Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates
  • C12P19/28—N-glycosides
  • C12P19/30—Nucleotides
  • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/93—Ligases (6)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
  • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor

Definitions

• the present invention relates to a method of preparing nucleic acids from a sample of the environment, more particularly to a method of obtaining a collection of nucleic acids from a sample.
• the invention also relates to nucleic acids or collections of nucleic acids obtained according to the process and their application to the synthesis of new compounds, in particular new compounds of therapeutic interest.
• a subject of the invention is also the new means used in the process for obtaining the above nucleic acids, such as new vectors and new methods for preparing such vectors or also recombinant host cells comprising an acid. nucleic acid of the invention.
• the invention also relates to methods for detecting a nucleic acid of interest within a collection of nucleic acids obtained according to the above method, as well as the nucleic acids detected by such a method and the polypeptides encoded by such methods. nucleic acids.
• the invention also relates to nucleic acids obtained and detected according to the above methods, in particular nucleic acids coding for an enzyme participating in the biosynthetic pathway of antibiotics such as ⁇ -lactams, aminoglycosides, nucleotides heterocyclic or polyketides as well as the enzyme encoded by these nucleic acids, the polyketides produced by the expression of these nucleic acids and finally pharmaceutical compositions comprising a pharmacologically active amount of a polyketide produced by the expression of such nucleic acids.
• nucleic acids coding for an enzyme participating in the biosynthetic pathway of antibiotics such as ⁇ -lactams, aminoglycosides, nucleotides heterocyclic or polyketides as well as the enzyme encoded by these nucleic acids, the polyketides produced by the expression of these nucleic acids and finally pharmaceutical compositions comprising a pharmacologically active amount of a polyketide produced by the expression of such nucleic acids.
• antibiotics penicillin, erythromycin, actinomycin, tetracycline, cephalosporin
• anticancer drugs cholesterol-lowering agents or even pesticides.
• the products of therapeutic interest of microbial origin known to date come mainly (around 70%) from the group of actinomycetes and more particularly from the genus Streptomyces.
• the rediscovery rate of previously known antibiotics is around 99%.
• fluorescence microscopy techniques have made it possible to count more than 10 10 bacterial cells in 1 g of soil, while only 0.1 to 1% of these bacteria can be isolated after seeding on culture media.
• Methods have thus been developed which include a step of extracting DNA from telluric organisms, if necessary after prior isolation of the organisms contained in the soil samples.
• the DNA thus extracted after lysis of the bacterial cells without prior stage of culture ' in vitro, is cloned into vectors used to transfect host organisms, in order to constitute DNA libraries originating from soil bacteria.
• Such techniques have been used on samples from aquatic environments, whether fresh or marine. They comprise a first stage of prior concentration of the cells present freely or in the form of particles, generally consisting of filtration of large volumes of water on different filtration devices, for example conventional filtration on a membrane, tangential or rotational filtration or even ultrafiltration. .
• the pore size is between 0.22 and 0.45 mm and often requires prefiltration in order to avoid clogging due to the treatment of large volumes.
• the harvested cells are lysed directly on the filters in small volumes of solutions, by enzymatic and / or chemical treatment.
• WISCONSIN ALUMNI RESEARCH FOUNDATION describes a step of lysis of bacteria in situ using an extraction buffer containing a protease and SDS.
• Bacteria lysis techniques have also been used using a succession of sonication steps, microwave heating and thermal shock (PICARD et al. (1992).
• the in situ chemical or enzymatic treatments of the sample have the disadvantage of only lysing certain categories of microorganisms due to the selective resistance of the various indigenous microorganisms at the lysis stage due to their heterogeneous morphology .
• Gram-positive bacteria resist heat treatment with SDS detergent while almost all Gram-negative cells are lysed.
• some of the direct extraction protocols described above promote the adsorption of the extracted nucleic acids on the mineral particles of the sample, thus significantly reducing the amount of accessible DNA.
• the quantity of DNA directly usable for cloning into recombinant vectors is also dependent on the purification steps subsequent to its extraction.
• the extracted DNA is then purified, for example by the use of polyvinylpolypyrrolidone, by precipitation in the presence of ammonium or potassium acetate, by centrifugations on a cesium chloride gradient, or else chromatographic techniques, in particular on a hydroxyapatite support, on an ion exchange column or molecular sieving or by electrophoresis techniques on agarose gel.
• Another object of the invention was to overcome the drawbacks of the previous purification protocols and to develop a DNA purification step making it possible to optimally maintain the diversity of the DNA of the initial sample, on the one hand, and, quantitatively promoting its obtaining, on the other.
• Such techniques use a first step of separation of the different organisms from the telluric microflora from the other constituents of the starting sample, prior to the step of extraction of the DNA itself.
• the prior separation of a microbial fraction from a soil sample most often comprises a physical dispersion of the sample by grinding the latter in a liquid medium, for example using devices of the type Waring Blender or a mortar.
• Chemical dispersions have also been described, for example on ion exchange resins or else dispersions using non-specific detergents such as sodium deoxycholate or polyethylene glycol. Whatever the mode of dispersion, the solid sample must be suspended in water, phosphate buffer or saline solution.
• the physical or chemical dispersion step can be followed by centrifugation on a density gradient allowing the separation of the cells contained in the sample and the particles of the latter, it being understood that the bacteria have densities lower than those of most soil particles.
• the physical dispersion step can also be followed alternately by a low speed centrifugation step or even a cell elutriation step.
• the DNA can then be extracted from the separated cells by all available lysis methods and can be purified by numerous methods, including the purification methods described in paragraph 1.1 above.
• the inclusion of cells in agarose with a low melting point can be carried out in order to protect the lysis.
• the methods described in the state of the art known to the applicant are not satisfactory because of the presence, in the fractions containing the extracted DNA, of undesirable constituents of the starting sample having a significant influence on the quality and the amount of final DNA.
• the present invention proposes to solve the technical difficulties encountered in the processes of the prior art as will be described below.
• the objective of such a molecular characterization of the extracted and purified DNA is to obtain profiles representing the proportions of the different bacterial taxa present in this DNA extract.
• the molecular characterization of the extracted and purified DNA makes it possible to determine whether artefacts were introduced during the implementation of the various extraction and purification stages and, if necessary, whether the diversity of origin of the DNA. extracted and purified is representative of the microbial diversity initially present in the sample, especially in the soil sample.
• MPN-PCR is of complex use due to the multiplication of dilutions and repetitions which makes it unsuitable for a large number of samples or of pairs of primers.
• viral vectors phages, plasmids, phagemids, cosmids, phosmids, vectors of the BAC type (bacterial artificial chromosome) or else can be used.
• bacteriophage P1 PAC type vectors (artificial chromosome based on bacteriophage P1)
• YAC type vectors artificial yeast chromosome
• yeast plasmids or any other vector capable of maintaining and stably expressing genomic DNA.
