# Patent

## Title
Bacterial strains, plasmids, method of producing bacterial strains capable of chemolithotrophic arsenites oxidation and uses thereof

## Abstract

The invention provides novel strains Agrobacterium tumefaciens KKP 2039p and Paracoccus alcaliphilus KKP 2040p, the plasmid pSinA and its functional derivative, method for producing bacterial strains capable of chemolithotrophic arsenite oxidation and novel bacterial strains produced by this method. The invention also relates to the composition, comprising the novel bacterial strain or the plasmid pSinA and the use of these novel strains, as well as the method of bioaugmentation of an arsenic contaminated environment, particularly the method for the removal of arsenic from waters.

## Background
RELATED APPLICATIONS AND INCORPORATION BY REFERENCE
This application is a divisional of U.S. patent application Ser. No. 14/163,565 filed Jan. 24, 2014 which is a continuation-in-part application of international patent application Serial No. PCT/IB2013/055577 filed Jul. 8, 2013, which published as PCT Publication No. WO 2014/009867 on Jan. 16, 2014, which claims benefit of Polish patent application Serial No. P.399883 filed Jul. 10, 2012.
The foregoing applications, and all documents cited therein or during their prosecution (“appln cited documents”) and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.


FIELD OF THE INVENTION
The invention provides novel strains Agrobacterium tumefaciens KKP 2039p and Paracoccus alcaliphilus KKP 2040p, the plasmid pSinA and its functional derivative, a method for producing bacterial strains capable of chemolithotrophic arsenite oxidation, and novel bacterial strains produced by this method. The invention also provides a composition comprising a novel bacterial strain or plasmid pSinA or its functional derivative and the use of these novel strains as well as the method of bioaugmentation of an arsenic-contaminated environment, particularly the method for the removal of arsenic from waters.
BACKGROUND OF THE INVENTION
Arsenic is among the elements which are widely distributed in the Earth's crust, where it is present in trace amounts, mainly in the soil and minerals. Under the influence of natural processes and human activities, arsenic is also released to waters and air. The presence of arsenic compounds in drinking water sources poses a threat to human and animal health. The most dramatic effects of the influence of arsenic are observed in Bangladesh and in Western Bengali in India, where, according to the World Health Organization (WHO), over 50 million inhabitants are exposed to the consumption of drinking water contaminated with this toxic element.
Biological removal of arsenic from contaminated areas seems to be a necessary complement to many traditional, chemical methods of remediation. The use of such methods as coagulation or filtration is associated with the removal of not only arsenic, but also other elements present in the treated environment. Current studies on biological systems for arsenic removal, mainly focus on the use of the potential of microorganisms and plants (Kostal et al., 2004, Tripathi et al., 2007).
Effective purification of an arsenic-contaminated waters is associated with the removal of both inorganic forms of arsenic (As III and As V). While arsenates can be efficiently and selectively precipitated on strong adsorbents (Pattanayak et al., 2000), in the case of arsenites there is no possibility of using selective oxidants without side effects to the environment. Microbial oxidation of As (III) becomes therefore an alternative to chemical oxidation. Lievermont et al. (2003) proposed an efficient, low input, two-step technology for arsenic removal from waters with the use of Herminiimonas arsenicoxidans ULPAs1 bacteria. The authors have demonstrated that the strain ULPAs1, immobilised on alginate deposit, can efficiently oxidise even 100 mg/L of As (III) and may be applied in technologies for the removal of arsenic, where initial oxidation of contaminated waters is required.
The known applications of arsenite-oxidising bacteria in bioremediation processes are so far limited to laboratory studies and ex situ methods. The known ways of bioremediation of areas contaminated with arsenic by in situ methods do not fulfill their functions, because bacteria introduced into the “new” environment are not able to survive in the new conditions. This is mainly due to the existence of physico-chemical conditions other than laboratory and to the interspecific competition with the indigenous microflora. The proposed solution to this problem is the biostimulation of indigenous microflora or the use of genetically modified organisms.
Yang et al. (2010) relates to a lab constructed vector, derivative of the plasmid pBBR1MCS-5, carrying genes for the large and small subunits of arsenite oxidase. This vector contains the gene for resistance to gentamicin and its use requires an application of selection pressure of gentamicin at concentration of 60 mg/L. Because of this, an introduction of bacteria harbouring such plasmid into the environment carries the risk of dissemination of genes for gentamicin resistance, and also involves the risk of instability of such strains in the environment. The vector of Yang et al. (2010) is used for constructing strains useful in bioremediation of arsenic, but it only works when introduced into strains originally capable of arsenite oxidation, and it only increases the efficiency of the already existing process. This vector does not cause the acquisition of a new ability, which is the possibility of catalysing the oxidation reaction of As (III) to As (V).
The proposed use of genetically modified organisms involves the introduction of foreign genes carried by them, such as marker genes for antibiotic resistance or encoding the green fluorescent protein (Gfp) into the natural environment, which is unacceptable for social reasons and undesirable for environmental reasons, as well as causing the loss of plasmids in case of the absence of selection pressure for the chosen markers in the natural environment.
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.
SUMMARY OF THE INVENTION
It is desirable for the microorganisms capable of arsenite oxidation to also show resistance to the presence of other heavy metals in the environment.
Sinorhizobium sp. M14 strain was isolated from microbial mats from a gold mine in Zloty Stok (Drewniak et al., 2008). This strain can grow chemolithoautotrophically using arsenites as the source of energy and can mobilise arsenic from arsenopyrite (Drewniak et al. 2010). Strain M14 carries two megaplasmids: 109 kbp plasmid named pSinA and about 300 kbp plasmid named pSinB (Drewniak, 2009). Partial sequence of the plasmid pSinA was revealed in the GenBank NCBI database under the accession number GU990088.1 (the revealed sequence corresponded only to nucleotides 21498 to 48497 of SEQ ID NO: 1 according to the present invention).
The aim of the present invention is to overcome the indicated inconveniences and to provide novel bacterial strains, plasmids, and methods enabling the introduction of a plasmid into a bacterial strain, especially an indigenous strain, in order to produce stable, improved strains, capable of arsenite oxidation, which, are furthermore characterized by an increased resistance to other heavy metals. Such strains may be simultaneously deprived of undesirable marker genes, such as antibiotic resistance genes. The aim of the invention is also to provide novel bacterial strains capable of arsenite oxidation, but not accumulating arsenic, compositions comprising them, and their use.
The essence of this invention is thus based on an unexpected finding, that it is possible to use the natural plasmid pSinA of Sinorhizobium sp. M14 to produce stable bacterial strains of various species of bacteria, capable of arsenite oxidation, preferably not bearing any undesirable marker genes, as well as on the development of a method for producing novel bacterial strains, using strains comprising this plasmid or plasmid pSinA. Surprisingly, it has been found that plasmid pSinA introduced into bacterial strains and species other than Sinorhizobium sp. is fully functional and stably maintained in them and enables such bacteria to chemolithotrophically oxidize arsenites. Moreover, it was unexpectedly found that unlike the Sinorhizobium sp. M14 strain, the new obtained strains comprising the plasmid do not accumulate arsenic inside their cells, but allow it to be processed, leading to the obtaining of biomass free of harmful arsenic.
Accordingly, it is an object of the invention to not encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product.
It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.
These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.
DEPOSITS
The Deposits with the IAFB Collection of Industrial Microorganisms of the Institute of Agricultural and Food Biotechnology in Warsaw, Poland, under deposit accession numbers KKP2039p and KKP2040p were made pursuant to the terms of the Budapest Treaty. Upon issuance of a patent, all restrictions upon the deposit will be removed, and the deposit is intended to meet the requirements of 37 CFR §§1.801-1.809. The deposit will be maintained in the depository for a period of 30 years, or 5 years after the last request, or for the effective life of the patent, whichever is longer, and will be replaced if necessary during that period.



BRIEF DESCRIPTION OF THE DRAWINGS
The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.
FIG. 1. Shows the genetic organization of the plasmid pSinA. In the diagram, different modules of the plasmid backbone and phenotypic regions have been described: REP/STA and REP/STA2—replication-stabilization modules, TRA/TRB—conjugation module, ARS—arsenic metabolism module, HMR—arsenic resistance module, and TOXIN/ANTITOXIN module. RepABC system (replication and partitioning system—active separation) MRS system (multimer resolution system) and PHD-DOC system (addiction system—toxin/antitoxin) are located within the REP/STA module.
FIG. 2A-C. Shows a comparison of the ability to oxidise arsenites by wild-type strains (wt) Agrobacterium tumefaciens LBA288 and Paracoccus alcaliphilus JCM7364R and their derivatives Agrobacterium tumefaciens, deposited as KKP 2039p (D10), and Paracoccus alcaliphilus, deposited as KKP 2040p (C10) harbouring the plasmid pSinA. In order to compare the abilities of the investigated strains to oxidise arsenites to arsenates, cultures were carried out in minimal MSM medium containing 5 mM (375 ppm) of sodium arsenite. (A) and (B) show the content of As(III) and As(V) in culture fluids collected from the cultures every 24 hours. (A) shows a comparison of kinetics of arsenite oxidation carried out by the A. tumefaciens LBA288 strain and its derivative, the A. tumefaciens (D10) strain with pSinA; (B) shows a comparison of kinetics of arsenite oxidation carried out by the P. alcaliphilus JCM7364R strain and its derivative P. alcaliphilus (C10) with pSinA; (C) shows a comparison between the minimal inhibitory concentration (MIC) values for As(III) of the wild-type strains A. tumefaciens LBA288 and P. alcaliphilus JCM7364R, and their respective derivatives harbouring the plasmid pSinA: A. tumefaciens KKP 2039p (D10) and P. alcaliphilus KKP 2040p (C10).
FIG. 3. Shows a graph illustrating the frequency of conjugative transfer of the plasmid pSinA from the cells of the Sinorhizobium sp. M14 strain to the cells of indigenous bacteria. In the experiment, two soil samples were used: (I) coming from the Zloty Potok area and designated as ZP and (II) coming from Potok Trujaca and designated as PT. ▪—indicates the Sinorhizobium sp. M14 strain (donor of the plasmid pSinA); ▪—indigenous microflora, capable of arsenite oxidation, comprising bacteria of the genera: Brevundimonas sp., Stenotrophomonas sp., and Pseudomonas sp. for the soil I (ZP) and (ii) Achromobacter sp., Acidovorax sp., Acinetobacter sp. Brevundimonas sp., Microbacterium sp., Pseudomonas sp., and Stenotrophomonas sp. for the soil II (PT); —transconjugants harbouring the plasmid pSinA—derivatives of the indigenous bacteria, including bacteria of the genus Sinorhizobium sp. and Pseudomonas sp. for the soil I (ZP) and Brevundimonas sp., Sinorhizobium sp. and Pseudomonas sp. for the soil II (PT).
FIG. 4. Shows a comparison of the efficiency of arsenite removal out of the cell carried out by the wild-type strain Sinorhizobium sp. M14 and the newly created strains harbouring pSinA plasmid: Agrobacterium tumefaciens KKP 2039p (D10) and Paracoccus alcaliphilus KKP 2040p (C10). In order to compare the efficiency of the investigated strains to oxidize As(III) to As(V) and to remove the resulting arsenates, cultures were carried out in minimal MSM medium containing 5 mM (375 ppm) of sodium arsenite. As(V) content in culture fluids collected from the cultures every 24 hours is shown on the graph.
FIG. 5A-B. Photograph from the observations and analysis of granules of high electron density in the cells of Sinorhizobium sp. M14. A—Transmission Electron Microscopy. B—X-ray analysis.
FIG. 6. Shows a graph illustrating the frequency of conjugative transfer of the plasmid pSinA from the produced strains harbouring this plasmid: Agrobacterium tumefaciens KKP 2039p (D10) and Paracoccus alcaliphilus KKP 2040p (C10) to the cells of indigenous bacteria. In the experiment, a soil sample from the Zloty Potok area was used. The frequency of conjugal transfer was assessed after 15 days of incubation at room temperature. ▪—indicates the rate of conjugal transfer of the plasmid pSinA when Agrobacterium tumefaciens KKP 2039p (D10) was used as the donor; ▪—indicates the rate of conjugal transfer of the plasmid pSinA when Paracoccus alcaliphilus KKP 2040p (C10) was used as the donor;
FIG. 7. Shows a comparison of the efficiency of arsenite removal out of the cell carried out by wild-type strains (wt) Escherichia coli TOP10, Agrobacterium tumefaciens LBA288 and Paracoccus aminovorans JCM7685, and their derivatives Escherichia coli AIO, Agrobacterium tumefaciens AIO1 and Paracoccus aminovorans AIO2 harbouring the plasmid pAIO1. In order to compare the efficiency of the investigated strains to oxidize As(III) to As(V) and to remove the resulting arsenates, cultures were carried out in minimal MSM medium containing 2 mM (150 ppm) of sodium arsenite.
FIG. 8. Shows a comparison of MICs—minimal concentration of As(III), inhibiting the growth of the wild-type strains, and their derivatives harbouring the plasmid pARS1. In order to compare the MICs for As(III), cultures were carried out in LB medium, with various concentrations of sodium arsenite (up to 20 mM). After 48 h of cultivation at 30° C., optical density of the cultures (OD600nm measurements of absorbance at 600 nm) was monitored.



