AU2008201183A1 - Array of nucleotidic sequences for the detection and identification of genes that codify proteins with activities relevant in biotechnology present in a microbiological sample, and method for using this array - Google Patents

Description

P/00/011 Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Array of nucleotidic sequences for the detection and identification of genes that codify proteins with activities relevant in biotechnology present in a microbiological sample, and method for using this array The following statement is a full description of this invention, including the best method of performing it known to us: 00 O ARRAY OF NUCLEOTIDIC SEQUENCES FOR THE DETECTION AND C IDENTIFICATION OF GENES THAT CODIFY PROTEINS WITH ACTIVITIES RELEVANT IN BIOTECHNOLOGY PRESENT IN A MICROBIOLOGICAL SAMPLE, AND METHOD FOR USING THIS ARRAY Scope of the Invention 00 present invention makes known an array of nucleotidic sequences for rapidly c and simultaneously identifying the presence of certain genes that codify proteins 00 oO Swith relevant activities in biotechnology present in a microbiological sample, and Sthe method with which this array is used in the identification of the above mentioned genes. Specifically speaking, genes that codify for proteins relevant in the biofilm formation, in CO2 fixation, in energetic metabolism, for chemiotaxis and mobility, in iron oxidizing, in nitrogen metabolism, in sulphur assimilation, and in oxidation/reduction of sulphide compounds, are identified. This array of nucleotidic sequences is presented as a useful tool in biotechnology whenever evaluating the quality of a microbiological community is required.

Background Information of the Invention Biotechnology is, generally speaking, any technological application that uses biological systems (such as a complex microbiological community), living organisms (microorganisms, animals or plants) or their derivates (metabolites, proteins, nucleic acids) in the creation or modification of products or processes for specific uses. In this invention, we will focus on the biotechnology that employs microorganisms, especially biomining and microbiological bioremediation.

Many processes of microbiological biotechnology are carried out strengthening the development of the microbiological community originally present in the substrate to be treated, for instance the community present in ore in the case of biomining, or present in contaminated soil or water in the case of bioremediation. In these 005099352 00 0 cases having a tool that that would allow us to evaluate the metabolic characteristics of the community present, would be of great use, for example, in judging whether the process we are interested in can be carried out with the community present, or if we should inoculate other adequate strains.

Up to now, the most common approach used in evaluating microbiological 00 communities has been the identification of species present, by means of a variety of techniques such as selective cultures, fluorescent in situ hybridization (FISH),

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N conjugated with specific markers such as detection probes, polymerase chain 00 Sreaction (PCR) and macro and micro DNA arrays. Among the previously C mentioned techniques, all the ones pertaining to molecular biology, that is to say FISH, detection probes, PCR, and macro and micro DNA arrays, are based on hybridizing the DNA of the unknown sample with known sequences that are specific for the microorganism or microorganisms that need to be detected or identified, for example, a certain specie or genus.

The problem with the previous approach is that there are many types of different microorganisms that may be present in these microbiological communities, so that many tests should be carried out to establish their composition, and then correlate these acquired data with the characteristics of each identified taxon, to finally establish whether the function of interest is present in the sample. If there were a microorganism not previously described, this correlation could not be carried out, even if it's metabolic activities were known, and even important for the process.

So it rises the need for a method oriented for directly evaluating and controlling the metabolic characteristics of a microbiological community, and not for identifying what microorganisms it is composed of.

This technical problem has been resolved by means of the present invention, by creating an array of nucleotidic sequences for detecting and identifying genes that codify proteins with relevant activities in biotechnology present in a microbiological sample, and a Method for employing this array.

005099352 00 O When changing the focus of the approach from the taxonomy of the studied Scommunity to the identification of proteins with relevant activities present in this community, we focus directly on the essential of the biotechnological process: the metabolic functions which ultimately carry out these processes. For example, each time the presence of Leptospirillum spp. is sought for in a bioleaching process, what really is being sought for is iron oxidization, and it is so for each of the 00 microorganisms that are relevant in some biotechnological process.

c The new approach, designed by us, makes it possible to respond to exactly what oo 00 Sthe operator of a biotechnological process requires: Does this community oxidize CI iron?, does it oxidize sulfur?, does it fix 00 2 does it fix nitrogen? With the approach that currently exists in the technique, the operator first knows what species are present, and then he or she must correlate this information with the characteristics of each of these species in order to answer the same questions.

In each case, we have designed fragments of DNA for identifying the genes that codify proteins with relevant activities in biotechnology that comply with an essential condition: the specificity of the mentioned fragment must correspond solely to the target gene. On the other hand, whenever it has been possible, regions have been sought which being specific for each target gene, and orthologically conserved. This is with the idea of detecting not only the genes that have been sequenced, but also genes that have not been described, that are orthologues of each target gene. The result of this search corresponds to fragments that are specific for a certain gene or function within a specific taxon in a specific region.

With these DNA fragments designed we have developed DNA fragment arrays which allow us to carry out the identification of the presence of these genes that codify proteins with relevant activities in biotechnology, which are present in a microbiological sample.

005099352 00 O A good definition of DNA arrays is the one proposed by Schena and colleagues: "a microscopic and methodical arrangement of nucleic acids that allow simultaneous analysis of complex DNA samples" (Schena Heller, Theriault, Konrad, Lachenmeier, E. and Davis, R.W. Trends Biotechnol. 16, 301-306, 1998).

Depending on the diameter of the printed DNA dot there are 2 types of arrays, macro arrays (300 microns or more), and micro arrays (less than 100 microns).