• Example 1 of PCT application No. WO 99 / 20,799 describes the construction of a genomic DNA library by cloning into a vector of the BAC type.
• PCT application No. WO 99 / 20,799 cites numerous appropriate cellular hosts, such as Escherichia coli, in particular the strain DH 10B or even the strain 294 (ATCC 31446, the strain E. coli B, E. Coli X 1776 (ATCC N ° 31.537), E.coli DH5 ⁇ and E.coli W3110
• This PCT application also cites other appropriate host cells such as Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, Serratia, Schigella or also strains of the bacillus type such as B. subtilis and ⁇ . licheniformis as well as bacteria of the genus
• US Patent No. 5, 824,485 cites in particular the strain of Streptomyces lividans TK66 or also yeast cells such as those of Saccharomyces pombe.
• This technique made it possible to detect a chromosomal DNA fragment of about 26 kb carrying part of the operon for biosynthesis of surfactin.
• the article by HONG-FU et al. (1995) describes the construction of expression cassettes containing the different open reading phases of the operon responsible for the biosynthesis of polyketides, the different expression cassettes having been constructed artificially by combining the open reading phases which are not found together naturally in the genome of Streptomyces coelicolor.
• This article shows that the combination, in artificial expression cassettes, of open reading frames originating from different bacterial strains allows the production of polyketides with different structural characteristics and more or less large antibiotic activities against Bacillus subtilis and Bacillus cereus.
• Polyketides are part of a large family of natural products of variable structure and with a wide variety of biological activities. Polyketides include, for example, tetracyclines and erythromycin (antibiotics), FK506 (immunosuppressant), doxorubicin (anti-cancer agent), monensin (a coccidiostatic agent) as well as avermectin (an antiparasitic agent).
• polyketide synthases which catalyze repeated condensation cycles between acyl thioesters (in general acetyl, propionyl, malonyl or methylmalonyl thioesters). Each condensation cycle results in the formation, on a growing carbon chain, of a ⁇ -keto group which can then undergo, if necessary, one or more series of reducing steps.
• New artificial polyketides have thus been produced by genetic engineering, such as mederrhodin A or dihydrogranatirhodin.
• genetic engineering such as mederrhodin A or dihydrogranatirhodin.
• the vast majority of new polyketide molecules obtained by genetic engineering are very different, from a structural point of view, from the corresponding natural polyketides.
• the invention relates first of all to a method for the construction of DNA libraries originating from a sample of the environment, such a sample being able either to be an aquatic medium (fresh or marine water), a soil sample (layer surface, soil or sediment), or a sample of eukaryotic organisms containing an associated microflora, such as for example a sample from plants, insects or even marine organisms and having an associated microflora.
• a sample of the environment such a sample being able either to be an aquatic medium (fresh or marine water), a soil sample (layer surface, soil or sediment), or a sample of eukaryotic organisms containing an associated microflora, such as for example a sample from plants, insects or even marine organisms and having an associated microflora.
• the development of a process for constructing a DNA library of an environmental sample, and in particular of a soil sample includes critical steps, the implementation of which must necessarily be optimized for obtaining a DNA bank whose content in nucleic acids of interest meets the objectives initially set.
• a first critical step consists in the extraction and subsequent purification of the nucleic acids initially contained in the sample, that is to say mainly of the nucleic acids contained in the various organisms making up the microflora of this sample.
• the quality of the purification of the extracted DNA is decisive on the result obtained.
• a second important step in a process for building a library of nucleic acids from an environmental sample is the evaluation of the genetic diversity of the extracted and purified nucleic acids.
• the development of a simple and reliable production step of pre-screening the extracted and purified DNA in order to verify that it accounts, at least partially, for the phylogenetic diversity of the organisms initially present in the sample starting point in fact makes it possible to determine the advantage or not of using the initial source of DNA extracted and purified for the construction of the bank of nucleic acids proper or on the contrary not to continue the construction of the bank d nucleic acids due to excessive artefacts introduced during the extraction and purification of nucleic acids.
• a third critical step is the insertion of the nucleic acids extracted and purified into vectors capable of integrating nucleic acids of selected length, on the one hand, and, on the other hand, of allowing their transfection or even the integration into the genome in specific cell hosts as well as, if necessary, allowing expression thereof in such cell hosts.
• the vectors capable of integrating large nucleic acids that is to say of size greater than 100 kb when the objective pursued consists in cloning and identification of one .
• complete operon capable of directing a complete pathway for biosynthesis of a compound of industrial interest, in particular of a compound of pharmaceutical or agronomic interest.
• nucleic acids means both DNA and RNA sequences as well as hybrid RNA / DNA sequences of more than 2 nucleotides, indifferently under the single strand or double strand shape.
• library or “collection” is used in the present description with reference to either a set of extracted and, where appropriate purified, nucleic acids, coming from a sample of the environment, to a set of recombinant vectors, each of the recombinant vectors of the set comprising a nucleic acid originating from the abovementioned set of nucleic acids extracted and, where appropriate purified, as well as to a set of recombinant host cells comprising one or more nucleic acids originating from the all of the abovementioned extracted and, where appropriate, purified nucleic acids, said nucleic acids being either carried by one or more recombinant vectors, or integrated into the genome of said recombinant host cells.
• environment sample is used to designate either a sample of aquatic origin, for example fresh or saline water, or a telluric sample originating from the surface layer of a soil, from sediments or from lower layers of the soil. (underground), as well as samples of eukaryotic organisms, possibly multicellular, of plant origin, from marine organisms or even insects and having an associated microflora, this associated microflora constituting organisms of interest .
• operon means a set of open reading frames whose transcription and / or translation is co-regulated by a single set of signals for regulating transcription and / or translation.
• an operon can also comprise said signals for regulating transcription and / or translation.
• metabolic pathway for the purposes of the invention or also “biosynthetic pathway” is meant a set of anabolic or catabolic biochemical reactions effecting the conversion of a first chemical species into a second chemical species.
• an antibiotic biosynthesis pathway is made up of all of the biochemical reactions converting primary metabolites into antibiotic intermediates, and subsequently into antibiotics.
• regulation sequence placed "in phase" in English operably linked
• transcription regulation sequence s
• the transcription regulation sequence are localized, relative to the nucleotide sequence d interest whose expression is sought, so as to allow the expression of said sequence of interest, the regulation of said expression being dependent on factors interacting with the regulatory nucleotide sequences.
• nucleotide sequence of interest whose expression is sought is placed "under the control" of the nucleotide sequences which regulate transcription.
• isolated in the sense of the present invention designates a biological material which has been removed from its original environment (the environment in which it is naturally located).
• a polynucleotide or a polypeptide naturally present in an organism is not isolated.
• the same polypeptide separated from its natural environment or the same polynucleotide separated from the adjacent nucleic acids in which it is naturally inserted into the genome of the organism, is isolated.