DETAILED DESCRIPTION OF THE INVENTION
The present invention concerns the novel strains Agrobacterium tumefaciens (D10) deposited under the number KKP2039p on the 30 Mar. 2012 and Paracoccus alcaliphilus (C10) deposited under the number KKP2040p on the 30 Mar. 2012 in the IAFB Collection of Industrial Microorganisms of the Institute of Agricultural and Food Biotechnology in Warsaw, Poland and functional derivatives (variants) thereof.
The term variant (derivative) of the novel strain or strains produced by the method according to the invention is to be understood a mutant strain or strain obtained by culturing the deposited strain or the strains produced by the method according to the invention as the starting material, which may comprise the plasmid pSinA shown in SEQ ID NO: 1 and is capable of chemolithotrophic arsenite oxidation.
Furthermore, the invention relates to the isolated plasmid pSinA shown in SEQ ID NO: 1 or its functional derivative.
The term ‘derivative of the plasmid’ or ‘functional derivative of the plasmid’ may comprise plasmids having a nucleotide sequence coding for open reading frames, encoding products comprising an amino-acid or a nucleotide sequence identical or highly homologous to the sequences coded by the original plasmid e.g. pSinA, wherein the coding sequences or other plasmid sequences which have been modified e.g. by substitution, replacement, deletion or insertion, such that it does not essentially alter the activity of the products of these open reading frames, and enables the maintenance of functional features carried by the original plasmid e.g. pSinA, such as the ability to chemolithotrophically oxidize arsenites and the resistance to arsenates [As(V)] and arsenites [As(III)]. A highly homologous sequence means that the sequence is homologous, preferably identical in at least 70%, preferably 80%, more preferably 90%, the most preferably, in at least 95%.
The invention relates to the use of novel strains: Agrobacterium tumefaciens KKP 2039p and Paracoccus alcaliphilus KKP 2040p, harbouring the natural plasmid pSinA of Sinorhizobium sp. M14 and the use of the plasmid pSinA of Sinorhizobium sp. M14 alone or its functional derivative, carrying: (i) all the genes necessary for chemolithoautotrophic arsenite oxidation, (ii) heavy metal resistance genes, and (iii) genes coding for the replication-stabilization system (with partitioning-active separation), multimer resolution system, and addiction toxin-antitoxin system providing stable maintenance of the plasmid in bacterial cells, for constructing bacterial strains capable of chemolithotrophic oxidation of arsenites. Such strains or the plasmid are useful in bioremediation, including the direct application in the process of bioaugmentation of the microflora of an arsenic-contaminated environments. Such strains may also be used to produce other strains capable of chemolithoautotrophic oxidation of arsenites or to improve the strains that already possess such a characteristic. The complete sequence of the plasmid pSinA of Sinorhizobium sp. M14 has been shown in SEQ ID NO: 1. The presented solution enables the construction of strains useful for the removal of arsenic from the contaminated environments, without the use of genetic manipulations and introduction of common risk genes (e.g. resistance to antibiotics) into circulation in the environment. By the invention, it is possible to introduce the plasmid pSinA to the cells of indigenous strains isolated from given environment and to construct stable strains capable of arsenite oxidation. Moreover, the invention allows for the conduction of a method for selection and monitoring of the strains harbouring the pSinA plasmid.
The invention therefore relates to the method for producing bacterial strains capable of chemolithotrophic arsenite oxidation, comprising the following steps: a) obtaining the recipient strain, and b) introduction of the plasmid pSinA, shown in SEQ ID NO: 1 or its functional derivative into the recipient strain. In the preferred method, step b) is carried out by:

    
    
        (i) triparental mating using a donor strain, containing the plasmid pSinA shown in SEQ ID NO: 1 or its functional derivative and a helper strain carrying a helper plasmid, or,
        (ii) biparental mating using a donor strain, containing the plasmid pSinA shown in SEQ ID NO: 1 or its functional derivative.
    
    


The preferred donor strain in this method is Agrobacterium tumefaciens (D10) deposited under the number KKP 2039p or Paracoccus alcaliphilus (C10) deposited under the number KKP 2040p.
In the preferred method for producing bacterial strains capable of chemolithotrophic arsenite oxidation in step a) of obtaining the recipient strain, a gene encoding an additional selection marker, preferably, coding for resistance to antibiotics, is additionally introduced into the recipient strain. More preferably, the gene coding for an additional selection marker is introduced on a plasmid, preferably by triparental mating with a bacterial strain harbouring the plasmid containing a gene coding for the additional selection marker and the helper strain, containing a helper plasmid.
In the preferred method for producing bacterial strains capable of chemolithotrophic arsenite oxidation the recipient is a bacterial strain isolated from the natural environment, preferably from an arsenic-contaminated environment, a particularly preferred recipient strain being a bacterial strain belonging to Alphaproteobacteria and Gammaproteobacteria.
The invention relates to the construction of strains capable of chemolithotrophic oxidation of As(III). By the use of the pSinA plasmid, its derivative or the strains: Agrobacterium tumefaciens KKP 2039p, Paracoccus alcaliphilus KKP 2040p, it is possible to construct bacterial strains capable of carrying out such reactions, starting from the strains which originally did not possess the entire gene apparatus, necessary for arsenite oxidation.
The invention provides for the construction of strains basing on bacteria isolated from various arsenic-contaminated environments, without limitation by the latitude. Due to the fact that the plasmid pSinA is capable of replication in bacterial cells belonging to Alphaproteobacteria and Gammaproteobacteria, it may be used in practically any environment. It is commonly known that the bacteria belonging to Alphaproteobacteria and Gammaproteobacteria are generally found in every environment studied.
The invention also relates to the composition, comprising the novel bacterial strain Agrobacterium tumefaciens KKP 2039p, Paracoccus alcaliphilus KKP 2040p, a novel bacterial strain capable of chemolithotrophic arsenite oxidation, produced by the method according to the invention or the plasmid pSinA shown in SEQ ID NO: 1, or its functional derivative.
In another aspect, the invention relates to the use of the novel bacterial strain Agrobacterium tumefaciens KKP 2039p, Paracoccus alcaliphilus KKP 2040p, a novel bacterial strain capable of chemolithotrophic arsenite oxidation, produced by the method according to the invention or the plasmid pSinA shown in SEQ ID NO: 1, or its functional derivative or a combination thereof, for constructing bacterial strains capable of chemolithotrophic arsenite oxidation.
Furthermore, the invention relates to the use of the novel bacterial strain Agrobacterium tumefaciens KKP 2039p, Paracoccus alcaliphilus KKP 2040p, a novel bacterial strain capable of chemolithotrophic arsenite oxidation, produced by the method according to the invention, the plasmid pSinA shown in SEQ ID NO: 1, or its functional derivative, the composition according to the invention, or a combination thereof, in the processes of biological removal of arsenic.
In the preferred embodiment, biological removal of arsenic may comprise bioremediation or biometallurgy of arsenic.
By “bioremediation” it is to be understood the conversion of harmful substances present in the environment to less toxic or completely safe metabolites, using microorganisms or higher organisms.
According to the invention, “bioaugmentation” means the introduction into the natural or degraded environment, of selected strains/a composition of microorganisms in order to increase the performance and capabilities of the course of a given process.
By “biometallurgy” it is to be understood the technology for metal recovery from metal ores and metal industry wastes.
In another aspect, the invention relates to the method of bioaugmentation of an arsenic-contaminated environment, which may comprise the step of introducing the novel bacterial strain Agrobacterium tumefaciens KKP 2039p, Paracoccus alcaliphilus KKP 2040p, a novel bacterial strain capable of chemolithotrophic arsenite oxidation, produced by the method according to the invention, or the plasmid pSinA, shown in SEQ ID NO: 1, or its functional derivative, the composition according to the invention or a combination thereof, into the arsenic contaminated environment.
The invention therefore relates to the method of introducing the plasmid pSinA directly into an environment as a part of bioaugmentation with the strain Agrobacterium tumefaciens KKP 2039p, Paracoccus alcaliphilus KKP 2040p, Sinorhizobium sp. M14, a bacterial strain capable of chemolithotrophic arsenite oxidation, obtained by the method according to the invention, comprising the plasmid pSinA shown in SEQ ID NO: 1, or its functional derivative, the plasmid pSinA or the composition according to the invention.
In case there is no possibility of directly constructing arsenite oxidizing strains based on the indigenous microflora, the plasmid can be introduced into the environment through the methods of bioaugmentation. A strain harbouring the plasmid pSinA or its derivative, or the composition according to the invention, is introduced into the soil and/or water contaminated with arsenic compounds and as a result of natural conjugation, the plasmid is transferred to the cells of indigenous microorganisms (autochthonous microorganisms).
The advantage of the bacterial strains comprising the plasmid pSinA, shown in SEQ ID NO: 1, or its functional derivative produced by the method according to the invention, is their stable maintenance of the plasmid introduced. Such strains are unable to get rid of it even in the absence of selection pressure i.e. in the absence of arsenic in the medium, as a result of possession of genes encoding the toxin and antitoxin system on the plasmid, providing for stable maintenance of the plasmid in bacteria. Particularly preferred in bioaugmentation, is the use of the Agrobacterium tumefaciens KKP 2039p strain, a derivative of A. tumefaciens—a bacteria recognised as environmentally safe and approved for use in soil and water environments. Moreover, an advantage of newly produced bacterial strains comprising the plasmid pSinA, like Agrobacterium tumefaciens KKP 2039p (D10), Paracoccus alcaliphilus KKP 2040p (C10), in contrast to the parental strain—Sinorhizobium sp. M14, is the ability to oxidize (up to  ̃400 mg/L) arsenites to arsenates with 100% efficiency or close to 100%, as well as the lack of accumulation of arsenic inside the cells.
The invention also relates to the method of removing or recovering arsenic through chemolithotrophic arsenite oxidation, in which the chemolithotrophic arsenite oxidation step is carried out by the novel strain Agrobacterium tumefaciens KKP 2039p, Paracoccus alcaliphilus KKP 2040p, a novel bacterial strain capable of chemolithotrophic arsenite oxidation, produced by the method according to the invention, the composition according to the invention, containing strains capable of chemolithotrophic arsenite oxidation, or a combination thereof.
In the preferred method of removing or recovering arsenic, the step of chemolithotrophic arsenite oxidation is followed by the step of arsenate removal e.g. by precipitation of the resulting arsenates in the form of an insoluble, stable precipitant or by adsorption of arsenates. For the precipitation or adsorption and effective removal of arsenates, among others, burnt lime (CaO) (Twidwell et al. 1999), calcium hydroxide Ca(OH)2 (Bothe, Brown 1999) or bog iron ores may be used.
The invention also relates to the method of selection and identification of transconjugants, obtained as the result of bi- and triparental mating, based on the phenotypic characteristics encoded by the plasmid pSinA.
In another aspect, the invention relates to a plasmid comprising the nucleotide sequence corresponding to nucleotides 24376-34453 of SEQ ID NO: 1 or its functional derivative.
Such plasmid is a derivative of the plasmid pSinA, which may comprise the nucleotide sequence corresponding to nucleotides 24376-34453 of SEQ ID NO: 1, i.e. the aio module, comprising aioXSRABmoeA genes, and may be used as a plasmid or as a sequence fragment integrated into the bacterial genome for constructing strains capable of arsenite oxidation.
The invention also relates to a bacterial strain comprising a plasmid, which may comprise the nucleotide sequence corresponding to nucleotides 24376-34453 of SEQ ID NO: 1 or its functional derivative, or a bacterial strain comprising such a nucleotide sequence, comprising the fragment 24376-34453 of SEQ ID NO: 1 or its functional derivative integrated into the bacterial genome of the strain. The strains containing the nucleotide sequence corresponding to nucleotides 24376-34453 of SEQ ID NO: 1 or its functional derivative will be capable of arsenite oxidation and/or arsenate production.
The invention also relates to the use of a plasmid comprising the nucleotide sequence corresponding to nucleotides 24376-34453 of SEQ ID NO: 1 or its functional derivative, or a bacterial strain, which may comprise the nucleotide sequence corresponding to nucleotides 24376-34453 of SEQ ID NO: 1 or its functional derivative, or a bacterial strain comprising such a nucleotide sequence, comprising the fragment 24376-34453 of SEQ ID NO: 1 or its functional derivative integrated into the bacterial genome, for arsenite oxidation and arsenate production.
In a further aspect, the invention relates to a plasmid comprising the nucleotide sequence corresponding to nucleotides 43229-50772 of SEQ ID NO: 1 or its functional derivative.
Such plasmid is a derivative of the plasmid pSinA, which may comprise the nucleotide sequence corresponding to nucleotides 43229-50772 of SEQ ID NO: 1, i.e. the ars module, comprising arsR1C1C2BtrkAmsfarsHarsR2 genes, and may be used as a plasmid or as a sequence fragment integrated into the bacterial genome, for constructing strains resistant to arsenic, both As (III) and As (V), and for increasing resistance to arsenic, particularly in relation to the original strain, into which such a sequence is to be introduced.
The invention also relates to a bacterial strain comprising a plasmid comprising the nucleotide sequence corresponding to nucleotides 43229-50772 of SEQ ID NO: 1 or its functional derivative, or a bacterial strain comprising such a nucleotide sequence, comprising the fragment 43229-50772 of SEQ ID NO: 1 or its functional derivative integrated into the bacterial genome of the strain. The strains comprising the nucleotide sequence corresponding to nucleotides 43229-50772 of SEQ ID NO: 1 or its functional derivative will have an increased resistance to arsenic and/or will acquire the resistance to arsenic, both As (III) and As (V), particularly in comparison with the original strain.
The invention also relates to the use of a plasmid comprising the nucleotide sequence corresponding to nucleotides 43229-50772 of SEQ ID NO: 1 or its functional derivative, or a strain comprising a plasmid, which may comprise the nucleotide sequence corresponding to nucleotides 43229-50772 of SEQ ID NO: 1 or its functional derivative, or a bacterial strain comprising such a nucleotide sequence, comprising the fragment 43229-50772 of SEQ ID NO: 1 or its functional derivative integrated into the bacterial genome, for producing a strain with an increased resistance to arsenic, both As (III) and As (V), particularly in comparison with the original strain.
The following examples are presented merely to illustrate the invention and to clarify its various aspects, but are not intended to be limitative, and should not be equated with all its scope, which is defined in the appended claims.
In the following examples, unless it was otherwise indicated, standard materials and methods described in Sambrook and Russell. 2001. Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, New York. were used, or the manufacturers' instructions for specific materials and methods were followed.
Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims.
The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.
EXAMPLES
Example 1
Characteristics of Plasmid pSinA and Determination of Its Complete Sequence
Plasmid pSinA, of the size of 109 kbp, was isolated from the Sinorhizobium sp. M14 strain (Drewniak et al., 2008, Drewniak et al. 2010). In order to sequence the plasmid, plasmid pSinA was isolated from 200 ml of overnight culture of Sinorhizobium sp. M14 by alkaline lysis method. Plasmid pSinA was sequenced by pyrosequencing method, using “shotgun” strategy on the GS FLX Titanium (454) sequencer (in the Oligo Pl. centre). For the construction of DNA library, approx. 5 μg of pSinA DNA was used and reagent kits provided by the manufacturer were applied (GS FLX Titanium Library Preparation Kit, Roche). The constructed library was sequenced and assembled using the software from the Newbler de novo assembler package (Roche). The obtained sequences were then assembled into contigs using Seqman software from Lasergene package (DNAStar) Annotation of the plasmid (identification of the open reading frames and determination of their potential functions) were performed using Artemis program and BLAST programs (from the NCBI database).
Sequencing of the plasmid pSinA showed that it is a DNA particle of the size of 108 938 bp and the GC-content of 59.5%. It may comprise 103 open reading frames (ORF), which constitute 89% of the sequence of the plasmid. Table 1, below, features a detailed description of the identified ORFs within SEQ ID NO: 1.