00 The former can be made manually in the laboratory and the dots can be visualized without the help of special equipment. The latter require an automated printing C system (normally a robotic printing platform) and specialized acquisition and 00oo Simage-processing equipment.

In the present invention, DNA fragment arrays include an ordered series of dots printed on a flat surface such as a sheet of glass, silicone or nylon, where each dot contains a large quantity of copies of a DNA fragment that is known and specific for a certain gene of interest in biotechnology.

The detection method that employs DNA fragment arrays includes simultaneous hybridization of the set of 'dots' of the array with an extract of labeled DNA from the sample under study. Normally, the DNA of the sample which has been labeled and in given case fragmented, is submitted to a denaturing stage in which the double strand of DNA separates, for example, with heat. As the temperature is lowered, the DNA, due to its physical-chemical characteristics, will tend to hybridize with its most exact complementary. When this DNA is in contact with the array, if there is a coincidence between the DNA of the sample and the fragment of DNA contained in the dot, the copies of the sample's labeled DNA will most probably remain linked specifically to that dot. This occurs due to the higher number of copies of complementary DNA contained in the dot of the array. In the image acquisition and processing stage of the hybridized array, this label will allow the detection of microorganisms present in the sample under study.

DNA labeling can be done with any known labeling technique, the most common of which are fluorescent or radioactive label.

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The arrays and their use are known, and in the state of the art we find examples of arrays that detect the presence of microorganisms in a sample, but none of them focus on the detection of genes that codify proteins with relevant activities in biotechnology.

oo00 There are currently various protocols published for manufacturing DNA fragment as well as laboratories that render services in the manufacture of this kind 0 N of arrays. As a result, only the selection of the genes and the design of the 00 Ofragments of DNA used, is what defines the specificity and usefulness of an array, C because manufacture may vary in regard to the support, the method with which the fragments of DNA are bound to the support, the spatial distribution of the dots on this support, etc., depending on the company entrusted with the array manufacturing or the protocol employed for doing it in the laboratory itself. (Ye et al Journal of Microbiological Methods 47(2001) 257-272).

Summarized Description of the Invention The present invention makes known an array for rapidly and simultaneously identifying the presence of certain genes that codify proteins with relevant activities in biotechnology that are present in a microbiological sample, and the method for using this array in the identification of said genes.

We have designed DNA fragments 100 or less nitrogenated bases long, that make it possible to identify the presence of genes that codify for proteins relevant in biofilm formation, in CO2 fixation, in energetic metabolism, in chemiotaxis and mobility, in iron oxidization, in nitrogen metabolism, in sulfur assimilation, and in oxide-reduction of sulfide compounds. All the designed fragments are specific for genes that codify particular proteins, which are related to the function of interest, and therefore we say they are specific for certain metabolic functions. In some cases, the genes based on which the oligonucleotides have been designed are specific for only one specie, and consequently, the oligonucleotide is specific both 005099352 00 O for the function and the specie. In other cases, the genes of interest are found orthologically conserved. In these cases the oligonucleotides were designed within the region of consensus among the described orthologues, and are thus specific for the function and taxon under discussion.

When there is at least one of these fragments of DNA in an array, it is possible to 00 evaluate the metabolic characteristics of a microbiological community. Arrays several of the designed DNA fragments that make it possible to identify c simultaneously and in only one process the presence of several or all of the genes oo 00 Sthat codify for proteins relevant in biofilm formation, in CO 2 fixation, in energetic metabolism, in chemiotaxis, in nitrogen metabolism, in sulfur assimilation and in oxide-reduction of sulfide compounds, are preferably provided.

This method is presented as a tool that is useful in biotechnology whenever evaluating or controlling the qualities of a microbiological community regarding its specific metabolic functions is required.

Brief Description of Figures.

Figure 1 corresponds to the result of hybridization of biomining sample 1 with a microarray of the invention. The fragments of the invention correspond to subfragments of the fragments described in Table 2. In Table 1, the contents of each of the positions of the microarray are detailed, and Table 3 sums up the positive or negative results of the hybridization.

The results show the presence of genes that codify for proteins relevant in the following functions: metabolism of sulfide compounds, CO 2 fixation, iron oxidizing, nitrogen fixation, and in key enzymes of energetic metabolism. The sample doesn't seem to have the genes evaluated for chemiotaxis and biofilm formation.

Positive controls showed signs of hybridization, and negative controls remained unlabeled.

005099352 00 O Figure 2 corresponds to the result of hybridization of a second biomining sample Swith a microarray of the invention. The microarray used is the same one used for Ssample 1, whose contents are declared in Table 1. The fragments used in the example are contained in the sequences described in Table 2, and Table 4 sums up the positive or negative results of the hybridization.

00 The results show the presence of genes that codify for proteins relevant for all the evaluated functions it seems to be the case of a mixed culture. Unlike the c previous sample, we observe high representation of genes for cellular movement 00 and chemiotaxis, and for biofilm formation. Positive controls showed signs of c hybridization and negative controls remained unlabeled.

Detailed Description of the Invention.

Being provided with an array that allows rapid and simultaneous detection of the presence of genes that codify proteins with activities relevant in biotechnology that are present in a microbiological sample would have applications in various areas of industrial business, and the method for using this array. For example, evaluation of wild microbiological communities present in biotechnological process substrates, such as minerals, for biomining processes; contaminated soils or water, for bioremediation processes, or any other of biotechnological application. A second aspect is that it would also allow microbiological communities already involves in a biotechnological process to be controlled.