• Such a polynucleotide can be included in a vector and / or such a polynucleotide can be included in a composition and nevertheless remains in an isolated state, because the vector or the composition does not constitute its natural environment.
• purified does not require that the material be present in a form of absolute purity, exclusive of the presence of other compounds. Rather, it is a relative definition.
• a polypeptide or a polynucleotide is in the purified state after purification of the starting material of at least one order of magnitude, preferably 2 or 3 and preferably 4 or 5 orders of magnitude.
• the "percentage of identity" between two nucleotide or amino acid sequences can be determined by comparing two optimally aligned sequences, through a comparison window.
• the part of the nucleotide or polypeptide sequence in the comparison window can thus include additions or deletions (for example "gaps") with respect to the reference sequence (which does not include these additions or these deletions) so as to obtain an optimal alignment of the two sequences.
• the percentage is calculated by determining the number of positions at which an identical nucleic base or amino acid residue is observed for the two sequences (nucleic or peptide) compared, then by dividing the number of positions at which there is identity between the two bases or amino acid residues by the total number of positions in the comparison window, then multiplying the result by 100 to obtain the percentage of sequence identity.
• the optimal alignment of the sequences for the comparison can be achieved by computer using known algorithms contained in the package of the company WISCONSIN GENETICS SOFTWARE PACKAGE, GENETICS COMPUTER GROUP (GCG), 575 Science Doctor, Madison, WISCONSIN.
• the percentage of sequence identity may be carried out using the BLAST software (BLAST versions 1.4.9 of March 1996, BLAST 2.0.4. Of February 1998 and BLAST 2.0.6. Of September 1998 ), using only the default settings (SF AltschuI et al., J. Mol. Biol. 1990 215: 403-410, SF AltschuI et al., Nucleic Acids Res. 1997 25: 3389-3402).
• the query sequence and the databases used can be peptide or nucleic, any combination being possible.
• microparticles from samples of dry soil combine physicochemical properties favorable to the extraction of an optimal quantity of nucleic acids which, in their nature, could be representative of the genetic diversity of organisms initially present in the starting soil sample. It has been shown in particular that the method of direct extraction of nucleic acids according to the invention allows the extraction of DNA from rare microorganisms, such as certain rare Streptomyces or spore-forming microorganisms.
• micro-particles of the soil sample for the purposes of the present invention is meant particles derived from the sample having an average size of approximately 50 ⁇ m, that is to say on average between 45 and 55 .mu.m /.
• the micro-particles are obtained from soil samples previously dried or desiccated and then ground until micro-particles of average size between 2 ⁇ m and 50 ⁇ m, before resuspension in a liquid buffer medium of the microparticles obtained.
• Such a liquid buffer medium can consist of a nucleic acid extraction buffer, in particular a conventional DNA extraction buffer well known to those skilled in the art.
• the grinding of the soil sample into microparticles has the dual function of mechanically lysing the majority of the organisms present in the initial soil sample and of making non-lysed organisms accessible by this mechanical treatment at optional later stages of lysis. chemical and / or enzymatic.
• a first object of the invention consists of a method of preparing a collection of nucleic acids from a soil sample containing organisms, said method comprising a first step (l- (a)) of obtaining micro-particles by grinding the soil sample previously dried or desiccated, then suspending the micro-particles in a liquid buffer medium.
• the grinding step is carried out using an agate or tungsten ball device or even using a tungsten ring device.
• agate or tungsten ball device or even using a tungsten ring device.
• These devices are preferred because the hardness of materials such as agate or tungsten significantly facilitates the production of microparticles of the size specified above. For this reason, we will not preferentially choose, or even avoid, recourse to a glass ball grinding device, which has proved to be much less effective.
• the drying or classification of the soil sample can be carried out by any method known to those skilled in the art. For example, the raw soil sample can be dried at room temperature for 24 to 48 hours.
• the liquid buffer medium can consist of a medium for extracting the DNA present in the microparticles.
• An extraction buffer designated TENP containing 50 mM tris, 20 mM EDTA, 100 mM NaCl and 1% (weight / volume) of polyvinylpolypyrrolidone, at pH 9.0, will be used most preferably.
• the method of preparing a collection of nucleic acids from a soil sample is further characterized in that the step of obtaining microparticles by grinding the previously dried or desiccated soil sample is followed by a step 1- (b) of extraction of the nucleic acids present in the microparticles.
• a step of purifying the DNA extracted from the microparticles of the soil sample meeting the selectivity and yield criteria defined above comprises a treatment of the extracted DNA by a combination of two successive chromatography steps, respectively a molecular sieve chromatography and an anion exchange chromatography.
• step I- (b) of extraction of the nucleic acids is followed by a step l- (c) of purification of the nucleic acids extracted using the two steps of chromatography following:
• the chromatographic medium of the type "molecular sieve" of the nucleic acid purification step above consists of a Sephacryl type of chromatographic support ®
• the chromatographic medium anion exchange used in the second stage of purification of the extracted DNA is a media type Elutip ® d, or a chromatographic support with equivalent characteristics.
• Contaminants such as humic acids severely affect the analyzes and subsequent uses of the nucleic acids whose purification is sought.
• the micro-particles of the soil sample can be the subject of subsequent steps of chemical, enzymatic or physical lysis treatment, or else of a combination of chemical, enzymatic or physical treatments.
• step l- (a) is followed by the following steps:
• a sonication treatment of a device of the titanium micro-tip type, such as the 600 W Vibracell Ultrasound icator device sold by the company Bioblock or a Cup Horn type sonicator.
• the sonication stage is carried out at a power of 15 W for a period of 7 to 10 min and comprises successive sonication cycles, the actual sonication being carried out for 50% of the duration of each cycle.
• step l- (a) is followed by the following steps:
• the incubation step in the presence of lysozyme and achromopeptidase will be carried out at a final concentration of 0.3 mg / ml of each of the two enzymes, preferably for 30 minutes at 37 ° C.
• the SDS will be used at a final concentration of 1% and for an incubation time of 1 hour at a temperature of 60 ° C before centrifugation and precipitation.
• step l- (a) is followed by the following steps:
• the suspensions of soil micro-particles are vortexed and then homogenized by gentle agitation on a circular rotation agitator for a period of two hours before being frozen at ⁇ 20 ° C.
• the suspensions are again agitated violently by vortexing for 10 minutes, after thawing and before the sonication step.
• nucleic acids extracted by the embodiments of the direct nucleic acid extraction process described above are preferably purified according to the purification step consisting of a first pass on molecular sieve and then a subsequent pass. elution fractions obtained after chromatography on a molecular sieve on an anion exchange chromatographic support.
• said sample of the environment undergoes a first treatment such as to allow separation organisms contained in this sample, other macroconstituents in the sample.