TABLE 1







Determination of the potential coding sequences of the plasmid pSinA in reference to SEQ ID NO: 1.












Coding






sequence
Protein




ORF
(start-stop
size

The greatest similarity (BLASTP program)













No
codon)*
(aa)
Predicted protein function
Identity (%)
Organism
GenBank number
















1
 189-1463
424
Replication initiation protein
90 (380/424)
Agrobacterium rhizogenes
NP_066713





(RepA)

(pRi1724)



2
1567-2571
334
Replication initiation protein
71 (236/335)
Agrobacterium rhizogenes
NP_066714





(RepB)

(pRi1724)



3
2740-3951
403
Replication initiation protein
73 (288/399)
Rhizobium etli CFN 42
YP_471771





(RepC)

(p42a)



4
 4297-5457c
386
Integrase family protein (Cre-like
82 (305/372)
Rhizobium leguminosarum
YP_770909





recombinases)

bv. trifolii WSM2304(pRL8)



5
5535-5813
92
Prevent-host-death family protein
84 (77/92) 
Rhizobium leguminosarum
YP_002279248







bv. trifolii WSM2304(pRL8)



6
5800-6231
143
Hypothetical protein
75 (107/143)
Brucella ovis ATCC 25840
YP_001257527





(PemK-like protein)





7
6467-7597
376
Protein of unknown function
67 (253/380)
Agrobacterium radiobacter K84
YP_002546559





(DUF1612)





8
7688-8281
197
Hypothetical protein
97 (179/185)
Agrobacterium tumefaciens (pTi)
NP_053264


9
8345-8731
128
Predicted nucleic acid-binding
91 (100/111)
Agrobacterium tumefaciens (pTi)
NP_053265





protein (PilT protein-like protein)





10
 8768-12582c
1271
Putative protein involved in cell
 28 (326/1181)
Rhizobium etli CIAT 652
YP_001984284





division and chromosome partitioning





11
12767-13393
208
Hypothetical protein
85 (177/209)
Rhodopseudomonas palustris BisB18
YP_532912


12
13414-14253
279
Hypothetical protein
34 (87/256) 
Bacillus thuringiensis serovar
ZP_04087111







huazhongensis BGSC 4BD1



13
14474-16219
581
Predicted ATPase (COG5293)
45 (258/585)
Nostoc sp. PCC 7120
NP_485895


14
16235-17032
265
Hypothetical protein (transposon)
80 (211/265)
Pseudomonas aeruginosa
ACD39332


15
17092-17913
273
Hypothetical protein
49 (97/202) 
Agrobacterium tumefaciens (pTi)
NP_059784


16
18097-21072
991
Hypothetical protein (ATPase
86 (837/978)
Labrenzia alexandrii DFL-11
ZP_05114452





involved in DNA repair)





17
21082-21768
228
Putative siderophore biosynthesis-
72 (131/182)
Methylobacterium extorquens DM4
YP_003066102





associated protein





18
21761-22699
312
Hypothetical protein
84 (244/293)
Methylobacterium extorquens DM4
YP_003066101


19
22735-23241
168
Hypothetical protein
68 (108/161)
Xanthobacter autotrophicus Py2
YP_001415141


20
23283-23777
164
Hypothetical protein
92 (150/164)
Oceanicola granulosus
ZP_01157550







HTCC2516



21
 23783-24139c
118
Hypothetical protein
96 (113/118)
Rhizobium etli CIAT 894
ZP_03526252


22
 24660-25877c
405
Molybdenum-biosynthesis protein
84 (338/404)
arsenite-oxidising bacterium NT-26
ABC18312





(MoeA)





23
 26035-26418c
127
c-type cytochrome c552
97 (122/127)
Agrobacterium tumefaciens
ABB51926


24
 26508-29045c
845
large subunit of Arsenite oxidase
99 (831/845)
Agrobacterium tumefaciens
ABB51928





AioB (previously AoxB))





25
 29058-29585c
175
Small subunit of Arsenite oxidase
98 (171/175)
Agrobacterium tumefaciens
ABB51929





(AioA (previously AoxA)))





26
 29723-31051c
442
Putative transcriptional regulator
97 (428/442)
Agrobacterium tumefaciens
ABB51925





(AioR (previously AoxR)))





27
 31041-32507c
488
Putative sensor histidine kinase
97 (470/488)
Agrobacterium tumefaciens
ABB51924





(AioS(previously AoxS)))





28
 32504-33424c
306
Phosphate/phosphonate ABC
57 (167/295)
Xanthobacter autotrophicus Py2
YP_001418827





transporter (AioX (previously PhnD)





29
 33604-34296c
230
Phosphate regulon transcriptional
83 (187/227)
Agrobacterium vitis S4
YP_002548344





regulatory protein (PhoB)





30
34661-35698
345
Phosphate-binding protein (PstS)
75 (229/306)
Alcaligenes faecalis
AAQ19844


31
35756-36679
307
Phosphate ABC transporter, inner
75 (227/305)
Alcaligenes faecalis
AAQ19845





membrane subunit (PstC)





32
36679-37626
315
Phosphate ABC transporter, inner
70 (212/303)
Alcaligenes faecalis
AAS45094





membrane subunit (PstA)





33
37644-38486
280
phosphate ABC transporter,
75 (195/262)
Alcaligenes faecalis
AAS45095





ATPase subunit (PstB)





34
38502-39179
231
Phosphate transport system
49 (108/224)
Pseudovibrio sp. JE062
ZP_05085295





regulatory protein (PhoU)





35
39203-39865
220
Phosphate regulon transcriptional
40 (88/225) 
Pseudovibrio sp. JE062
ZP_05085350





regulatory protein (PhoB)





36
 39872-40963c
273
Bifunctional protein: N-terminal
47 (130/280)
Nitrobacter hamburgensis
YP_571847





transcriptional regulator (ArsR) and

X14 (pPB12)






C-terminal arsenate reductase (ArsC)