In order to fulfill these biotechnological industrial business needs we have designed DNA fragments 100 or less hydrogenated bases long, that are specific for genes that codify for proteins relevant in biofilm formation, in C02 fixation, in energetic metabolism, in chemiotaxis and mobility, in iron oxidization, in nitrogen metabolism, in sulfur assimilation, and in oxidation-reduction of sulfide compounds. These fragments, distributed in DNA arrays, make it possible to identify the presence of these genes in a sample obtained from a microbiological community, whether wild, or already involved in a biotechnological process.

005099352 00 SAll the fragments designed are specific for genes that codify specific proteins related to the function of interest and thus, we say they are specific for certain metabolic functions, specifically for the following functions: biofilm formation, CO 2 fixation, energetic metabolism, chemiotaxis and mobility, iron oxidization, nitrogen metabolism, sulfur assimilation, and oxide reduction of sulfide compounds.

t'q 00 In some cases, the genes based on which the oligonucleotides have been c designed are specific for only one specie, and consequently, the oligonucleotide is 00oO Sspecific both for the function and the specie. In other cases, the genes of interest c are orthologically conserved in different species within a specific genus. In these cases, the oligonucleotides were designed within the region of consensus between the described orthologues, and are thus specific for the function and for the taxon under discussion.

For a better understanding of how we developed this method, we will briefly describe the strategy employed.

In the first place, we established the functions of interest in biotechnology we wanted to detect, and the key genes for each of these functions.

In the second place, microorganisms with genes related to the functions of interest which should comply with two basic conditions, on one hand to possess any of the functions of interest, and on the other, to be completely or partially sequenced, were chosen.

SIn the third place, a list of reference sequences that codify for the protein or function of interest in well known microorganisms, as for example in E. coil was generated, and homology between the said known genes and the genomes of interest was inspected. In the cases in which homology was found, it was defined that the 'homologous' zone corresponds to the gene of interest in the microorganism of interest.

005099352 00 A start-up data base containing the sequence of each of the genes of interest in each of the microorganisms of interest was obtained this way.

Obviously, not all the genes of interest were present in all the microorganisms of interest (this is a known microorganism/sequenced genome).

00 The sequences of the genes contained in the start-up data base were 00 compared with all the genes of the microorganisms of interest, selecting 00 Sonly the regions that are specific for the genes of interest, in other words, the regions that do not exhibit homology with genes that have other functions. This selection is made by a search algorism into which these restrictions are entered.

The specific regions thus selected are grouped together by high similarity, which is understood as a coincidence of about 80% of the final length of the designed oligonucleotide, with the purpose of detecting possible consensus regions.

These specific regions are validated as regions useful for detection, taking physical-chemical variables of the oligonucleotides (GC composition level, hybridization temperature, possible secondary structures) into consideration. The regions that comply with these conditions are considered to be oligonucleotide candidates.

The oligonucleotide candidates are compared by homology with all the known gene sequences, excluding from this comparison the gene of interest based on which the oligonucleotide was designed with which there would obviously be homology.

The oligonucleotides adopted as specific, are the ones we will employ to detect the function of interest.

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c As it was previously indicated, the first step was to select the functions of interest.

We selected 8 of the metabolic functions that exist in a microbiological community and are useful in biotechnology to include in our DNA array: biofilm formation, CO 2 fixation, energetic metabolism, chemiotaxis and mobility, iron oxidization, nitrogen metabolism, sulfur assimilation, and oxide reduction of sulfide compounds. The 00 importance of each of them is evident for any person with thorough knowledge of Sthe technique.

00 O Some of these functions provide us with general and relevant information on any c microbiological community, for example, both energetic metabolism and whether the community can or cannot fix CO 2 or nitrogen, are relevant in establishing the nutrients a community needs. Knowing the community's biofilm-formation capability, or the presence of microorganisms capable of moving within the community, is likewise essential in establishing the culture conditions of the biotechnological process that we want to carry out. In another direction, iron oxidization, sulfur assimilation, and sulfide compound oxidation/reduction processes are important processes in specific biotechnological processes of great importance, such as biomining and bioremediation.

In the second place, the microorganisms of interest based on which the oligonucleotides that will be included in the microarray were designed, were determined. The selection was carried out keeping two relevant aspects in mind: first that the microorganisms possess at least one of the functions of interest, and second, that their genome be totally or partially sequenced. Considering these factors, the strains that are property of Biosigma: Wenelen (DSM 16786) and Licanantay (DSM 17318), and possess one or more of the mentioned metabolic functions and for which there is a sequenced genome, were selected. The Acidithiobacillus ferrooxidans ATCC 23270 species which possesses iron and sulfide compound oxidizing functions was also included. The Burkholderia pseudomallei species for which two published sequences were used: Burkholderia pseudomallei 1710b and Burkholderia pseudomallei K96243. Desulfovibrio 005099352 00 0 desulfuricans was selected for the sulfur-oxidizing activity, and the published t c Desulfovibrio desulfuricans G20 series was used; Thiobacillus denitrificans ATCC 25259, whose function is sulfide compound oxidization, was also included.

Biomining microorganisms Ferroplasma, Leptospirillum, Sulfolobus and Thermoplasma, in which the published sequences correspond to the following microorganisms; Ferroplasma acidarmanus, Ferroplasma sp.(ll), Leptospirillum sp.

oo Leptospirillum sp. (II) Sulfolobus acidocaldarius DSM 639, Sulfolobus solfataricus, Sulfolobus tokodaii, Thermoplasma acidophilum and Thermoplasma CI volcanium, were also included.

00 c Once it is clear which the functions of interest and the microorganisms with which we will work are, we determine what genes we will employ to identify the presence of the said function. A brief description of each of the functions of interest and the genes chosen to represent them is presented below.

Description of functions of interest.