• This second embodiment of the method for preparing a collection of nucleic acids according to the invention promotes the obtaining of large nucleic acids, which are practically impossible to obtain according to the first embodiment of the method according to the invention described above, the mechanical lysis step carried out to obtain micro-particles also having the effect of physically breaking the nucleic acids of the soil sample or of the nucleic acids contained in the organisms of the sample of ground.
• nucleic acids Obtaining large nucleic acids has been sought by the applicant with the aim of isolating and characterizing nucleic acids comprising, at least partially, all of the coding sequences belonging to the same operon capable of directing biosynthesis a compound of industrial interest.
• nucleic acids having a size greater than 100 are obtained by implementing the second embodiment of the method for preparing a collection of nucleic acids from a soil sample according to the invention.
• kb preferably greater than 200, 250 or 300 kb, and very preferably nucleic acids of size greater than 400, 500 or even 600 kb.
• nucleic acids from a sample of the environment according to the invention consists of a combination of four successive steps intended for obtaining nucleic acids having the characteristics described above.
• the environmental sample is a soil sample
• a first step of obtaining a suspension by dispersion of the soil sample in liquid medium favored the accessibility of the organisms contained in the sample without causing significant mechanical lysis of the cells.
• the first step of obtaining a dispersion of the above soil sample makes the sample organisms accessible to the outside environment and also allows a partial dissociation of the sample organisms and the macro-constituents. It thus makes possible a subsequent separation of the organisms initially contained in the sample from the other constituents of the latter.
• a preliminary treatment by grinding is necessary in order to make the organisms of the associated microflora accessible at the later stages of the process.
• the present process comprises a step of separation of the organisms from the other mineral and / or organic constituents obtained previously by centrifugation on a density gradient.
• the organisms thus separated are then subjected to a step of lysis and then extraction of the nucleic acids.
• the centrifugation step on a density gradient surprisingly made it possible to separate the cells of organisms from the soil particles contained in the suspension of the sample.
• a density gradient centrifugation of a soil sample made it possible to find, at the aqueous phase / gradient interface, a population of organisms representative of the diversity organisms present in the initial sample, because these organisms are of extremely variable volume, density and shape.
• a density gradient centrifugation of a soil sample made it possible to find, at the aqueous phase / gradient interface, a population of organisms representative of the diversity organisms present in the initial sample, because these organisms are of extremely variable volume, density and shape.
• a step of germination of the spores, in particular of actinomycetes is carried out, which has the effect of significantly increasing the amount of actinomycete DNA recovered.
• the last step consists of a step of purification of the nucleic acids thus extracted on a cesium chloride gradient.
• the purification of nucleic acids on the cesium chloride gradient allows a substantial, even complete elimination, of the substances making up the density gradient. This characteristic is decisive with regard to the subsequent use of the purified nucleic acids since the density gradient is known as a powerful enzymatic inhibitor, capable if necessary of inhibiting the catalytic activity of the enzymes used to prepare the insertion of the acids. nucleic acids extracted into vectors.
• the method for preparing a collection of nucleic acids from a sample of the environment containing organisms according to the invention comprises the following sequence of steps:
• step (ii) separation of the organisms from the other mineral and or organic constituents of the homogeneous suspension obtained in step (i) by centrifugation on a density gradient;
• step (iii) lysis of the microorganisms separated in step (ii) and extraction of the nucleic acids; (iv) purification of the nucleic acids on a cesium chloride gradient.
• the suspension of the soil sample is obtained by dispersing this sample by grinding using a device of the Waring Blender type or a device with equivalent characteristics.
• the sample suspension is obtained after three successive grindings of a duration of one minute each in a device of the Waring Blender type.
• the ground sample will be cooled in ice between each of the grindings.
• the organisms are then separated from the soil particles by centrifugation on a density cushion of the "Nycodenz” type, sold by the company Nycomed Pharma AS. (Oslo, Norway).
• the preferred centrifugation conditions are 10,000 g for 40 minutes at 4 ° C., advantageously in a rotor with movable bowls of the "TST 28.38 rotor” type sold by the company KONTRON.
• the ring of organisms localized, after centrifugation, at the interphase of the upper aqueous phase and the lower phase of Nycodenz is then removed and washed by centrifugation before resumption of the cell pellet in an appropriate buffer.
• Step (iii) of lysis of the organisms separated in step (ii) described above can be carried out in any manner known to those skilled in the art.
• the cells are lysed in a solution
• the actual DNA extraction can advantageously be carried out by adding a solution of lauryl sarcosyl (1% of the final weight of the solution) in the presence of proteinase K and incubation of the final solution at 37 ° C. for 30 minutes .
• the nucleic acids extracted in step (iii) are then purified on a cesium chloride gradient.
• the step of purification of the nucleic acids on a cesium chloride gradient is carried out by centrifugation at 35,000 revolutions / minute for 36 hours, for example on a rotor of the Kontron 65.13 type.
• said nucleic acids consist mainly, if not exclusively, of DNA molecules.
• the nucleic acids can be recovered after inclusion of the organisms, separated on a density gradient, in an agarose block and lysis, for example chemical and / or enzymatic, of the organisms included in the agarose block.
• Another subject of the invention consists of a collection of nucleic acids consisting of the nucleic acids obtained in step ll- (iv) of the process for preparing a collection of nucleic acids according to the invention or also obtained in the step (c) or a subsequent step of the process for preparing a collection of nucleic acids according to the invention.
• the invention also relates to a nucleic acid characterized in that it is contained in a collection of nucleic acids as defined above.
• such a nucleic acid constituting a collection of nucleic acids according to the invention is characterized in that it comprises a nucleotide sequence coding for at least one operon, or part of an operon. Most preferably, such an operon codes for all or part of a metabolic pathway.
• Example 9 describes the construction of a genomic DNA library from a strain of Streptomyces alboniger and its cloning in the shuttle cosmids pOS700l and pOS700R respectively. It has been shown according to the invention that in the DNA library produced in the integrative vector pOS700l nine clones contain nucleotide sequences belonging to the operon responsible for the puromycin biosynthetic pathway. Likewise, it could be identified within the DNA library produced in the replicative vector pOS 700R twelve. clones containing nucleotide sequences of the operon responsible for the puromycin biosynthetic pathway.
• certain integrative and replicative cosmids of the libraries produced present, after digestion with the restriction endonucleases Clal and EcoRV, a fragment with a size of 12 kb capable of containing all the sequences of the operon responsible for the biosynthesis pathway puromycin.
• a nucleic acid according to the invention contains, at least in part, nucleotide sequences of the operon responsible for the puromicyne biosynthetic pathway.
• Example 2 describes the construction of a DNA library according to a process in accordance with the present invention in a pBluescript SK ' vector from a soil contaminated with lindane.