37
40850-41803
317
Phosphate/phosphonate ABC
78 (232/300)
Xanthobacter autotrophicus Py2
YP_001418843





transporter (PhnD)





38
41877-42716
279
Phosphonate ABC transporter,
76 (190/251)
Roseobacter sp. AzwK-3b
ZP_01904969





ATP-binding protein (PhnC)





39
42716-43531
271
Phosphonate uptake ABC type
75 (197/266)
Fulvimarina pelagi
ZP_01438481





transporter (PhnE)

HTCC2506



40
43531-44349
272
Phosphonate uptake ABC type
70 (187/268)
Vibrio metschnikovii CIP 69.14
ZP_05881311





transporter (PhnE)





41
44496-44855
119
ArsR family transcriptional regulator
72 (80/112) 
Rhizobium etli CIAT 894
ZP_03530366


42
45085-45612
175
Tyrosine arsenate reductase (ArsC)
89 (147/166)
Rhizobium etli CIAT 894
ZP_03530368


43
45717-46151
144
Arsenate reductase (ArsC)
80 (114/143)
Sinorhizobium medicae WSM419
YP_001313767


44
46237-47307
356
Arsenite efflux transporter (AsrB)
89 (314/356)
Sinorhizobium medicae WSM419
YP_001313766


45
47324-48367
347
FAD-dependent pyridine nucleotide-
62 (206/336)
Burkholderia vietnamiensis G4
YP_001114753





disulphide oxidoreductase (TrkA)





46
 48317-49537c
406
Major facilitator superfamily
68 (272/404)
Rhizobium leguminosarum
YP_002976231





(MFS_1) protein

bv. trifolii WSM1325



47
 49534-50241c
235
NADPH-dependent FMN
84 (196/235)
Rhizobium leguminosarum
YP_768473





reductase (ArsH)

bv. viciae 3841



48
 50290-50622c
110
ArsR family transcriptional
65 (63/97) 
Agrobacterium vitis S4
YP_002547788





regulator





49
 50781-51596c
271
Putative universal stress response
34 (92/271) 
Rhizobium etli CFN 42
YP_472650





protein (UpsA)





50
 51610-52875c
421
Putative phosphopyruvate
70 (291/421)
Methylococcus capsulatus str.
YP_114366





hydratase (enolase)

Bath



51
 52931-53116c
61
Hypothetical protein
51 (30/59) 
Rhizobium leguminosarum
YP_002279234







bv. trifolii WSM2304



52
 53176-53514c
112
Hypothetical protein
92 (94/103) 
Rhizobium etli IE4771
ZP_03517489


53
 53752-55569c
605
ClC sycA-like chloride
80 (343/433)
Agrobacterium vitis S4
YP_002548815





channel protein





54
 55761-56153c
130
Hypothetical protein
48 (30/63) 
Burkholderia phytofirmans PsJN
YP_001893937


55
56215-56706
163
Putative Co/Zn/Cd efflux system
71 (109/155)
Methylobacterium nodulans
YP_002497120





component (CzcD)

ORS 2060



56
 56763-57551c
262
Predicted permease (DUF81)
76 (177/233)
Methylobacterium extorquens DM4
YP_003068171


57
 57710-58918c
402
pH-dependent sodium/proton
74 (287/392)
Rhizobium etli CFN 42
YP_468132





antiporter





58
59238-59546
102
Hypothetical protein (probable
50 (38/77) 
Agrobacterium vitis S4
YP_002542648





helicase)





59
 59529-59969c
146
MerR family transcriptional
100 (146/146) 
Ochrobactrum anthropi
YP_001371693





regulator

ATCC 49188



60
60055-60480
141
Mercuric transporter (MerT)
100 (141/141) 
Ochrobactrum anthropi
YP_001371694







ATCC 49188



61
60501-60794
97
Mercuric transport protein
100 (97/97)  
Ochrobactrum anthropi
YP_001371695





periplasmic component (MerP)

ATCC 49188



62
61040-63277
745
Mercuric reductase (MerA)
100 (745/745) 
Ochrobactrum anthropi
YP_001371697







ATCC 49188



63
 63661-65805c
714
Hypothetical protein (putative
99 (713/714)
Ochrobactrum anthropi
YP_001371699





phage integrase)

ATCC 49188



64
 65802-67610c
602
Hypothetical protein (putative
100 (602/602) 
Ochrobactrum anthropi
YP_001371700





phage integrase)

ATCC 49188



65
 67610-69049c
479
Putative XerD integrase
100 (479/479) 
Ochrobactrum anthropi
YP_001371701







ATCC 49188



66
69480-70514
344
Putative RecA relaxase
62 (209/341)
Rhizobium etli CFN 42
YP_471728


67
70577-71182
201
Protein of unknown function
73 (145/201)
Agrobacterium rhizogenes
NP_066672





(DUF1419)





68
 71278-71571c
97
Hypothetical protein
37 (35/95) 
Agrobacterium tumefaciens
NP_053284


69
71747-76921
1724
S-adenosylmethionine-dependent
 86 (1445/1687)
Rhizobium leguminosarum
YP_770997





methyltransferase

bv. viciae 3841



70
77344-79101
585
Partitioning protein ParBC
71 (408/578)
Agrobacterium rhizogenes
YP_001961038


71
79098-79991
297
Hypothetical protein
61 (171/282)
Agrobacterium rhizogenes
YP_001961040


72
79998-80299
103
Hypothetical protein
43 (38/89) 
Ochrobactrum anthropi
YP_001373171







ATCC 49188



73
80356-80589
77
Hypothetical protein
68 (27/40) 
Rhizobium etli IE4771
ZP_03514174


74
80683-81270
195
Hypothetical protein
83 (161/194)
Agrobacterium rhizogenes
YP_001961043


75
81548-82471
307
Conjugal transfer antirestriction
82 (244/300)
Rhizobium leguminosarum
YP_771003





protein (ArdC)

bv. viciae 3841












76
82799-83125
108
Hypothetical protein
No significant similarities found













77
83142-83450
102
Protein of unknown function
99 (100/102)
Sinorhizobium meliloti
YP_001965632





(DUF736)





78
83600-83938
112
Hypothetical protein
61 (69/114) 
Agrobacterium vitis S4
YP_002551439











79
84033-84302
89
Hypothetical protein
No significant similarities found













80
 84441-84893c
150
Putative nuclease
64 (96/150) 
Rhizobium leguminosarum
YP_771010







bv. viciae 3841



81
 85731-87668c
645
Conjugal transfer coupling protein
82 (508/627)
Rhizobium etli CFN 42
YP_471745





(TraG)





82
 87655-87870c
71
Conjugal transfer protein (TraD)
80 (56/70) 
Rhizobium leguminosarum
YP_771013







bv. viciae 3841



83
 87875-88171c
98
Conjugal transfer protein (TraC)
69 (67/98) 
Agrobacterium tumefaciens
BAB47248


84
88422-91745
1107
Dtr system oriT relaxase (TraA)
 78 (855/1109)
Agrobacterium tumefaciens
BAB47249


85
91742-92308
188
Conjugal transfer pilin processing
55 (102/188)
Rhizobium leguminosarum
YP_771016





protease (TraF)

bv. viciae 3841



86
92298-93464
388
Conjugal transfer protein (TraB)
62 (239/388)
Rhizobium sp. NGR234
NP_443826


87
93482-94093
203
Conjugal transfer protein (TraH)
71 (144/205)
Rhizobium leguminosarum
YP_770822







bv. viciae 3841



88
 94126-94746c
206
Hypothetical protein
71 (144/205)
Rhizobium leguminosarum
YP_770822







bv. viciae 3841



89
 94747-95502c
251
Hypothetical protein
65 (165/255)
Rhodopseudomonas palustris
ZP_06357667







DX-1



90
96193-96897
234
Putative LuxR-type transcrip-
51 (119/234)
Sinorhizobium meliloti
YP_001965652





tional regulator protein (TraR)





91
 96912-97211c
99
TraR antiactivator (TraM)
52 (51/99) 
Agrobacterium tumefaciens
NP_053353


92
97764-98483
239
AHL-dependent transcriptional
59 (134/231)
Rhizobium leguminosarum
AF210630_2





regulator similar to LuxR





93
98601-99263
220
Conjugation factor synthetase (TraI)
62 (136/221)
Rhizobium etli Brasil 5
ZP_03504772


94
 99303-100598c
431
Conjugal transfer protein (TrbI)
76 (328/432)
Sinorhizobium meliloti SM11
YP_001965654


95
 100611-101054c
147
Conjugal transfer protein (TrbH)
70 (100/143)
Rhizobium leguminosarum
YP_771025







bv. viciae 3841



96
 101058-101888c
276
Conjugal transfer protein (TrbG)
86 (237/276)
Sinorhizobium meliloti SM11
YP_001965656


97
 101904-102566c
220
Conjugal transfer protein (TrbF)
92 (201/220)
Rhizobium leguminosarum
YP_771027







bv. viciae 3841



98
 102588-103769c
393
Conjugal transfer protein (TrbL)
87 (327/380)
Rhizobium etli IE4771
ZP_03519561


99
 103944-104747c
267
Conjugal transfer/entry exclusion
87 (205/238)
Rhizobium leguminosarum
AAO21104





protein (TrbJ)

bv. viciae



100
 104740-107175c
811
Conjugal transfer protein (TrbE)
89 (719/809)
Rhizobium leguminosarum
YP_771030







bv. viciae 3841



101
 107186-107485c
99
Conjugal transfer protein (TrbD)
78 (77/99) 
Rhizobium etli CFN 42
YP_471765


102
 107478-107870c
130
Conjugal transfer protein (TrbC)
74 (97/132) 
Rhizobium leguminosarum
YP_771032







bv. viciae 3841



103
 107860-108825c
321
Conjugal transfer protein (TrbB)
90 (288/321)
Sinorhizobium meliloti SM11
YP_001965665





*The numbers in the coding sequence correspond to the nucleotide numbers in SEQ ID NO: 1.