Iron oxidization. Iron oxidization from Fe+2 to Fe+ 3 is fundamental for the development of bioleaching, because the iron (III) generated is a great oxidizing agent, which can oxidize the sulfides present or any compound that requires oxidizing, thus releasing the metals of interest into the solution.

In order to identify the presence of the 'iron oxidization' function, the following genes codifying for proteins that are relevant within the process, based on A. ferrooxidan, were chosen: rus, cycl, cyc2, cycA y cycA2 Definition of the sequences for all these genes within the microorganisms of interest (known microorganisms/sequenced genome) was available, on account of which it was not necessary to employ reference sequences.

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Biofilm formation. The capacity to form biofilm or silt, is important for the development of any bacterial community, because it assures permanence of the biomass attached to a support.

In order to identify the presence of the 'biofilm formation' function, the following 00 genes that codify for proteins relevant within the process were chosen.

c0 galE, galU, rfbA, rfbB, rfbC, rfbD, epsD, gdh, manB, manC, pgm, uppS, 00 Swza, wzc There was no information available on these genes in the microorganisms of interest, so work was done with reference genes looking for homology between the said genes and the genomes of the microorganisms of interest, to determine the 'probable' presence of these genes. In most cases, the gene of E. Coli was used as reference, except with epsD, in which the gene of Lactobacillus lactis was used as reference.

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2 fixation. CO2 fixation is important in microbiological biotechnological processes because it allows evaluating whether the community is autotrophous, which grants economic advantages to the process, because it is not necessary to add carbonated substrates to the community.

In order to identify the presence of the 'C0 2 -fixation' function, the following genes that codify for proteins relevant within the process were chosen: cbbL1, cbbL2, cbbM, cbbS1, cbbS2, cbbArchea, ppc, prk 005099352 00 S Information for only some of these genes in the microorganisms of interest, cbbL1, C cbbL2, cbbM, cbbS1 y cbbS2 was available. In the other cases, work was done with reference genes, and homology between these reference genes and the genomes of the microorganisms of interest was sought in order to determine the 'probable' presence of these genes. For gene cbb of Archaeas, Methanosarcina acetivorans was used as reference, for gene ppc, the gene of E. coli was used as 00 reference, and for gene prk, the gene of Synechococcus was used.

00 0 Chemiotaxis and mobility. Microorganism mobility is given by the presence of Cl flagellums; in given cases, movement develops as a response to the concentration of a metabolite in the medium, which is known as chemiotaxis. Knowing whether a microbiological community has the characteristics of chemiotaxis and mobility makes it possible to establish in a better way the culture conditions for the biotechnological process to be developed.

In order to identify the presence of the 'chemiotaxis and mobility' function, the following genes that codify for proteins relevant in the process were chosen: flhA, flhB, fliF, fliG, fliM, fliN, motA, motB No information on any of these genes in the microorganisms of interest was available, so work was done with reference genes, and homology between these reference genes and the genomes of the microorganisms of interest was sought in order to determine the 'probable' presence of the said genes. In every case, the gene of E. coli was used as reference.

Nitrogen metabolism. Regarding the metabolism of nitrogenated compounds, there are two processes which can be considered to be important. One of them is nitrogen fixation in which the essential component is the enzyme nitrogenase. The 005099352 00 O other is the degradation of nitrogenated compounds, in which nitrate reductases c participate.

In order to identify the presence of the 'nitrogen metabolism' function, the following genes that codify for proteins relevant within the process were chosen: 00 nifK, narH, nirA 00 0 No information on these genes in the microorganisms of interest was available, so Cl work was done with reference genes, and homology between these reference genes and the genomes of the microorganisms of interest was sought in order to determine the 'possible' presence of these genes. In the case of narH, the gene of E. coi; was used as reference; for nifK, Azotobacter vinelandii was used as reference, and in the case of nirA, the reference was Synechococcus.

Sulfur assimilation. Sulfur assimilation is the process through which environmental sulfur is fixed as organic sulfur for its use in cell metabolism. The two main final products of this process are the essential amino acids cysteine and methionine.

In order to identify the presence of the 'sulfur assimilation' function, the following genes that codify for proteins relevant within the process were chosen: cysl, cysJ No information on these genes in the microorganisms of interest was available, so work was done with reference genes, and homology between these reference genes and the genomes of the microorganisms of interest was sought in order to determine the 'probable' presence of the said genes. For genes cysl, and cysJ, the gene of E. coli was used as reference.

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Oxidation/reduction of sulfide compounds. A highly relevant process in bioleaching is the oxidization of sulfur compounds. For example, microorganisms of the Acidithiobacillus genus are capable of catalyzing the oxidization of reduced sulfur compounds (such as sulfide, elemental sulfur, thionates, etc.) using oxygen 00 as an electronic acceptor and generating sulfuric acid as a final product, and reducing species such as sulfite and thiosulfate as intermediate products which

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N makes solubilization of the metal associated to sulfides in the ore possible.

oO c In order to identify the presence of the 'sulfur oxidization' function, the following genes that codify for proteins relevant within the process were chosen: doxA, doxD, doxDA1, doxDA2, dsrA, dsrB, dsrC, dsrE, dsrF, dsrK, dsrL, dsrM, dsrN, dsrO, dsrP, dsrS, soxB There was only information available for the doxDA1, doxDA2 genes in the microorganisms of interest. In other cases, work was done with reference genes, and homology between these reference genes and the genomes of the microorganisms of interest was sought in order to determine the 'probable' presence of these genes. For genes doxA, doxD, Acidarmanus Ambivalens was used as reference; for gene soxB, the gene of Paracoccus denitrificans was used as reference, and for all the dsr genes, genes of Allochromatium vinosum were used.