• the recombinant vectors were transfected into cherscherichia coli DH10B cells and then the transformed cells were cultured in an appropriate culture medium in the presence of lindane. Screening of the transformed cell clones of the library made it possible to show that, out of 10,000 clones screened, 35 of them exhibited a degradation phenotype of lindane. The presence of the linA gene in these clones could be confirmed by PCR amplification using primers specific for this gene.
• the invention also relates to a nucleic acid containing a nucleotide sequence of the metabolic pathway causing the biodegradation of lindane. It is therefore clearly demonstrated, as described above, that a method of preparing a collection of nucleic acids from a soil sample containing organisms according to the invention as well as a method of preparing a collection of recombinant vectors containing the nucleic acids constituting the above-mentioned collection of nucleic acids was entirely suitable for the isolation and characterization of nucleotide sequences included in an operon.
• a further demonstration of the ability of a method according to the invention to identify coding nucleotide sequences involved in a biosynthetic pathway regulated in the form of an operon is further described below: this is cloning and some characterization of sequences coding for polyketide synthases involved in the biosynthetic pathway of polyketides, which belong to a family of molecules of which certain representatives are of major therapeutic interest, in particular antibiotic.
• the present invention therefore further relates to a nucleic acid constituting a collection of nucleic acids according to the invention, characterized in that it comprises the whole of a nucleotide sequence coding for a polypeptide.
• a nucleic acid constituting a collection of nucleic acids according to the invention is of prokaryotic origin.
• a nucleic acid constituting a collection of nucleic acids according to the invention comes from a bacterium or a virus.
• a nucleic acid constituting a collection of nucleic acids according to the invention is of eukaryotic origin.
• such a nucleic acid is characterized in that it comes from a fungus, a yeast, a plant or an animal.
• the method according to the invention thus consists in universally amplifying a 700 bp fragment located inside a 16 S ribosomal DNA sequence, then hybridizing the amplified DNA with an oligonucleotide probe of variable specificity and finally to compare the intensity of hybridization of the sample compared to an external standard range of DNA of known sequence or origin.
• the amplification prior to hybridization with the oligonucleotide probe makes it possible to quantify genera or species of microorganisms which are not abundant.
• amplification with universal primers makes it possible, during hybridization, to use a large series of oligonucleotide probes.
• the subject of the invention is also a method for determining the diversity of nucleic acids contained in a collection of nucleic acids, and very particularly of a collection of nucleic acids originating from a sample of the environment, preferably a sample of the soil, said method comprising the following steps:
• nucleic acids of the collection of nucleic acids to be tested with a pair of oligonucleotide primers hybridizing to any bacterial 16 S ribosomal DNA sequence
• oligonucleotide probe or a plurality of oligonucleotide probes, each probe specifically hybridizing with a 16 S ribosomal DNA sequence common to a kingdom, an order, a subclass or a bacterial genus;
• a first pair of primers hybridizing with universally conserved regions of the 16 S ribosomal RNA gene consists respectively of the primers FGPS 612 (SEQ ID No 12) and FGPS 669 (SEQ ID No 13).
• a second embodiment of a pair of primers preferred according to the invention consists of the pair of universal primers 63 f (SEQ ID No. 22) and 1387r (SEQ ID No. 23).
• the amplification step using a pair of universal primers can be carried out on a collection of recombinant vectors into each of which has been inserted a nucleic acid from the collection of nucleic acids considered, prior to the hybridization step with oligonucleotide probes specific for a kingdom, an order, a subclass or a particular bacterial genus.
• Such a method for determining the diversity of nucleic acids contained in a collection is very particularly applicable to collections of nucleic acids obtained in accordance with the teaching of the present description.
• Example 3 details a method for preparing a collection of nucleic acids from a soil sample containing organisms, comprising a step of indirect DNA extraction by dispersing a soil sample before separation of the cells on a Nycodenz gradient, lysis of the cells then purification of the DNA on a cesium chloride gradient.
• the nucleic acid collection thus obtained was used as such or in the form of inserts in cosmid-type vectors in an amplification process using the aforementioned universal primers of the 16 S rDNA, then the DNAs. amplified were subjected to a detection step using oligonucleotide probes of sequences SEQ ID No. 14 to SEQ ID No. 21 which are presented in Table 4.
• nucleic acids 106 also form part of the invention, as well as the nucleic acids having at least 99%, preferably 99.5% or 99.8% identity. nucleic acids with the nucleic acids comprising the sequences SEQ ID No. 60 to SEQ ID No. 106. Such sequences can be used in particular as probes to screen clones of a DNA library and thus identify those, among the clones of the library, which contain such sequences, these sequences being likely to be in the vicinity of coding sequences of interest, such as sequences coding for enzymes involved in the pathway for biosynthesis of antibiotic metabolites, for example polyketides.
• nucleic acids contained in a collection of nucleic acids according to the invention come from ⁇ -proteobacteria, ⁇ -proteobacteria, ⁇ -proteobacteria, ⁇ -proteobacteria, actinomycetes as well as a genus related to acidobacterium.
• Each of the nucleic acids contained in a collection of nucleic acids prepared in accordance with the invention can be inserted into a cloning and / or expression vector.
• vectors known from the prior art can be used, such as viral vectors, phages, plasmids, phagemids, cosmids, phosmids, BAC type vectors, P1 bacteriophages, BAC type vectors, YAC type vectors, yeast plasmids or any other vector known to the state of technique by those skilled in the art.
• vectors allowing stable expression of the nucleic acids of a DNA library.
• such vectors preferably include transcription regulation sequences which are located in phase ("operably linked") with the genomic insert so as to allow the initiation and / or regulation of the expression of at least part of said DNA insert.
• the invention also relates to a process for the preparation of a collection of recombinant vectors characterized in that the nucleic acids obtained in step ll- (iv) or in step l- (c ) or any other subsequent step in a process for preparing a collection of nucleic acids from a soil sample containing organisms according to the invention are inserted into a cloning and / or expression vector.
• the nucleic acids constituting a collection of nucleic acids according to the invention can be separated according to their size, for example by electrophoresis on a gel. agarose, if necessary after digestion using a restriction endonuclease.
• the average size of the nucleic acids constituting a collection of nucleic acids according to the invention can be made of a substantially uniform size by the implementation of a physical rupture step prior to their insertion into the cloning and / or expression vector.
• a step of physical or mechanical rupture of the nucleic acids can consist of successive passages of the latter, in solution, in a metal channel of approximately 0.4 mm in diameter, for example the channel of a syringe needle having a such diameter.
• the average size of the nucleic acids can in this case be between 30 and 40 kb in length.
• the construction of the preferred vectors according to the invention is shown in FIGS. 25 (integrative cosmid conjugate) and 26 (integrative BAC).
• Cloning and / or expression vectors which can advantageously be used for the insertion of nucleic acids contained in a DNA collection or library according to the invention are in particular the vectors described in European patent N ° EP-0 350 341 and in US Pat. No. 5,688,689, such vectors being specially adapted for the transformation of strains of actinomycetes.