The determined genetic organization of the plasmid pSinA has been presented in FIG. 1. The obtained, complete sequence of plasmid pSinA has been shown in SEQ ID NO: 1.
Example 2
Construction of the Agrobacterium tumefaciens and Paracoccus alcaliphilus Strains Capable of Chemolithotrophic Arsenite Oxidation
In order to demonstrate that the plasmid pSinA can be used for constructing strains capable of arsenite oxidation, the plasmid pSinA was introduced into two strains belonging to Alphaproteobacteria. For the construction, two strains have been selected: Agrobacterium tumefaciens LBA288 and Paracoccus alcaliphilus JCM7364R, incapable of arsenite oxidation and susceptible to As (III) (1 mM of sodium arsenite inhibits the growth of both strains). As a method for introducing plasmid DNA, bi- and triparental mating, described in Sambrook and Russel (2001), was used.
In order to allow the introduction of the pSinA plasmid into the selected strains, one must know their phenotypic characteristics that can be used as markers for selection, enabling the elimination of the cells of the plasmid donor. In case none of the phenotypic traits encoded by the recipient strain can be used, it should be appropriately modified (example 2A) or an appropriate method for identification of transconjugants should be applied (example 2B).
Example 2A
Construction of Strains Resistant to Tetracycline
The A. tumefaciens LBA288 strain does not carry any phenotypic characteristics that enable the use of an appropriate selection pressure to eliminate the cells of the plasmid donor. In accordance with the above, in order to establish an adequate method for selection, plasmid pBBR1MCS3 (Kovach et al., 1995), carrying a gene for tetracycline resistance, was introduced into its cells. The Sinorhizobium sp. M14 strain is susceptible to tetracycline, which allows for the removal of the cells of the donor strain in conjugation. The plasmid pBBR1MCS3 (introduced into Escherichia coli TG1 cells beforehand) was introduced into the cells of the A. tumefaciens LBA288 strain, by triparental mating, in which the pRK2013 helper plasmid (Ditta et al. 1980) (introduced into Escherichia coli TG1 cells beforehand) was used. The helper plasmid facilitates conjugation in case of strains, carrying genes responsible for the transfer only, and not for mobilization to the transfer. The conjugation was carried out according to Sambrook and Russel (2001), and for the selection of transconjugants, LB medium supplemented with tetracycline (20 μg/ml) (eliminating the cells of the recipient) and rifampicin (50 μg/ml) (eliminating the cells of the donor strain and of the strain harbouring the helper plasmid—in both cases Escherichia coli TG1) was used. The prepared donor cultures (E. coli TG1 with the plasmid pBBR1MCS3), the helper strain (E. coli TG1 with the plasmid pRK2013) and the recipient (A. tumefaciens LBA288) were mixed in a ratio 1:1:2, and then 100 μl of the mixture were plated on LB medium. After 24-hour incubation at 30° C., bacterial colonies were washed off the surface of the petri dish with 2 ml of saline solution, and appropriate dilutions (100-10−3) were plated on selective LB medium, supplemented with tetracycline and rifampicin, and then incubated for 48 h at 30° C. As a result of conjugation, transconjugants, derivatives of A. tumefaciens LBA288 harbouring the plasmid pBBR1MCS3, were obtained. For further analysis, one strain, named A. tumefaciens PBBR-Tc, was selected. The obtained strain was then used as the recipient strain in conjugation with Sinorhizobium sp. M14 strain.
Introduction of the Plasmid pSinA into the Cells of Strains Resistant to Tetracycline. Production of A. tumefaciens D10 Strain (Deposited as KKP2039p)
In order to introduce the plasmid pSinA into the cells of the A. tumefaciens PBBR-Tc strain, triparental mating was applied again (with the use of the pRK2013 helper plasmid, introduced into E. coli TG1 cells) and additionally, biparental mating. In both of these types of conjugation, the Sinorhizobium sp. M14 strain was used as the donor, capable of arsenite oxidation and resistant to As (III) (up to 20 mM) and susceptible to tetracycline. For the selection of transconjugants, LB medium (Sambrook and Russel, 2001), supplemented with 2.5 mM As(III) and tetracycline (20 μg/ml) was used. The prepared cultures of the donor (Sinorhizobium sp. M14 with the plasmid pSinA), the helper strain (E. coli TG1 with the plasmid pRK2013) (in case of triparental mating) and the recipient (A. tumefaciens PBBR-Tc) were mixed in a ratio 1:1:2, and then 100 μl of the mixture were plated on LB medium (Sambrook and Russel, 2001). After 24-hour incubation at 30° C., bacterial colonies were washed off the surface of the petri dish with 2 ml of saline solution, and dilutions (100-10−3) were plated on selective LB medium, supplemented with tetracycline and sodium arsenite, and then incubated for 48 h at 30° C. Potential transconjugants were subjected to the following analyses:

    
    
        1. physiological analysis to determine the ability to oxidize As (III) in modified MSM medium (Drewniak et al., 2008)—in order to determine the ability to oxidize As (III), potential transconjugant strains were cultivated in MSM medium supplemented with arsenites (the sole energy source) at 30° C. After 5 days of incubation under aerobic conditions, 500 μl of the culture were collected and added to 500 μl of 0.1 M solution of silver nitrate. The result of the reaction between AgNO3 and As (III) or As (V) is the formation of a coloured precipitate. A brown precipitate indicates the presence of Ag3AsO4 (silver orthoarsenate), while a yellow precipitate indicates the presence of Ag3AsO3 (silver arsenite). In case of testing for the ability to oxidise arsenites, the presence of a brown precipitate indicates that As (III) was oxidised to As (V).
        2. DNA-DNA hybridization (Southern blot)—in order to identify plasmid pSinA genes in the genomes of potential transconjugants. Fragments of the genes located on the plasmid pSinA, amplified by PCR (using the primers shown in Table 2) and labelled with digoxigenin were used as probes. Hybridization was carried out against the plasmid DNA isolated from transconjugants, obtained by alkaline lysis and visualised by DNA electrophoresis.
        3. PCR analyses—in order to identify plasmid pSinA genes in the genomes of potential transconjugants, PCR was performed using primers, described in Table 2.
        4. visualization of plasmids of potential transconjugants obtained by alkaline lysis and visualized by DNA electrophoresis.
    
    


For the hybridization analysis and PCR analysis, genes and primers presented in Table 2 were used.







TABLE 2







Sequences of the primers used in PCR amplification of plasmid pSinA


genes and chromosomal 16S rRNA genes










Gene


Position in the genome of plasmid pSinA,


name
Primer
Sequence
in reference to SEQ ID NO: 1





aoxB
aoxBF
CCACTTCTGCATCGTCGGCT
26701-26721



aoxBR
GTCGGTGTCGGATAGGCCAT
28954-28974


 


repA
repAF
CGTGCGCTATCTTCAGACGG
188-208



repAR
GCTTGAGTTCTTCGTAGTCC
1709-1729


 


traI
traIF
GTGCTCATCGGAGTGAATGG
98200-98220



traIR
GACATCAAGGATCTCGGCTA
99912-99932


 


orf12
12F
GCAATCGGTCTCACAAGAGG
12122-12142



12R
AAGGCGCACATCAGCTCGAA
14139-14159


 


16SrRNA
27F
AGAGTTTGATCMTGGCTCAG
Universal primers for amplification of



1492R
GGTTACCTTGTTACGACTT
bacterial 16S rRNA genes









In both types of conjugation (bi- and tri-parental), transconjugants harbouring the plasmid pSinA were obtained. For further analysis, the A. tumefaciens D10 strain from biparental mating (deposited as KKP2039p) was chosen. This strain was capable of arsenite oxidation and of using them as an electron donor (energy source) (FIG. 2A). In addition, this strain has increased its tolerance to As(III) (FIG. 2C). To verify whether the constructed strain stably maintains the plasmid, a series of passages (4-6 times) in media without selection pressure was performed. The obtained results showed that the constructed A. tumefaciens D10 strain stably maintains the plasmid pSinA. After about 60 generations of growth in conditions without selection pressure (without arsenic) no plasmid-less cells were observed.
Example 2B
In case we do not want to apply selection pressure associated with the use of antibiotics, there is a possibility of indirect selection of transconjugants harbouring plasmid pSinA or its derivative. For this purpose, bi- or triparental mating is carried out using minimal MSM medium as the selection medium, and sodium arsenite as the sole compound for the selection of potential transconjugants. Subsequently, an identification of approx. 100-200 randomly selected colonies of potential transconjugants is performed. Identification of the appropriate strains is performed using the analyses described in Example 2A.
Example 3
Construction of the Paracoccus alcaliphilus Strain, Capable of Chemolithotrophic Arsenite Oxidation
The strains into which the plasmid pSinA was introduced (e.g. A. tumefaciens deposited as KKP2039p (D10)) can also be used to construct further strains capable of arsenite oxidation. In order to confirm this assumption, the A. tumefaciens D10 strain was used for the transfer of the plasmid pSinA to the Paracoccus alcaliphilus JCM7364R strain (Bartosik et al., 2002). This strain is incapable of arsenite oxidation and is susceptible to As (III) (1 mM of sodium arsenite inhibits its growth). As the method for introducing plasmid DNA, biparental mating, described in Sambrook and Russel (2001) was used.
Construction of the P. alcaliphilus Strain, Resistant to Kanamycin
Because the P. alcaliphilus JCM7364R strain carries no phenotypic characteristics that allow for the application of an adequate selection pressure to eliminate the cells of the plasmid donor, genetic manipulations were performed, involving introduction of the plasmid pBBR1MCS2 (Kovach et al., 1995), carrying resistance to kanamycin, into the cells of the P. alcaliphilus JCM7364R strain. The A. tumefaciens KKP2039p (D10) strain that was used as the donor in conjugation, is susceptible to kanamycin, which allowed for the removal of the cells of the donor strain in conjugation.
The plasmid pBBR1MCS2 introduced beforehand, into Escherichia coli TG1 cells was introduced into the cells of the P. alcaliphilus JCM7364 strain using triparental mating, in which the pRK2013 helper plasmid (Ditta et al. 1980) (introduced into Escherichia coli TG1 cells, beforehand) was used. Conjugation was carried out according to Sambrook and Russel (2001), and LB medium supplemented with kanamycin (50 μg/ml), which eliminates the cells of the recipient, and with rifampicin (50 μg/ml), which allows for the elimination of the cells of the donor strain and of the strain harbouring the helper plasmid—in both cases Escherichia coli TG1, was used for the selection of transconjugants. The prepared donor cultures (E. coli TG1 with the pBBR1MCS2 plasmid), the helper strain (E. coli TG1 with the pRK2013 plasmid) and the recipient (P. alcaliphilus JCM7364R) were mixed in a ratio 1:1:2, and then 100 μl of the mixture were plated on LB medium (Sambrook and Russel, 2001). After 24-hour incubation at 30° C., bacterial colonies were washed off the surface of the petri dish with 2 ml of saline solution, and appropriate dilutions (100-10−3) were plated on selective LB medium, supplemented with kanamycin and rifampicin, and then incubated for 48 h at 30° C. As a result of conjugation, transconjugants, derivatives of P. alcaliphilus JCM7364R harbouring the plasmid pBBR1MCS2, were obtained. For further analysis, one strain, named P. alcaliphilus PBBR-Km, was selected. The obtained strain was then used as the recipient strain in conjugation with A. tumefaciens D10 (deposited as KKP 2039p).
Introduction of the Plasmid pSinA of A. tumefaciens KKP 2039p (D10) to P. alcaliphilus PBBR-Km. Production of the Paracoccus alcaliphilus KKP 2040p (C 10) Strain.
In order to introduce the pSinA plasmid into the cells of the constructed P. alcaliphilus PBBR-Km strain, triparental mating was applied (using the pRK2013 helper plasmid, introduced into E. coli TG1 cells). The A. tumefaciens D10 strain, capable of arsenite oxidation and resistant to As (III) (up to 15 mM) and susceptible to kanamycin was used as the donor. For the selection of transconjugants, LB medium supplemented with 2.5 mM As(III) and kanamycin (50 μg/ml) was used. The prepared cultures of the donor (A. tumefaciens D10 with the plasmid pSinA), the helper strain (E. coli TG1 with the pRK2013 plasmid), and the recipient (P. alcaliphilus PBBR-Km) were mixed in a ratio 1:1:2, and then 100 μl of the mixture were plated on LB medium. After 24-hour incubation at 30° C., bacterial colonies were washed off the surface of the petri dish with 2 ml of saline solution, and dilutions (100-10−3) were plated on selective LB medium, supplemented with kanamycin and sodium arsenite, and then incubated for 48 h at 30° C. Potential transconjugants were subjected to analyses analogous to those in Example 2A.
As a result of conjugation, P. alcaliphilus transconjugants harbouring the plasmid pSinA were obtained. For further analysis, the P. alcaliphilus C10 strain was chosen. This strain acquired the ability to oxidise arsenites and to use them as an electron donor (energy source) (FIG. 2B). In addition, this strain has increased its tolerance to As(III) (FIG. 2C). To verify whether the constructed strain stably maintains the plasmid, a series of passages (4-6 times) in media without selection pressure was performed. The obtained results showed that the constructed P. alcaliphilus C10 strain stably maintains the plasmid pSinA. After about 60 generations of growth in conditions without selection pressure (without arsenic) no plasmid-less cells were observed.
Example 4
Introduction of the Plasmid to the Cells of Indigenous Microflora of Arsenic Contaminated Environments by Means of Bioaugmentation with Sinorhizobium sp. M14 Strain
In order to demonstrate that the plasmid pSinA can be used in bioaugmentation of indigenous microflora of arsenic contaminated environments, an experiment was conducted on two different soil samples coming from the gold mine area in Zloty Stok. The soil designated as ZP (I) came from the vicinity of the Zloty Potok and contained from 1149.3 to 1241 mg of As/kg of soil. The soil designated as PT (II) came from the vicinity of the Potok Trujaca and contained from 528 to 532 mg of As/kg of soil. The experiment was carried out for 60 days in microcosms, supplemented with 100 g of non-sterile soil, to which the Sinorhizobium sp. M14 strain was added. The soil not enriched with the M14 strain was used as the control. At the beginning of the experiment, and every 15 days, samples of soil were collected, and the bacteria were plated on solid MSM medium (Drewniak et al., 2008) with 5 mM sodium arsenite. The grown cultures were passaged to LB medium with 5 mM As(III) and to liquid MSM medium with 5 mM of As(III). In order to verify whether the grown colonies (potential transconjugants) harbour the plasmid pSinA, the following analyses were performed: (i) physiological analysis to determine the ability to oxidize As(III) on the modified MSM medium; (ii) DNA-DNA hybridization (Southern blot) in order to identify pSinA plasmid genes in the genomes of potential transconjugants; (iii) PCR analyses, in order to identify plasmid pSinA genes in the genomes of potential transconjugants; (iv) visualization of plasmids and megaplasmids of potential transconjugants. For the hybridization analysis and PCR analysis, genes and primers presented in Table 2 were used.
After 60 days of cultivation, in both soil samples, transconjugants harbouring the plasmid pSinA were identified. Depending on the type of soil, transconjugants constituted for 25-40% of all arsenite-oxidising bacteria isolated from microcosms (FIG. 3). In Table 3 below, a list of identified strains, to which the plasmid pSinA has been introduced, has been presented.







