulation génétique. En outre, cette bactérie a été modifiée pour exprimer sur sa surface, au moins une protéine ou une séquence peptidique pouvant unir un ou plusieurs métaux lourds. Dans un mode de réalisation, la bactérie adaptée au sol et résistante aux métaux lourds de manière naturelle est Ralstonia eutropha tandis que la protéine pouvant unir un ou plusieurs métaux lourds est une métalothionéine de souris. Ces bactéries sont utiles pour la récupération d'un milieu, solide ou liquide, contaminé par des métaux lourds.
PCT/ES2001/000214 2000-05-31 2001-05-25 Bacteries pouvant unir des metaux lourds et leur utilisation dans la detoxication de milieux contamines par des metaux lourds Ceased WO2001092471A1 (fr)

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CN107227260A (zh) * 2017-05-12 2017-10-03 广州大学 一种吸附铊离子的生物吸附剂及其制备方法与应用
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WO2006072845A3 (fr) * 2004-12-02 2007-03-01 Csir Proteine de recombinaison gram-positives pour la production de bacteries
US7888064B2 (en) 2004-12-02 2011-02-15 Csir Gram positive bacterial cells comprising a disrupted flagellin gene, flagellin-based fusion proteins and use in removal of metal ions from a liquid
CN106479914A (zh) * 2016-09-23 2017-03-08 北京林业大学 一株抗锑假单胞菌xks1及其应用
CN106479914B (zh) * 2016-09-23 2019-09-17 北京林业大学 一株抗锑假单胞菌xks1及其应用
US11939605B2 (en) 2016-12-14 2024-03-26 Wageningen Universiteit Thermostable CAS9 nucleases
US11976306B2 (en) 2016-12-14 2024-05-07 Stichting Voor De Technische Wetenschappen Thermostable CAS9 nucleases
US12071639B2 (en) * 2016-12-14 2024-08-27 Wageningen Universiteit Thermostable Cas9 nucleases
CN107227260A (zh) * 2017-05-12 2017-10-03 广州大学 一种吸附铊离子的生物吸附剂及其制备方法与应用
CN107227260B (zh) * 2017-05-12 2020-07-14 广州大学 一种吸附铊离子的生物吸附剂及其制备方法与应用
CN111019855A (zh) * 2019-11-27 2020-04-17 福建省农业科学院农业生态研究所 一种重金属抗性菌及其应用
CN111019855B (zh) * 2019-11-27 2021-12-31 福建省农业科学院农业生态研究所 一种重金属抗性菌及其应用

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