• Such vectors contain, in addition to a DNA sequence of the insert, an att attachment sequence as well as a DNA sequence coding for an integrase (int sequence) functional in the strains of actinomycetes.
• the applicant first sought to increase expression of the integrase gene by substituting for the initial transcription promoter a transcription promoter capable of significantly increasing the number of integrase transcripts.
• the applicant was able to show that the lack of stability of the integrase transcripts was caused by deficits in the termination of the transcription of the corresponding messenger RNA.
• the applicant then inserted a terminator site placed downstream of the sequence coding for the integrase of the vector so as to obtain a messenger RNA of determined size.
• the insertion of an additional termination signal downstream of the nucleotide sequence coding for the integrase of the vector has made it possible to obtain a family of integrative vectors of cosmid type and of BAC type.
• the terminating site is placed downstream of the att attachment site.
• the applicant has developed new conjugate vectors and new replicative vectors of the cosmid type and new conjugate vectors of the BAC type which can advantageously be used for the insertion of the nucleic acids constituting a collection of nucleic acids. prepared according to the process of the invention.
• vectors of the cosmid type capable of receiving inserts having a maximum size of approximately 50 kb.
• cosmid vectors are very particularly suitable for the insertion of nucleic acids constituting a collection of nucleic acids obtained according to the method of the invention comprising a first step of direct DNA extraction by mechanical lysis of the organisms contained in the initial soil sample.
• BAC type vectors capable of receiving DNA inserts of such size.
• Such BAC type vectors are very particularly suitable for the insertion of the nucleic acids constituting a collection of nucleic acids obtained in accordance with the method according to the invention in which the first step consists of an indirect extraction of DNA. by prior separation of the organisms contained in the initial soil sample and elimination of the macro-constituents of said soil sample.
• vectors of the BAC type are advantageously used for the insertion of large nucleic acids containing, at least partially, the nucleotide sequence of an operon.
• the process for preparing a collection of recombinant cloning and / or expression vectors according to the invention is further characterized in that the cloning and / or expression vector is of the plasmid type.
• such a method is characterized in that the cloning and / or expression vector is of the cosmid type.
• it may be a replicative cosmid in E. coli and integrative in Streptomyces.
• An entirely preferred cosmid vector corresponding to such a definition is the cosmid pOS700l described in Example 3.
• the cosmid vector is conjugative and integrative in Streptomyces.
• conjugate vectors of the cosmid type or of the BAC type which include in their nucleotide sequences a motif recognized by the cellular enzymatic machinery called "origin of conjugation" are used whenever it is desired to avoid the use of techniques. heavy and not very automated transformation.
• the transfection of vectors initially hosted by E. coli cells into Streptomyces cells conventionally requires a stage of recovery of the recombinant vector contained in Esche chia coli cells, and its purification prior to the stage of transformation of protoplasts of Streptomyces. It is commonly accepted that a transfection of a set of 1000 clones of Escherichia coli in Streptomyces requires obtaining approximately 8000 clones so that each clone of E. coli has a chance to be represented.
• a transfection step by conjugation of a vector hosted by E.coli to Streptomyces cells requires the same number of clones of each of the microorganisms, the conjugation step taking place "clone to clone" and also not understanding the technical difficulties associated with the step of transferring genetic material by transformation of protoplasts, for example in the presence of polyethylene glycol.
• novel conjugation vectors of cosmid type and of BAC type have been developed according to the invention so as to allow maximum efficiency of the conjugation step.
• the new conjugative vectors according to the invention have been constructed by placing a selection marker gene at the end of the DNA of the vector which is lastly transferred to the receptor bacteria.
• Conjugative and integrative cosmids in Streptomyces preferred according to the invention are the cosmids pOSV303, pOSV306 and pOSV307 described in Example 5.
• a process for preparing a collection of recombinant vectors according to the invention is implemented. works with a replicating cosmid both in E. coli and in Streptomyces. Such a cosmid is advantageously the cosmid pOS 700R described in Example 6.
• the above method can be implemented with a replicative cosmid in E. coli and Streptomyces and conjugative in Streptomyces.
• Such a replicative and conjugative cosmid can be obtained from a replicative cosmid according to the invention, by the insertion of a appropriate transfer origin, such as RK2, as described in Example 5 for the construction of the vector pOSV303.
• a transfer origin such as RK2
• BAC-type cloning and / or expression vector use is made of a BAC-type cloning and / or expression vector.
• the vector of the BAC type is integrative and conjugative in Streptomyces.
• such an integrative and conjugative BAC vector in Streptomyces is the BAC vector pOSV 403 described in Example 8, or else the BAC vectors pMBD-1, pMBD-2, pMBD-3, pMBD-4, pMBD-5 and pMBD-6 described in Example 15.
• the invention further relates to a recombinant vector characterized in that it is chosen from the following recombinant vectors: a) a vector comprising a nucleic acid constituting a collection of nucleic acids according to the invention; b) a vector as obtained according to a method eliminating any recourse to the action of a restriction endonuclease on the DNA fragment to be inserted, as described previously.
• the invention also relates to a vector chosen from the following vectors:
• the invention further relates to a collection of recombinant vectors as obtained according to any one of the methods according to the invention.
• a method for preparing a recombinant cloning and / or expression vector according to the invention is characterized in that the insertion of a nucleic acid into the cloning and / or expression vector , includes the following steps:
• the first homopolymeric nucleic acid is of poly (A) or poly (T) sequence
• - the second homopolymeric nucleic acid is of poly (T) or poly (A) sequence.
• the homopolymeric nucleic acids have a length of between 25 and 100 nucleotide bases, preferably between 25 and 70 nucleotide bases.
• the process for the preparation of a recombinant cloning and / or expression vector described above is particularly suitable for the construction of DNA libraries in BAC type vectors.
• said method is further characterized in that the size of the nucleic acid to be inserted is at least 100 kb, and preferably at least 200, 300, 400, 500 or 600 kb.
• Such a preparation process is therefore particularly suitable for the insertion of the nucleic acids contained in a collection of nucleic acids obtained according to the process of the invention.
• Such a method of preparing a recombinant cloning and / or expression vector according to the invention is characterized in that the step of inserting a nucleic acid in said cloning and / or expression vector comprises the following steps:
• the elimination of the outgoing 3 ′ sequences is carried out using an exonuclease, such as the Klenow enzyme.
• the filling of the outgoing 5 ′ sequences is carried out using a polymerase, and very preferably T4 polymerase, in the presence of the four nucleotide triphosphates.
• a process for preparing a recombinant cloning and / or expression vector by eliminating the outgoing 3 'sequences and filling in the outgoing 5' sequences as described above is particularly suitable for the construction of DNA libraries from of cosmid-like vectors.