TABLE 3







Taxonomic classification of the obtained soil


transconjugants harbouring the plasmid pSinA









Name

Similarity to the sequences


of the

deposited in the GenBank database


strain
Taxonomic group
(GenBank no) and identity[%]










Soil transconjugants harbouring the plasmid pSinA,


isolated from the soil ZP (I)









SZP1
Alphaproteobacteria
Ensifer adhaerens strain REG34




(EU647697.1) [100%]


SZP2
Alphaproteobacteria
Sinorhizobium sp. S1-2B




(AY505137.1) [99%]


SZP3
Alphaproteobacteria
Sinorhizobium sp. TB8-2




(AY505141.1) [99%]


SZP4
Gammaproteobacteria
Pseudomonas marginalis strain




LMG 2238 (HE586396.1) [97%]







Soil transconjugants harbouring the plasmid pSinA,


isolated from the soil PT (II)









SPT1
Gammaproteobacteria
Pseudomonas sp. PSA A4(4)




(DQ628969.1) [97%]


SPT2
Gammaproteobacteria
Pseudomonas jessenii strain Gd4F




(GU391474.1) [99%]


SPT3
Gammaproteobacteria
Pseudomonas sp. BIHB 813




(EF437218.1) [99%]


SPT4
Alphaproteobacteria
Brevundimonas sp. sp. CCBAU




(JF772569.1) [99%]


SPT5
Gammaproteobacteria
Pseudomonas sp. OS8




(EF491958.1) [99%]









Among the transconjugants harbouring the pSinA plasmid, there are strains classified as Alpha- and Gammaproteobacteria. All the constructed strains were capable of arsenite oxidation and of using them as an electron donor (energy source), and stably maintained the plasmid pSinA (after about 60 generations of growth in a medium without selection pressure).
The obtained results indicate the possibilities of a horizontal transfer of arsenic metabolism genes using the plasmid pSinA. This plasmid can be transferred between species belonging to Alphaproteobacteria and Gammaproteobacteria due to the presence of a broad host range replication system and conjugational transfer system. Due to the presence of a set of genes responsible for the arsenite metabolism, the strains harbouring the plasmid pSinA are characterised by high tolerance to arsenic compounds and are capable of arsenite oxidation.
Example 5
Analysis of the Accumulation of Arsenic by the Strains Harbouring the Plasmid pSinA and Oxidation Performance Analysis
Oxidation performance analysis was carried out for the Sinorhizobium sp. M14, A. tumefaciens KKP 2039p (D10) and P. alcaliphilus KKP 2040p (C10) strains. Growth experiment and the performance analysis were carried out in MSM medium, enriched with arsenites as the sole source of energy, at 22° C. for 120 hours. From culture fluids, initially containing 5 mM (375 ppm) of sodium arsenite, samples were collected every 24 hours, and As(III) and As(V) content was determined (Drewniak et al., 2008).
The performance analysis of arsenite oxidation to arsenates revealed, that the initial Sinorhizobium sp. M14 strain completely oxidizes arsenites to arsenates, which are partially removed out of the cell, and partially accumulated inside the cell. Of the initial concentration of 388 mg/L of As(III), after 120 hours of incubation, 155 mg/L of As (V) remained (FIG. 4), which, as the As (III) content was zero, indicates that part of arsenic is accumulated in/on the Sinorhizobium sp. M14 cells. In order to verify whether the Sinorhizobium sp. M14 strain accumulates arsenic, cells cultured in MSM medium supplemented with arsenites were observed under transmission electron microscope (TEM) and were subjected to X-ray analysis. It was observed that, in the M14 cells, circular granules of high electron density are present (FIG. 5). All the cells cultured in medium supplemented with arsenic contained at least two “granules” each, and more than 90% contained three to five of them. No granules were observed in the cells cultured in medium without the addition of arsenic. The conducted analysis showed that the granules present in the Sinorhizobium sp. M14 cells contain mainly arsenic, iron and molybdenum (FIG. 5).
Oxidation performance analysis of the A. tumefaciens KKP 2039p and P. alcaliphilus KKP 2040p strains showed, that both strains, after 120 hours of cultivation, completely oxidize arsenites to arsenates, all of which are removed out of the cell (FIG. 4). On the basis of the data obtained, it has been found that, unlike the Sinorhizobium sp. M14 strain, from which the plasmid pSinA originates, the Agrobacterium tumefaciens KKP 2039p, and Paracoccus alcaliphilus KKP 2040p strains, do not accumulate arsenic in their produced biomass and they show increased efficiency of oxidation of As (III) to As (V).
Example 6
Introduction of the Plasmid to the Cells of Indigenous Microflora of Arsenic Contaminated Environments by Means of Bioaugmentation with the A. tumefaciens KKP 2039p (D10) Strain
In order to demonstrate that the newly constructed A. tumefaciens KKP 2039p strain and the plasmid pSinA introduced into its cells can be used in bioaugmentation of the indigenous microflora of arsenic contaminated environments, an experiment was conducted on soil samples coming from the gold mine area in Zloty Stok, designated as ZP (I). The experiment was carried out for 15 days in 100 ml of liquid MSM medium (Drewniak et al., 2008), supplemented with 10 g of non-sterile soil, to which A. tumefaciens KKP 2039p was added. After 15 days of incubation at room temperature, samples of soil were collected and the bacteria were plated on solid MSM medium with 5 mM sodium arsenite. The grown cultures were passaged to LB medium with 5 mM As(III) and to liquid MSM medium with 5 mM of As(III). In order to verify whether the grown colonies (potential transconjugants) harbour the plasmid pSinA, their ability to oxidize As(III) was tested in modified MSM medium. All strains [the donor (A. tumefaciens KKP 2039p) and potential transconjugants] capable of arsenite oxidation were then subjected to detailed analyses: (i) verification of the presence of the plasmid pSinA through the identification of plasmid pSinA genes (aoxB, repA, traI, orf12) in the genomes of potential transconjugants using PCR; (ii) identification of the donor strain (A. tumefaciens KKP 2039p) and transconjugants, by analysis of restriction fragments of 16S rRNA genes (iii) visualization of plasmids and megaplasmids of potential transconjugants. For PCR analysis, genes and primers presented in Table 2 were used. The frequency of plasmid pSinA transfer from the cells of A. tumefaciens KKP 2039p to the cells of indigenous bacteria is shown in FIG. 6.
Example 7
Introduction of the Plasmid to the Cells of Indigenous Microflora of Arsenic Contaminated Environments by Means of Bioaugmentation with P. alcaliphilus KKP 2040p
In order to demonstrate that the newly constructed P. alcaliphilus KKP 2040p strain and the plasmid pSinA introduced into its cells can be used in bioaugmentation of the indigenous microflora of arsenic contaminated environments, an experiment was conducted on soil samples coming from the gold mine area in Zloty Stok, designated as ZP (I). The experiment was carried out for 15 days in 100 ml of liquid MSM medium (Drewniak et al., 2008), supplemented with 10 g of non-sterile soil, to which P. alcaliphilus KKP 2040p was added. After 15 days of incubation at room temperature, samples of soil were collected and the bacteria were plated on solid MSM medium with 5 mM sodium arsenite. The grown cultures were passaged to LB medium with 5 mM As(III) and to liquid MSM medium with 5 mM of As(III). In order to verify whether the grown colonies (potential transconjugants) harbour the plasmid pSinA, analyses were carried out as in Example 6. The frequency of plasmid pSinA transfer from the cells of P. alcaliphilus KKP 2040p to the cells of indigenous bacteria is shown in FIG. 6.
Example 8
Construction of the Vector Carrying a Gene Module Coding for the Proteins Involved in Arsenite Oxidation and Its Use for the Production of Strains Capable of Oxidizing Arsenites
In order to demonstrate, which genes located on plasmid pSinA (SEQ ID NO: 1) encode proteins responsible for arsenite oxidation, the aio module, comprising aioXSRABmoeA genes, was cloned in the vector pBBR1-MCS2 (Kmr), in the Escherichia coli TOP10 strain, and then its functionality was tested.
In order to clone the aio module, amplification of a DNA fragment of the size 10077 by (comprising the region from position 24376 to 34453 in the genome of pSinA) was performed on a DNA template of the plasmid pSinA, isolated by alkaline lysis. For PCR reaction, the following oligonucleotides were used as primers:

    
    
        AIOf_XbaI: ggtggcCAGCGGCTTCACACATAGTCCCCAG [position in the genome of plasmid pSinA: 24376-24400; the underlined sequence is the restriction site recognized by the enzyme XbaI (TCTAGA)], and
        AIOr_Bsu15: ggtGCACCCACGATGGCGAGAG [position in the genome of plasmid pSinA: 34430-34453; the underlined sequence is the restriction site recognized by the enzyme BsuRI (ClaI) (ATCGAT)] For the amplification, Phusion® High-Fidelity DNA Polymerase (Thermo Scientific) was used.
    