Energetic metabolism.

In order to identify the presence of the 'energetic metabolism' function, the following genes that codify for proteins relevant within the process were chosen: pfk, korA, korB, idh, pdhA, pdhB, pdhC, gitA, accA, accB, aaC, accD 005099352 00 There was only information available for the pdhC gene in the microorganisms of interest. In other cases, work was done with reference genes, and homology Sbetween these reference genes and the genomes of the microorganisms of Sinterest was sought in order to determine the 'probable' presence of these genes.

For genes pfk, idh, gltA, accA, accB, aaC and accD, the gene of E. col was used as reference. For genes korA and korB, Methanocaldococcus jannaschii was used 00 as reference and for genes pdhA and pdhB, the genes of Mycobacterium tuberculosis were used.

00 oO The array of nucleotide sequences for detecting and identifying genes that codify previously defined proteins with relevant activities in biotechnology present in a microbiological sample includes, adhered to its surface, a representative of one, several, or all of the following fragments of DNA: a. at least one fragment of DNA that specifically identifies a gene that codifies for a protein relevant in biofilm formation; b. at least one fragment of DNA that specifically identifies a gene that codifies for a protein relevant in the CO2 fixation process; c. at least one fragment of DNA that specifically identifies a gene that codifies for a protein relevant in energetic metabolism; d. at least one fragment of DNA that specifically identifies a gene that codifies a protein relevant for chemiotaxis and mobility; e. at least one fragment of DNA that specifically identifies a gene that codifies for a protein relevant in iron oxidization; f. at least one fragment of DNA that specifically identifies a gene that codifies for a protein relevant in nitrogen metabolism; g. at least one fragment of DNA that specifically identifies a gene that codifies for a protein relevant in sulfur assimilation; h. at least one fragment of DNA that specifically identifies a gene that codifies for a protein relevant in oxidation/reduction of sulfide compounds; 005099352 00 Swhere each fragment of DNA is present in hundreds of copies, forming dots of G homogeneous composition, spatially distributed over the surface of the support.

For each of these previously mentioned genes we have designed fragments of to 100 specific nitrogenated bases, preferably 100 specific nitrogenated bases, 00 which are made known in the list of sequences.

c In all, we designed a total of 232 fragments of DNA for identification of genes that 00 Scodify proteins with relevant activities in biotechnology, all of which are 100 Snucleotides long. The sequences of the 232 DNA fragments designed are included in the listing of sequences.

Out of the 232 sequences designed, sequences No 1 to 86 (SEQ ID NO: 1 to 86) are specific for the biofilm formation function.

Sequence Nos 87 to 101 (SEQ ID NO: 87 to 101) are specific for the C0 2 -fixation function.

Sequence Nos 102 to 156 (SEQ ID NO: 102 to 156) are specific for the energetic metabolism function.

Sequence Nos 157 to 192 (SEQ ID NO: 157 to 192) are specific for the chemiotaxis and mobility function.

Sequence Nos 193 to 197 (SEQ ID NO: 193 to 197) are specific for the iron oxidization function.

Sequence Nos 198 to 203 (SEQ ID NO: 198 to 203) are specific for the nitrogen metabolism function.

005099352 00 Sequence Nos 204 to 210 (SEQ ID NO: 2044 to 210) are specific for the sulfur assimilation function.

q And sequence nos 211 to 232 (SEQ ID NO: 211 to 232) are specific for the sulfide compound oxidation reduction function.

00 The fragments designed can be imprinted in the array, whether complete or in a fragment that includes it, or partially like any of the subfragments included in 00 the fragment, or as the reverse complement sequences of all the previous options.

Subfragments of 50 to 70 nucleotides are conveniently imprinted.

It is necessary to keep in mind that the arrays contained in the present invention are those that comprise at least one of the DNA fragments included in sequence Nos 1 to 232 (SEQ ID NO: 1 to 232), whether completely; in a larger region in which the said fragment represents over 20% of the region, just like a PCR product; or partially, as one of the subfragments contained in each of the fragments published here, or as the reverse complement sequences of all the previous options.

The previous aspect is of vital importance, because the specificity of a nucleotidic sequence is the same as that of its reverse complement sequence. It is this characteristic, specificity, which is the hardest to attain in the design of DNA fragments. It could happen that the stability of the complement sequence is not appropriate for use in an array, although a person with thorough knowledge of the technique could distinguish between thermodynamically stable and non-stable oligonucleotides using a series of tools that exist in the technique. All the reverse complement sequences of fragments No 1 to 232 (SEQ ID NO: 1 to 232) of the present invention, whether completely; in a larger region that contains it, such as a PCR product; or partially, as one of the subfragments contained in each of the fragments published here, are considered to be within the scope of the present invention.

005099352 00 O The array will preferentially contain at least one fragment or subfragment for each Sfragment of interest. It is also possible to prepare an array containing all the published fragments or subfragments of DNA, or in a given case, only one of them. All these options, as well as all the possible intermediate combinations, are included in the scope of the present invention.

00 Effectiveness of the arrays of the invention is given by the specificity and stability the fragments to be imprinted. These characteristics persist in each of the c subfragments contained in the designed fragments. This means that specificity 00 Skeeps the same if nucleotides 1 to 100, or 42 to 92 or 15 to 65 or any other Spossible selection is used. Every selection corresponds to subfragments and is included in the scope of the present invention.

It is also possible to obtain DNA fragments that contain the fragments or subfragments of the present invention flanked by other oligonucleotides, whether through synthesis or as products of PCR. These larger regions that contain the fragments of the present publication, in which specificity is given by the fragments or subfragments designed and made known in the present invention, are also found contained in the scope of the present invention.