• oligonucleotides comprising one or more rare restriction sites are added to the vector at the level of the cloning site of the DNA to be inserted, in accordance with the teaching of Example 10. This addition of oligonucleotides facilitates the subsequent recovery of the inserts without cleavage of the latter.
• any type of host cell can be used for transfection or transformation with a nucleic acid or a recombinant vector according to the invention, in particular a prokaryotic or eukaryotic host cell, use will preferably be made of host cells whose physiological, biochemical and genetic characteristics are well characterized, easily cultivable on a large scale and whose culture conditions for the production of metabolites are well known.
• the host cell receiving a nucleic acid or a recombinant vector according to the invention is phylogenetically close to the donor organisms initially contained in the sample from the environment from which the nucleic acids originate.
• a host cell according to the invention must have a use of codons similar to, or at least close to, the donor organisms initially present in the environmental sample, especially the soil sample.
• the size of the DNA fragments capable of carrying the nucleotide sequences of interest sought can be variable.
• enzymes encoded by genes of average size of 1 kb can be expressed from small inserts while the expression of secondary metabolites will require the maintenance in the host organism of fragments of much larger size, for example from 40 kb to more than 100 kb, 200 kb, 300 kb, 400 kb or 600 kb.
• Eschenchia coli host cells are a preferred choice for cloning large DNA fragments.
• strains of Eschenchia coli can be advantageously used for the construction of a DNA library according to the invention, such as the strains E.coli Sure, E.coli DH5 ⁇ , or even E.coli 294 ( ATCC No. 31446).
• E. coli host cells can in all cases constitute transient hosts in which the recombinant vectors according to the invention can be maintained with great efficiency, the genetic material being able to be easily manipulated and archived and stably.
• cellular hosts can also be advantageously used, such as cells of Bacillus, Pseudomonas, Streptomyces, Myxococcus, Aspergillus nidulans or even Neurospora crassa.
• Streptomyces lividans cells can be used with success and constitute expression systems complementary to Eschenchia coli.
• Streptomyces lividans is a model for studying the genetics of Streptomyces and has also been used as a host for heterologous expression of many secondary metabolites. Streptomyces lividans, has in common with other actinomycetes such as Streptomyces coelicolor, Streptomyces griseus, Streptomyces fradiae, as well as Streptomyces griseochromogenes, precursor molecules and regulatory systems necessary for the expression of all or part of the complex biosynthetic pathways, such as for example the pathway for biosynthesis of polyketides or the pathway for biosynthesis of non-ribosomal polypeptides representing classes of molecules of very diverse structures.
• actinomycetes such as Streptomyces coelicolor, Streptomyces griseus, Streptomyces fradiae, as well as Streptomyces griseochromogenes, precursor molecules and regulatory systems necessary for the expression of all or part of the complex biosynthetic pathways
• Streptomyces lividans also has the advantage of accepting foreign DNA with high transformation efficiencies.
• the invention also relates to a recombinant host cell comprising a nucleic acid according to the invention, constituting a collection of nucleic acids prepared according to a process in accordance with the invention, or a recombinant host cell comprising a recombinant vector as defined above.
• a recombinant cell according to the invention is a bacterium, and very preferably a bacterium chosen from E.coli and Streptomyces.
• a recombinant host cell according to the invention is characterized in that it is a yeast or else a filamentous fungus.
• the invention also relates to a collection of recombinant host cells, each of the constituent host cells of the collection comprising a nucleic acid originating from a collection of nucleic acids produced according to a method for preparing a collection of nucleic acids. from a soil sample containing organisms as described above.
• the invention also relates to a collection of recombinant host cells, each of the constituent host cells of the collection comprising a recombinant vector according to the invention. Due to the large size of the inserts it is necessary to have maximum processing efficiency. For this purpose, a Streptomyces lividans receptor strain constitutively expressing the integrase of pSAM2 in order to promote the site-specific integration of the vector is preferred. For this, the int gene under the control of a strong promoter is integrated into the chromosome. The overproduction of integrase does not induce excision phenomena (Raynal et al., 1998).
• a collection of recombinant host cells was obtained after transfection of the host cells by a collection of recombinant vectors each containing a nucleic acid insert from a collection of nucleic acids prepared according to the method according to the invention .
• DNA fragments obtained according to the method of the invention in which a step of indirect DNA extraction from the organisms contained in the soil sample is implemented were first of all cloned into the integrative cosmid pOS700l.
• the step of inserting the DNA fragments into the integrative cosmid pOS700l was carried out according to the method of the invention in which tails of homopolymeric polynucleotides poly (A) and poly (T) were added at the end 3 'respectively of the vector nucleic acid and DNA fragments to be inserted.
• the recombinant vectors thus constructed were packaged in lambda phage heads and the phages obtained were used to infect E. coli cells according to techniques well known to those skilled in the art.
• This bank of clones was screened with pairs of primers specific for a nucleotide sequence coding for a enzyme involved in the polyketide biosynthesis pathway, the PKS type I enzyme, also known as ⁇ -ketoacyl synthase.
• polyketides constitute a chemical class of great structural diversity comprising a large number of molecules of pharmaceutical interest such as tylosin, monensin, vermectin, erythromycin, doxorubicin or even FK506.
• Polyketides are synthesized by condensation of acetate molecules under the action of enzymes called polyketide synthases (PKSs).
• PKSs polyketide synthases
• Polyketide synthases type II are generally involved in the synthesis of polycyclic aromatic antibiotics and catalyze the condensation of acetate units iteratively.
• Polyketide synthases type I are involved in the synthesis of macrocyclic polyketides or macrolides and constitute multifunctional modular enzymes.
• nucleotide sequences coding for polyketide synthases of type I made it possible to identify recombinant clones containing DNA inserts comprising a sequence nucleotide encoding new polyketide synthases.
• the nucleotide sequences coding for these new polyketide synthases are referenced as the sequences SEQ ID No. 33 to SEQ ID No. 44 and SEQ ID No. 115 to SEQ ID No. 120.
• Another subject of the invention consists of a nucleic acid coding for a new polyketide synthase I, characterized in that it comprises one of the nucleotide sequences SEQ ID No. 34 to SEQ ID No. 44 and SEQ ID No. 115 at SEQ ID N ° 120.
• such a nucleic acid is in an isolated and / or purified form.
• the invention also relates to a recombinant vector comprising a polynucleotide comprising one of the sequences SEQ ID No. 34 to SEQ ID No. 44 and SEQ ID No. 115 to SEQ ID No. 120
• the invention also relates to a recombinant host cell comprising a nucleic acid chosen from polynucleotides comprising one of the nucleotide sequences SEQ ID No. 34 to SEQ ID No. 44 and SEQ ID No. 115 to SDEQ ID No. 120 as well than to a recombinant host cell comprising a recombinant vector into which is inserted a polynucleotide comprising one of the nucleotide sequences SEQ ID No. 34 to SEQ ID No. 44 and SEQ ID No. 115 to SEQ ID No. 120.