    


The obtained PCR product (10077 bp) was cloned into a plasmid vector: pBBR1MCS-2 (Kmr) (Kovach et al., 1995) digested (linearized) with SmaI. The ligation mixture of the PCR product and the vector pBBR1MCS2 digested with the enzyme SmaI (CCC↓GGG) was introduced, by means of chemical transformation, using the calcium-rubidium method according to Kushner (1978), into the cells of Escherichia coli Top10 strain [mcrA Δmrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL endA1 nupG]. As the selection medium, complete LB medium with kanamycin (30 μg/ml), IPTG (0.5 μg), and X-gal (40 μg/ml) was used.
From the pool of the obtained transformants (white colonies resistant to kanamycin) strains that were harbouring a plasmid of the appropriate size: 15221 bp [pBBR1MCS2 (5144 bp)+aio module−(10077 bp)] were selected. The presence of the constructed plasmid was confirmed by electrophoretic analysis and sequencing. The Escherichia coli AIO strain (derivative of the E. coli TOP10 strain), harbouring the plasmid pAIO1 (derivative of pBBR1MCS2 with cloned aio module), was selected for further analysis.
In order to demonstrate that the constructed plasmid pAIO1 can be used for constructing strains capable of arsenite oxidation, the plasmid pAIO1 was introduced into 5 strains belonging to Alphaproteobacteria, Betatproteobacteria and Gammaproteobacteria. For the construction, the following strains were selected:

    
    
        (i) Agrobacterium tumefaciens LBA288 and Paracoccus aminovorans JCM7685 (Alphaproteobacteria) as well as Stenotrophomonas sp. LM24R (Gammaproteobacteria) incapable of arsenite oxidation and susceptible to As (III) (1 mM of sodium arsenite inhibits the growth of these strains),
        (ii) Brevundimonas sp. OS24R (Alphaproteobacteria) and Pseudomonas sp. OS29R (Gammaproteobacteria) incapable of arsenite oxidation, but resistant to As (III).
    
    


As the method for introducing plasmid DNA, triparental mating, described in Sambrook and Russel, 2001, was used. The E. coli AIO strain, harbouring the plasmid pAIO1, carrying the genes for arsenite oxidase and determining kanamycin resistance, was used as the donor. The prepared cultures of the donor (E. coli AIO with the plasmid pAIO1), the helper strain (E. coli TG1 with the plasmid pRK2013) and the recipient (A. tumefaciens LBA288, P. aminovorans JCM7685, Stenotrophomonas sp. LM24R, Brevundimonas sp. OS24R, and Pseudomonas sp. OS29R) were mixed in a ratio 1:1:2, and then 100 μl of the mixture were plated on LB. After 24-hour incubation at 30° C., bacterial colonies were washed off the surface of the petri dish with 2 ml of saline solution, and appropriate dilutions (100-10−3) were plated on selective LB medium, supplemented with kanamycin (50 μg/ml), which eliminates the cells of the recipient, and rifampicin (50 μg/ml), which allows for the elimination of the cells of the donor strain and of the strain harbouring the helper plasmid. They were subsequently incubated for 48 h at 30° C. Potential transconjugants were subjected to the following analyses:

    
    
        (i) verification of the restriction pattern of 16S rRNA genes [isolation of DNA, amplification of 16S rRNA genes using primers 27F and 1492R (Lane, 1991), digestion with the restriction enzyme HaeIII, DNA electrophoresis],
        (ii) analysis for the presence of the plasmid pAIO1 in the transconjugant cells (alkaline lysis and visualization during DNA electrophoresis),
        (iii) PCR analysis for the presence of arsenite oxidase genes (DNA amplification using primers aoxBF and aoxBR, and electrophoretic analysis of DNA),
        (iv) physiological analysis to determine the ability to oxidize As (III) in modified MSM medium (Drewniak et al., 2008) according to the description presented in Example 2A.
    
    


In all the conjugations, transconjugants harbouring the plasmid pAIO1 were obtained. Physiological analysis with the AgNO3 test revealed that all derivatives of the wild-type strains, previously incapable of arsenite oxidation, acquired the ability to oxidize arsenites with the introduction of the plasmid pAIO1 [all strains oxidized As(III) to As(V) and a brown precipitate formed in the reaction with AgNO3].
In order to confirm that the newly constructed strains, harbouring the plasmid pAIO1, are capable of arsenite oxidation, an analysis of As(III) oxidation efficiency was carried out, on the example of Agrobacterium tumefaciens AIO1 (derivative of A. tumefaciens LBA288 harbouring the plasmid pAIO1) and Paracoccus aminovorans AIO2 (derivative of P. aminovorans JCM7685 harbouring the plasmid pAIO1). Wild-type strains were used as the control. The growth experiment and the performance analysis were carried out in MSM medium, enriched with arsenites as the sole source of energy, and with 0.004% yeast extract as the source of vitamins, at 30° C. for 96 hours. From culture fluids, initially containing 2 mM (150 ppm) of sodium arsenite, samples were collected every 24 hours, and As(III) and As(V) content was determined (Drewniak et al., 2008).
The performance analysis of oxidation of As(III) to As(V) (FIG. 7) revealed, that the strains harbouring the plasmid pAIO1 are capable of complete arsenite oxidation and production of arsenates already within 72 hours from the beginning of the culture. On the other hand, wild-type strains deprived of the plasmid pAIO1 are not capable of growth and arsenite oxidation, and thus, of producing arsenates.
The conducted experiments made it possible to confirm that the derivative of the plasmid pSinA, comprising the aio module (sequence from 24376 to 34453) can be used for constructing strains capable of arsenite oxidation.
Example 9
Construction of a Vector Carrying the Gene Module Coding for the Proteins Involved in Resistance to As (III) and Its Use for the Production of Strains Resistant to Arsenic
In order to demonstrate, which genes located on the plasmid pSinA (SEQ ID NO: 1) encode proteins responsible for the resistance to arsenites, the ars module, comprising arsR1C1C2BtrkAmsfarsHarsR2 genes, was cloned in the vector pBBR1-MCS2 (Kmr), in the Escherichia coli TOP10 strain, and then its functionality was tested.
In order to clone the ars module, amplification of a DNA fragment of the size 7544 by (comprising the region from position 43229 to 50772 in the genome of pSinA) was performed on a DNA template of the plasmid pSinA, isolated by alkaline lysis. For PCR reaction, the following oligonucleotides were used as primers:

    
    
        ArsF_Bsu15: ggtggtGAAAAGCAGGCAGAGGCC [position in the genome of the plasmid pSinA: 43229-43523; the underlined sequence is the restriction site recognized by BsuRI (ClaI) (ATCGAT), that is present in the sequence of plasmid pSinA, while the sequence not present in plasmid pSinA was indicated by lower-case letters], and
        ArsR_Xba: gttACACTTCTTGACGTAGCCGCAACTAACTC [position in the genome of plasmid pSinA: 50744-50772; the underlined sequence is the restriction site recognized by the enzyme XbaI (TCTAGA)] For the amplification, Phusion® High-Fidelity DNA Polymerase (Thermo Scientific) was used.
    
    


The obtained PCR product (7544 bp) was digested with the enzymes Bsu15I and XbaI, and subsequently, was cloned into the vector pBluescriptKSII(+) (Stratagene) previously cleaved with the restriction enzymes Bsu15I and XbaI. The ligation mixture of the PCR product and the vector pBluescriptKSII(+) was introduced, by means of chemical transformation, using the calcium-rubidium method according to Kushner (1978), into the cells of Escherichia coli TOP10F′ strain: F′ {lacIqTn10(TetR)} mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL endA1 nupG. As the selection medium, complete LB medium with ampicillin (150 μg/ml), IPTG (0.5 μg), X-gal (40 μg/ml) was used. From the pool of the obtained transformants (white colonies resistant to ampicillin), the strains that were harbouring a plasmid of the appropriate size: 10456 bp (pBluescriptKSII(+)−2912+ars module−7544 bp) were selected. The presence of the constructed plasmid was confirmed by electrophoretic analysis and sequencing. The Escherichia coli ARS1 strain (derivative of E. coli TOP10F′ strain) harbouring the plasmid pKS_Ars (derivative of pBluescriptKSII with cloned ars module), was selected for further analysis.
As the use of the plasmid pBluescriptKSII is limited to the strains of Escherichia coli as the only host, ars module was cloned into the broad-host-range plasmid pCM62, carrying resistance to tetracycline (Marx and Lindstrom, 2001). For this purpose, the plasmid pKS_Ars (isolated from Escherichia coli ARS1 by alkaline lysis) was digested with the restriction enzymes VspI, XbaI. Subsequently, the obtained DNA fragment of the size of 7742 bp, containing the module ars, was cloned into the vector pCM62 previously digested with the enzymes VspI and XbaI. The ligation mixture of the DNA fragment of the plasmid pKS-Ars (containing the ars module) and the vector pCM62 was introduced, by means of chemical transformation, into the cells of Escherichia coli TOP10F strain [F-mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZAM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ−]. As the selection medium, complete LB medium with tetracycline (10 μg/ml) was used. From the pool of the obtained transformants (colonies resistant to tetracycline), the strains that were harbouring a plasmid of the appropriate size: 14407 bp (pCM62−6863 bp+ars module−7544 bp) were selected. The presence of the constructed plasmid was confirmed by electrophoretic analysis and sequencing. The Escherichia coli ARS2 strain (derivative of E. coli TOP10F strain) harbouring the plasmid pARS1 (derivative of pCM62 with cloned ars module), was selected for further analysis.
In order to demonstrate that the constructed plasmid pARS1 can be used for constructing strains with increased resistance to arsenic, the plasmid pARS1 was introduced into Agrobacterium tumefaciens LBA288, susceptible to As (III) (1 mM of sodium arsenite inhibits the growth of LBA2888 cells), Paracoccus aminophilus JCM7686 showing low resistance to As(III) (5 mM of sodium arsenite inhibits the growth of the cells of the JCM7686 strain), and Brevundimonas sp. OS24R showing high resistance to As (III) (10 mM of sodium arsenite inhibits the growth of OS24R cells). As the method for introducing plasmid DNA, triparental mating, described in Sambrook and Russel, 2001, was used. The E. coli ARS1 strain, which harbours the plasmid pARS1, carrying the genes for arsenite oxidase and determining resistance to tetracycline, was used as the donor. The prepared cultures of the donor (E. coli ARS1 with the plasmid pARS1), the helper strain (E. coli TG1 with the plasmid pRK2013) and the recipient (Agrobacterium tumefaciens LBA288, Brevundimonas sp. OS24R, P. aminophilus JCM7686) were mixed in a ratio 1:1:2, and then 100 μl of the mixture were plated on LB. After 24-hour incubation at 30° C., bacterial colonies were washed off the surface of the petri dish with 2 ml of saline solution, and appropriate dilutions (100-10−3) were plated on selective LB medium, supplemented with tetracycline (10 μg/ml), which eliminates the cells of the recipient, and rifampicin (50 μg/ml), which allows for the elimination of the cells of the donor strain and of the strain harbouring the helper plasmid. They were subsequently incubated for 48 h at 30° C. Potential transconjugants were subjected to the following analyses:

    
    
        (i) verification of the restriction pattern of 16S rRNA genes [isolation of DNA, amplification of 16S rRNA genes using primers 27F and 1492R (Lane, 1991), digestion with the restriction enzyme HaeIII, DNA electrophoresis],
        (ii) analysis for the presence of the plasmid pARS1 in the transconjugant cells (alkaline lysis and visualization during DNA electrophoresis),
        (iii) PCR analysis for the presence of cytoplasmic arsenate reductase genes [DNA amplification using primers ParsH-L (TGACGTAGCCGCAACTAACT-position in the genome of pSinA:50745-50764) and ParsH-P (TGGCTTGTGCTGCGAATAAG-position in the genome of pSinA: 50155-50174) and electrophoretic analysis of DNA].
    