In order to carry out an array according to the invention, each fragment or subfragment selected must be synthesized, in several hundreds of copies, and imprinted, like a homogeneous dot, on a support appropriate for an array, such as glass, silicone, nylon, or any other that exists in the technique.

Just as we indicated when discussing the background information for this invention, DNA fragment synthesis and array-making techniques are known, and any of them can be used in making the arrays of the present invention.

Added to these specific DNA fragments for genes that codify proteins with relevant activities in biotechnology, it is convenient to include negative and positive controls in each array. Negative controls should correspond to nucleotidic sequences that 005099352 00 O should never be found in a biotechnological context. Positive controls should Scorrespond to nucleotidic sequences that are always present in the problem sample.

It will be appreciated that the 232 fragments of DNA designed for the present invention allow identification of genes that codify proteins with relevant activities in 00 biotechnology, not only when used in an array but also in other molecular biology techniques based on the hybridization of an unknown DNA with a specific c fragment. Among these molecular biology techniques we can mention detection 00 Sprobes, FISH, PCR and sequencing, for example.

Using the 232 fragments of DNA made known in this invention, whether completely; in a larger region in which the said fragment represents over 20% of the region, just like a product of PCR; or partially, as one of the subfragments contained in each of the fragments made known here; or as reverse complement sequences of all the previous options, in any molecular biology technique it is considered within the scope of the present invention.

Use of the array In order to detect and identify the presence of certain genes that codify proteins with relevant activities in biotechnology present in a microbiological sample using the arrays of the present invention, in the first place it is necessary to isolate the DNA of the sample we are interested in evaluating. It is also possible to work with cDNA where the only difference is that in this case the RNA of the sample is isolated in the first stage. In the technique, different methods for extracting DNA and RNA from environmental samples or from cultures are known, and any of them can be used, considering in each case the particular nature of the sample.

During a second stage, all the DNA or RNA of the sample must be converted to short fragments of labeled DNA which are appropriate for hybridizing with the fragment imprinted in the dots of the array. If the DNA of the sample has been 005099352 00 O isolated, it must be fragmented and labeled. When not working with the RNA of Sthe sample, it is not necessary to fragment and the marking stage should only be carried out to obtain labeled cDNA. A technique that allows both fragmentation and marking of the DNA is marking with aleatory splitters of 6 nucleotides of DNA.

Marking can be done employing nucleotides that are labeled or susceptible to being labeled with any of the existing techniques of the art, such as radioactivity, t'q 00 biotine, fluorescence, or others. If a macroarray is to be uses, marking should be preferably done with radioactivity, 32 p, and if a microarray is to be used, it will be c with fluorescence, for example, with Cy5 or Cy3.

00oo c The methods described for the preparation of DNA or cDNA for the array, are not limiting for the present invention, and any method existing in the state of the art for the preparation of DNA or cDNA can be used, which doesn't mean that the use of the array can break away from the scope of the present invention.

Once the DNA is prepared, it is submitted to a DNA denaturing stage. This denaturalized DNA is then incubated on the array, placing an aliquot of the DNA mixture on the array. The array is left to hybridize at a temperature within the range of 40 to 700C for at least an hour and preferably all night.

After hybridization, the array should be carefully washed. Washing is usually done with softening solutions at moderate temperatures, for example from 35 50 0C, and preferably from 40 45 OC Once the array is washed, it is preferentially dried, conveniently by centrifugation, for example, in a Falcon tube or something similar, for a short time and at a moderate speed (200-3000 x g per 1 to 5 minutes).

Finally, it is necessary to visualize which dots exhibit marking. The position of each labeled dot indicates the presence of the gene that codifies a protein with relevant activity in biotechnology based on which the fragment of DNA was designed.

005099352 00 It is necessary to check that the dot or dots corresponding to the negative control remain unlabeled, because if there is hybridization with these fragments of DNA, G this indicates that the reaction was unspecific and that the results obtained must t' be discarded because they could contain false positives.

The dots corresponding to the positive control should likewise be labeled, because 00 when no hybridization with this fragment of DNA takes place, this indicates that there was interference in the reaction and that therefore the dots with no signs can 00 correspond to false negatives.

According to the above, determining the presence of certain genes that codify proteins with relevant activities in biotechnology in a microbiological community would be reduced to the reading of the labeled dots in an array of the present invention.

Examples Example 1: Microarray to detect and identify the presence of certain genes that codify proteins with relevant activities in bio technology.

A microarray with 23 different fragments of DNA from the invention that identify genes that codify proteins with relevant activities in biotechnology was prepared, in which these activities are associated to the following functions: sulfur assimilation,

CO

2 fixation, iron oxidization, nitrogen metabolism, energetic metabolism, chemiotaxis and mobility, and biofilm formation.

Two positive controls and two negative controls were also included in the microarray. The contents of each of the positions in the microarray are detailed in Table 1 below.

005099352 Table 1 Function Position in microarray Sulfur assimilation A1-A3 Fixation of CO2 A4-A6 Iron oxidization A7-A9 Nitrogen metabolism B2-B4 Energetic metabolism B5-B7 Chemiotaxis and mobility B8, C1-C3 Biofilm formation C4-C7 Negative control B9, C8 Positive control B1, C9 All the imprinted fragments were 60 nucleotides long. The DNA fragments of the invention that were chosen correspond to subfragments of 60 oligonucleotides, of the fragments shown in Table 2 which are defined in the sequence list.

Table 2 Function Position SEQ ID NO Sulfur assimilation A1-A3 208-210 Fixation of C02 A4-A6 92 94 Iron oxidization A7-A9 193-195 Nitrogen metabolism B2-B4 201-203 Energetic metabolism B5-B7 113-115 Chemiotaxis and mobility B8, C1-C3 167-170 Biofilm formation C4-C7 Negatives B9, C8 Positives B1, C9 A company specialized in this service was entrusted with the preparation of the microarray.

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Example 2: Use of the microarrangement for detecting and identifying genes that codify proteins with relevant activities in biotechnology.

The microarray obtained in example 1 was used to determine the composition of the microbiological community in two samples of bioleaching heap effluents, oo sample 1 (S1) and sample 2 (S2).

Cl Total DNA present in S1 and S2 was extracted with traditional DNA extraction 00 Smethods.

2 IL of DNA were taken from the samples and placed in Eppendorf tubes. The following method was followed in each case: 36 1 L of ddH 2 0 and 3,3 mL of commercial aleatory splitters of 6 nucleotides (hexamers) were added. It was brought to a boil for 5 minutes and then worked in ice.

2 pL of a mixture of nucleotides, where nucleotide dUTP is labeled with the fluorphor Cy, was added. Cy5, which presents red fluorescence, was used for S1, while Cy3 which presents green fluorescence was used for S2. After this, 4 PL of a Klenow-type polymerase and 5 pL of buffer solution appropriate for the polymerase (according to the distributor) were added; it was incubated for 4 hours at 37 OC.

The reaction was stopped with 5 pL of AEDT 0,5 M pH8. The labeled DNA was recovered employing an ionic interchange column. The effluent containing the DNA was vacuum-dried.

The DNA was resuspended adding 100 piL of a buffer solution of Tris pH 7 as basic ingredient, and was submitted to 100 °C for a minute and a half to denature the DNA. Hybridizing was done on the microarray at 55 OC, all night, with movement.

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The following morning, each microarray was washed twice with 2X SSC, 0,1% SDS (sodium dodecyl sulfate), at 45 once with 0,2X SSC 0,1% SDS at 42°C and once with 0,2X SSC at 420C. (For 500 of SSC 20x, 87,6 g of NaCI, 50 g of sodium citrate (2H 2 0) and adjusted to pH 00 Each microarray was placed in a box with miliQ water for 15 minutes, and was later dried by centrifugation in a Falcon Tube or something similar during one

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oO 00 cI Finally it was possible to observe the result of the microarrays, which are shown in Figure 1 for S1 and in Figure 2 for S2.

In Table 3, the positions of the different fragments in the microarray are indicated, and the result of hybridization with the DNA obtained from S1, shown in Figure 1, is summed up. All the positive controls showed hybridization, and the negative controls remained unlabeled.

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Table 3, Sample 1 s(1) Position in Function Result microarray Al Sulfur assimilation A2 A3 A4 C02 Fixation A5 A6 A7 Iron oxidization A8 A9 B2 Nitrogen metabolism B3 B4 Energetic metabolism B6 B7 B8- C1 Chemiotaxis and mobility C2 C3 C4 Biofilm formation C6 C7 B9 Negatives C8 B1 Positives C9 Symbols: positive; negative.

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The results show the presence of genes that codify for proteins relevant in sulfur Sassimilation, CO 2 -fixation, iron oxidization, and nitrogen-fixation functions, and key C proteins in energetic metabolism. The sample does not seem to have the genes evaluated for chemiotaxis and biofilm formation.

00 In Table 4, the positions of the different fragments in the microarray are shown S again, and the result of hybridization with DNA obtained from S2, shown in Figure 0 2, is summed up. All the positive controls showed hybridization, and the negative controls remained unlabeled.

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Table 4, Sample 2 (S2) Function Position Result microarray Al Sulfur assimilation A2 A3 A4 C02 Fixation A5 A6 A7 Iron oxidization A8 A9 B2 Nitrogen metabolism B3 B4 Energetic metabolism B6 B7 B8 C1 Chemiotaxis and mobility C2 C3 C4 Biofilm formation C6 C7 B9 Negatives C8 B1 Positives C9 Symbols: positive; negative.

005099352 00 The results show the presence of genes that codify for proteins relevant for all the functions evaluated. Apparently it is the case of a mixed culture. Unlike the previous sample, we observed a high representation of cells for cellular movement and chemiotaxis, and for biofilm formation.

00 The present examples are of an illustrative character and should in no way be considered as limitative of the scope of the present invention, which is defined in 0 c the accompanying claims.

00oo SReference to any prior art in the specification is not, and should not be taken as, an acknowledgment, or any form of suggestion, that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art.

005099352

Claims (29)

1. Array of nucleotide sequences for detection and identification of genes that codify proteins with activities relevant in biotechnology present in a microbiological sample wherein the array includes, attached to its surface, a representative of either one, several or all the following fragments of DNA: a. at least one fragment of DNA that specifically identifies a gene that codifies for a protein relevant in biofilm formation t'q 00 00 b. at least one fragment of DNA that specifically identifies a codifies for a protein relevant in CO 2 fixation; c. at least one fragment of DNA that specifically identifies a codifies for a protein relevant in energetic metabolism; gene that gene that d. at least one fragment of DNA that specifically identifies a gene that codifies for a protein relevant for chemiotaxis and mobility; e. at least one fragment of DNA that specifically identifies a codifies for a protein relevant in iron oxidization; f. at least one fragment of DNA that specifically identifies a codifies for a protein relevant in nitrogen metabolism; g. at least one fragment of DNA that specifically identifies a codifies for a protein relevant in sulfur assimilation; gene that gene that gene that h. at least one fragment of DNA that specifically identifies a gene that codifies for a protein relevant in oxidation/reduction of sulfide compounds; where each fragment of DNA is present in hundreds of copies, forming dots of homogeneous composition, spatially distributed over the surface of the support.