    


In all the conjugations, transconjugants harbouring the plasmid pARS1 were obtained. To confirm that the newly constructed strains, harbouring the plasmid pARS1, have an increased resistance to arsenic, analysis of MIC—minimal concentration of As(III), inhibiting the growth of the following strains: Agrobacterium tumefaciens ARS3 (derivative of A. tumefaciens LBA288 harbouring the plasmid pARS1), Brevundimonas sp. ARS4 (derivative of Brevundimonas sp. sp. OS24R harbouring the plasmid pARS1), P. aminophilus ARS5 (derivative of P. aminophilus JCM7686 harbouring the plasmid pARS1) was carried out. Wild-type strains were used as the control. Growth experiment and MIC analysis for As(III) was carried out in LB medium, with various concentrations of sodium arsenite (up to 20 mM). After 48 h of cultivation at 30° C., optical density of cultures at OD600nm was monitored. The conducted analysis revealed that all the investigated strains harbouring the plasmid pARS1 increased their tolerance to the presence of sodium arsenite, in relation to their related wild-type strains (FIG. 8). MIC for As(III) for A. tumefaciens LBA288 strain was 1 mM, while for its derivative, comprising the plasmid pARS1, 20 mM; for P. aminophilus JCM7686R strain 5 mM, while for its derivative, containing the plasmid pARS1, 20 mM. In turn Brevundimonas sp. OS24R could tolerate the maximum of 10 mM of As(III), and its derivative harbouring the plasmid pARS1 was resistant to 20 mM of As(III). The conducted analyses allowed to confirm that the derivative of the plasmid pSinA, comprising the ars module (sequence from 43229 to 50772) can be used for constructing strains with increased resistance to arsenic.
In the presented embodiments, the inventors have demonstrated the possibility of using a natural, genetically unmodified plasmid pSinA of its functional derivatives for constructing strains capable of arsenite oxidation, particularly preferably strains not accumulating arsenic compounds. Novel strains were produced: Agrobacterium tumefaciens (D10), deposited under the number KKP 2039p and Paracoccus alcaliphilus (C10) deposited under the number KKP 2040p, which do not accumulate arsenic, and do not store it in their produced biomass, and are characterized by an increased efficiency of oxidation of As (III) to As (V). It was unexpectedly found, that the use of the pSinA plasmid or its functional derivatives is not limited to strains originally capable of arsenite oxidation. Strains that are completely incapable of arsenite oxidation, acquire this ability with the acquisition of the pSinA plasmid. Introduction of the plasmid pSinA into the cells of the host, ensures their acquisition of resistance to arsenites and arsenates, as well as to other heavy metals.
Moreover, it was demonstrated, that the application of the Agrobacterium tumefaciens KKP 2039p, Paracoccus alcaliphilus KKP 2040p or Sinorhizobium sp. M14 strains, and other strains harbouring the plasmid pSinA, in the removal of arsenic by in situ methods and based on oxidation of As (III) to As (V), ensures the stability of this process. If the introduced strains will not be able to survive in the new conditions, then through the horizontal gene transfer they will pass the plasmid to the cells of indigenous microflora, and this, in turn, will ensure their capability of arsenite oxidation in a specific environment.
It was also demonstrated that the nucleotide sequence corresponding to nucleotides 24376-34453 in SEQ ID NO: 1 or its functional derivative, which contains the aio module of pSinA, gives the bacterial strains, to which it was introduced, the ability to oxidize arsenites and/or produce arsenates, and therefore this derivative of the plasmid pSinA can be used for producing bacterial strains, which after the introduction of such a sequence acquire the ability to oxidize arsenites and/or produce arsenates and can be used in applications that require such strains.
It was also demonstrated that the nucleotide sequence corresponding to nucleotides 43229-50772 in SEQ ID NO: 1 or its functional derivative, which contains the ars module of pSinA, gives the bacterial strains, to which it was introduced, an increased resistance to arsenic, and therefore this derivative of the plasmid pSinA can be used for producing bacterial strains, which after the introduction of such a sequence increase their resistance to arsenic and can be used in applications that require such strains.
LITERATURE CITED IN THE DESCRIPTION, INCLUDED HEREIN AS REFERENCES


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The invention is further described by the following numbered paragraphs:

    
    
        1. A novel strain Agrobacterium tumefaciens deposited in the IAFB Collection of Industrial Microorganisms of Institute of Agricultural and Food Industry under the number KKP 2039p.
        2. A novel strain Paracoccus alcaliphilus deposited in the The IAFB Collection of Industrial Microorganisms of Institute of Agricultural and Food Industry under the number KKP 2040p.
        3. A plasmid pSinA shown in SEQ ID NO: 1 and its functional derivative.
        4. A method for producing bacterial strains capable of chemolithotrophic arsenite oxidation, comprising the following steps:
        
            a) obtaining the recipient strain;
            b) introduction of the plasmid pSinA, shown in SEQ ID NO: 1, or its functional derivative into the recipient strain.
        
        
        5. The method according to paragraph 4, characterized in that, the step b) is carried out by:
        
            (i) triparental mating with the use of a donor strain harbouring the plasmid pSinA, shown in SEQ ID NO: 1 or its functional derivative, and a helper strain harbouring a helper plasmid, or,
            (ii) biparental mating with the use of a donor strain harbouring the plasmid pSinA shown in SEQ ID NO: 1 or its functional derivative.
        
        
        6. The method according to paragraph 4 or 5, characterised in that the donor strain is Agrobacterium tumefaciens deposited under the number KKP 2039p or Paracoccus alcaliphilus deposited under the number KKP 2040p.
        7. The method for producing bacterial strains according to paragraph 4, wherein in the step a) of the obtaining the recipient strain, a gene encoding a selection marker is additionally introduced into the recipient strain, preferably encoding antibiotic resistance.
        8. The method for producing bacterial strains according to paragraph 7, wherein the gene encoding the additional selection marker is introduced on a plasmid, preferably by triparental mating with a bacterial strain harbouring the plasmid containing the gene encoding the additional selection marker and with a helper strain harbouring a helper plasmid.
        9. The method according to claims 4-8, wherein the recipient strain is a bacterial strain isolated from natural environment, preferably from arsenic contaminated environment.
        10. The method according to claims 4-9, wherein the recipient strain is a bacterial strain belonging to Alphaproteobacteria or Gammaproteobacteria.
        11. A novel bacterial strain capable of chemolithotrophic arsenite oxidation, produced by the method according to claims 4-10.
        12. A composition comprising the novel bacterial strain according to claims 1-2, the novel bacterial strain according to paragraph 11, the plasmid according to paragraph 3 or combination thereof.
        13. Use of the novel bacterial strain according to claims 1-2, the novel bacterial strain according to paragraph 11, the plasmid according to paragraph 3, the composition according to paragraph 12, or combination thereof, for constructing bacterial strains capable of chemolithotrophic arsenite oxidation.
        14. Use of the novel strain according to claims 1-2, the novel bacterial strain according to paragraph 11, the plasmid according to paragraph 3, the composition according to paragraph 12, or combination thereof, in the processes of biological removal of arsenic.
        15. The use according to paragraph 14, characterised in that, the biological removal of arsenic comprises bioremediation, preferably bioaugmentation or biometallurgy of arsenic.
        16. A method of bioaugmentation of arsenic contaminated environment, comprising the step of introducing the novel strain according to claim 1-2 or 11, the plasmid according to paragraph 3, the composition according to paragraph 12, or combination thereof, into an arsenic contaminated environment.
        17. A method for the removal or recovery of arsenic by chemolithotrophic arsenite oxidation, wherein the step of chemolithotrophic arsenite oxidation is carried out by the novel strain defined in claims 1-2, the novel strain defined in paragraph 11, the composition defined in paragraph 12, or combination thereof.
        18. The method for the removal or recovery of arsenic according to paragraph 17, wherein the step of chemolithotrophic arsenite oxidation is followed by precipitation of the resulting arsenates in the form of insoluble precipitate and/or adsorption of arsenates, wherein the precipitation or adsorption is preferably carried out using burnt lime (CaO), calcium hydroxide Ca(OH)2, bog iron ores or combination thereof.
        19. A plasmid comprising the nucleotide sequence corresponding to nucleotides 24376-34453 in SEQ ID NO: 1 or a functional derivative thereof.
        20. A bacterial strain comprising the plasmid defined in paragraph 19 or the nucleotide sequence comprising the fragment 24376-34453 of SEQ ID NO: 1 or a functional derivative thereof.
        21. Use of the plasmid defined in paragraph 19 or the strain defined in paragraph 20 for arsenite oxidation.
        22. A plasmid comprising the nucleotide sequence corresponding to nucleotides 43229-50772 in SEQ ID NO: 1 or a functional derivative thereof.
        23. A bacterial strain comprising the plasmid defined in paragraph 22 or the nucleotide sequence comprising the fragment 43229-50772 of SEQ ID NO: 1 or a functional derivative thereof.
    
    


24. Use of the plasmid defined in paragraph 22 or the nucleotide sequence comprising the fragment 43229-50772 of SEQ ID NO: 1 or a functional derivative thereof for the production of a strain with increased resistance to arsenic.
Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

## Claims

1. An isolated or novel bacteria strain Agrobacterium tumefaciens deposited in the IAFB Collection of Industrial Microorganisms of Institute of Agricultural and Food Industry under the number KKP 2039p.

2. A composition comprising the novel bacterial strain according to claim 1.

3. An isolated or novel bacteria strain Paracoccus alcaliphilus deposited in the The IAFB Collection of Industrial Microorganisms of Institute of Agricultural and Food Industry under the number KKP 2040p.

4. A composition comprising the novel bacterial strain according to claim 3.

5. An isolated, novel non-naturally occurring bacterial strain capable of chemolithotrophic arsenite oxidation, produced by a method for producing a bacterial strain capable of chemolithotrophic arsenite oxidation, comprising introducing a novel or isolated or non-naturally occurring plasmid pSinA having a nucleotide sequence shown in SEQ ID NO: 1 into the bacterial strain.

6. An isolated, novel non-naturally occurring bacterial strain capable of chemolithotrophic arsenite oxidation according to claim 5, wherein the introducing is carried out by a process comprising:
(i) triparental mating with the use of a donor strain harbouring the plasmid, and a helper strain harbouring a helper plasmid, or,
(ii) biparental mating with the use of a donor strain harbouring the plasmid.

7. An isolated, novel non-naturally occurring bacterial strain capable of chemolithotrophic arsenite oxidation according to claim 6 wherein the donor strain is Agrobacterium tumefaciens deposited under the number KKP 2039p or Paracoccus alcaliphilus deposited under the number KKP 2040p.

8. An isolated, novel non-naturally occurring bacterial strain capable of chemolithotrophic arsenite oxidation according to claim 5, for producing a bacterial strain capable of chemolithotrophic arsenite oxidation, wherein the method of producing includes introducing a gene encoding a selection marker into the bacterial strain.

9. An isolated, novel non-naturally occurring bacterial strain capable of chemolithotrophic arsenite oxidation of claim 8 wherein the selection marker comprises antibiotic resistance.

10. An isolated, novel non-naturally occurring bacterial strain capable of chemolithotrophic arsenite oxidation according to claim 8, wherein the introducing is by a plasmid.

11. An isolated, novel non-naturally occurring bacterial strain capable of chemolithotrophic arsenite oxidation according to claim 10 wherein the Previously Presented plasmid is introduced by triparental mating including a bacterial strain harbouring the plasmid containing the gene encoding the selection marker and a helper strain harbouring a helper plasmid.

12. An isolated, novel non-naturally occurring bacterial strain capable of chemolithotrophic arsenite oxidation according to claim 5 wherein the bacterial strain into which a novel or isolated or non-naturally occurring plasmid pSinA having a nucleotide sequence shown in SEQ ID NO: 1 is introduced is the strain isolated from an arsenic contaminated environment.

13. An isolated, novel non-naturally occurring bacterial strain capable of chemolithotrophic arsenite oxidation according to claim 5, wherein the bacterial strain is an Alphaproteobacteria or Gammaproteobacteria bacterial strain.

14. A composition comprising the isolated, novel, non-naturally occurring bacterial strain according to any one of claim 6-11 or 5, 12, 13.

15. A composition comprising a novel, non-naturally occurring, isolated bacteria containing a novel or isolated or non-naturally occurring plasmid pSinA having a nucleotide sequence shown in SEQ ID NO: 1 introduced into the bacterial strain.