2. Array according to claim 1, wherein the fragment of DNA that specifically identifies a gene that codifies for a protein relevant in biofilm formation, is chosen 005099352 00 O from among the fragments of DNA defined by SEQ ID NO: 1 to 86 and their reverse complement sequences.

3. Array according to claim 2, wherein the fragment of DNA is either complete, or within a larger sequence where specificity is given by the said fragment, or like any of the subfragments contained in it. 00

4. Array according to claim 3, wherein the subfragments preferentially C comprise 50 to 70 nucleotides. o00 C

5. Array according to claim 1, wherein the fragment of DNA that specifically identifies a gene that codifies for a protein relevant in the CO 2 -fixation process is chosen from among the fragments of DNA defined by SEQ ID NO: 87 to 101 and their reverse complement sequences.

6. Array according to claim 5, wherein the fragment of DNA is either complete, or within a larger sequence in which specificity is given by the said fragment, or like any of the subfragments contained in it.

7. Array according to claim 6, wherein the subfragments comprise from 50 to nucleotides.

8. Array according to claim 1, wherein the fragment of DNA that specifically identifies a gene that codifies for a protein relevant in energetic metabolism is chosen from among the fragments of DNA defined by SEQ ID NO: 102 to 156 and their reverse complement sequences.

9. Array according to claim 8, wherein the fragment of DNA is either complete, or within a larger sequence in which specificity is given by the said fragment, or like any of the subfragments contained in it. 005099352 00 O

10. Array according to claim 9, wherein the subfragments preferentially Scomprise 50 to 70 nucleotides.

11. Array according to claim 1, wherein the fragment of DNA that specifically identifies a gene that codifies for a protein relevant for chemiotaxis and mobility, is chosen from among the fragments of DNA defined by SEQ ID NO: 157 to 192 and 00 their reverse complement sequences. c

12. Array according to claim 11, wherein the fragment of DNA is either oO 00 Scomplete, or within a larger sequence in which specificity is given by the said Sfragment, or like any of the subfragments contained in it.

13. Array according to claim 12, wherein the subfragments preferentially comprise 50 to 70 nucleotides.

14. Array according to claim 1, wherein the fragment of DNA that specifically identifies the gene that codifies for a protein relevant in iron oxidization, is chosen from among the fragments of DNA defined by SEQ ID NO: 193 to 197 and their reverse complement sequences.

Array according to claim 14, wherein the fragment of DNA is either complete, or within a larger sequence in which specificity is given by the said fragment, or as any of the subfragments contained in it.

16. Array according to claim 15, wherein the subfragments preferentially comprise 50 to 70 nucleotides.

17. Array according to claim 1, wherein the fragment of DNA that specifically identifies a gene that codifies for a protein relevant in nitrogen metabolism, is chosen from among the fragments of DNA defined by SEQ ID NO: 198 to 203 and their reverse complement sequences. 005099352 00 O

18. Array according to claim 17, wherein the fragment of DNA is either Scomplete, or within a larger sequence in which specificity is given by the said fragment, or like any of the subfragments contained in it.

19. Array according to claim 18, wherein the subfragments preferentially comprise 50 to 70 nucleotides. 00

20. Array according to claim 1, wherein the fragment of DNA that specifically c identifies a gene that codifies fro a protein relevant in sulfur assimilation is chosen o00 Sfrom among the fragments of DNA defined by SEQ ID NO: 204 to 210 and their c reverse complement sequences.

21. Array according to claim 20, wherein the fragment of DNA is either complete, or within a larger sequence in which specificity is given by the said fragment, or like any of the subfragments contained in it.

22. Array according to claim 21, wherein the subfragments preferentially comprise 50 to 70 nucleotides.

23. Array according to claim 1, wherein the fragment of DNA that specifically identifies a gene that codifies for a protein relevant in oxide reduction of sulfide compounds, is chosen from among the fragments of DNA defined by SEQ ID NO: 211 to 232 and their reverse complement sequences.

24. Array according to claim 23, wherein the fragment of DNA is either complete, or within a larger sequence in which specificity is given by the said fragment, or like any of the subfragments contained in it.

Array according to claim 24, wherein the subfragments preferentially comprise 50 to 70 nucleotides. 005099352 00 O

26. Method for detection of genes that codify proteins with activities relevant in Sbiotechnology present in a microbiological sample, including: Sa. incubating a sample of labeled nucleic acid, obtained from a CC microbiological sample, on an array as defined in any one of claims 1 to Cc b. after incubation, washing the array to eliminate labeled DNA 00oo fragments that have not hybridized, c c. visualizing the dots of the array that exhibit marks, oo 00 d. establishing that genes that codify proteins with relevant activities in cthe following functions: biofilm forming, CO2 fixation, energetic metabolism, chemiotaxis and mobility, iron oxidizing, nitrogen metabolism, sulfur assimilation, and oxide reduction of sulfide compounds, are present in the sample.

27. Method for identification of genes that codify proteins with activities relevant in biotechnology present in a microbiological sample, according to claim 26, further including the previous steps of: a. extracting the nucleic acid from a microbiological sample; and/or b. fragmenting and marking a sample of nucleic acid, using nucleotides that are labeled or susceptible to being labeled, before incubating a sample of labeled nucleic acid on an array, as defined in any one of claims 1 to

28. An array according to claim 1, substantially as hereinbefore described.

29. A method according to claim 26, substantially as hereinbefore described. 005099352