• the recombinant vectors containing a DNA insert coding for a new polyketide synthase type I according to the invention are vectors for cloning and expression.
• a recombinant host cell as described above is a bacterium, a yeast or even a filamentous fungus.
• amino acid sequences of new polyketide synthases originating from organisms contained in a soil sample have been deduced from the nucleotide sequences SEQ ID No. 34 to SEQ ID No. 44 AND SEQ ID No. 115 to SEQ ID No. 120 below. above. These are polypeptides comprising one of the amino acid sequences SEQ ID No. 48 to SEQ ID No. 59 and SEQ ID No. 121 to 126.
• the invention also relates to new polyketide synthases comprising an amino acid sequence chosen from the sequences SEQ ID No. 48 to SEQ ID No. 59 and SEQ ID No. 121 to SEQ ID No. 126.
• nucleotide sequence SEQ ID No 114 which comprises six open reading frames which respectively encode the polypeptides of sequences SEQ ID No 121 to SEQ ID No 126.
• nucleotide sequence SEQ ID No. 113 of the cosmid a26G1 which contains the sequence complementary to the sequence SEQ ID No. 114.
• Genomic DNA was also extracted and amplified according to the invention from pure bacterial strains, such as
• PCR amplification of the DNA of each of the bacterial strains described above was carried out using pairs of primers specific for polyketide synthase type I nucleic sequences.
• New bacterial polyketide synthase type I genes were thus isolated and characterized. These are the nucleic sequences of sequences SEQ ID No. 30 to SEQ ID No. 32.
• a subject of the invention is therefore also nucleotide sequences coding for new type I polyketide synthases chosen from polynucleotides comprising one of the nucleotide sequences SEQ ID No. 30 to SEQ ID No. 32.
• Also part of the invention are recombinant vectors comprising the nucleotide sequences coding for new polyketide synthase type I defined above.
• the invention also relates to recombinant host cells characterized in that they contain a nucleic acid coding for a new polyketide synthase type I comprising a nucleotide sequence chosen from the sequences SEQ ID No. 30 to SEQ ID No. 32 as well as Recombinant host cells comprising a recombinant vector as defined above.
• the subject of the invention is also polypeptides encoded by sequences comprising the nucleic acids SEQ ID Nos. 30 to 32, and more precisely polypeptides comprising the amino acid sequences SEQ ID No. 47 to SEQ ID No. 50.
• the subject of the invention is also a process for the production of a polyketide synthase type I according to the invention, said process for producing comprising the following steps: - Obtaining a recombinant host cell comprising a nucleic acid coding for a polyketide synthase type I comprising a nucleotide sequence chosen from the sequences SEQ ID No. 33 to SEQ ID No. 44, SEQ ID No. 30 to SEQ ID No. 32 and SEQ ID No. 115 to SEQ ID No. 120;
• the new type I polyketide synthases obtained according to the method described above can be characterized by fixation on an immunoaffinity chromatography column on which antibodies recognizing these polyketide synthases have been immobilized beforehand.
• HPLC high performance liquid chromatography techniques
• reverse phase chromatography techniques such as reverse phase chromatography techniques.
• anion or cation exchange chromatography well known to those skilled in the art.
• polyketide synthases recombinant or non-recombinant, according to the invention can be used for the preparation of antibodies.
• the invention therefore also relates to an antibody specifically recognizing a polyketide synthase type I according to the invention or a peptide fragment of such a polyketide synthase.
• the antibodies according to the invention can be monoclonal or polyclonal.
• Monoclonal antibodies can be prepared from of hybridoma cells according to the technique described by KOHLER and MILSTEIN C. (1975), Nature, Vol. 256: 495.
• the polyclonal antibodies can be prepared by immunization of a mammal, in particular mice, rats or rabbits with a polyketide synthase of type I according to the invention, if necessary in the presence of an adjuvant compound of immunity, such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide or a compound from the family of muramyl peptides.
• an adjuvant compound of immunity such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide or a compound from the family of muramyl peptides.
• the antibody fragments such as the Fab, Fab ', F (ab') 2 fragments, or even the single chain antibody fragments containing the variable part (ScFv) described by MARTINEAU et al. (1998) J. Mol. Biol., Vol.
• the antibody preparations according to the invention are useful in particular in qualitative or quantitative immunological tests aimed either at simply detecting the presence of a polyketide synthase type I according to the invention, or at quantifying the amount of this polyketide synthase, for example in the culture supernatant or the cell lysate of a bacterial strain capable of producing such an enzyme.
• Another subject of the invention consists of a method for detecting a polyketide synthase type I according to the invention or a peptide fragment of this enzyme, in a sample, said method comprising the steps of:
• the invention also relates to a kit or kit for detecting a polyketide synthase type I according to the invention in a sample, comprising: a) an antibody according to the invention; b) where appropriate, reagents necessary for the detection of the antigen / antibody complex possibly formed.
• An antibody directed against a polyketide synthase type I according to the invention can be labeled using a detectable isotopic or non-isotopic marker, according to methods well known to those skilled in the art.
• the subject of the invention is therefore a method for detecting a nucleic acid of determined nucleotide sequence, or of nucleotide sequence structurally related to a determined nucleotide sequence, in a collection of recombinant host cells according to the invention, characterized in that it includes the following stages:
• the invention also relates to a method for detecting a nucleic acid, of determined nucleotide sequences, or of nucleotide sequences structurally related to a determined nucleotide sequence, in a collection of recombinant host cells according to the invention, characterized in that it comprises the following steps:
• the recombinant clones of interest were detected by their phenotype corresponding to their ability to degrade lindane.
• the isolated clones and / or clone sets of the prepared DNA library were cultured in a culture medium in the presence of lindane and the degradation of lindane was observed by the formation of a halo disorder in the immediate environment of the cells.
• the invention also relates to a method for identifying the production of a compound of interest by one or more recombinant host cells in a collection of recombinant host cells according to the invention, characterized in that it comprises the following steps:
• the subject of the invention is also a method for selecting a recombinant host cell producing a compound of interest from a collection of recombinant host cells according to the invention, characterized in that it comprises the following steps:
• the invention also relates to a process for the production of a compound of interest, characterized in that it comprises the following steps:
• a compound of interest according to the invention may consist of a polyketide produced by the expression of at least one nucleotide sequence comprising a sequence chosen from the sequences SEQ ID N ° 33 to 44, SEQ ID N ° 30 to 32 and SEQ ID N ° 1 15 to SEQ ID N ° 120.
• the invention also relates to a composition comprising a polyketide produced by the expression of at least one nucleotide sequence comprising a sequence chosen from the sequences SEQ ID No. 33 to SEQ ID No. 44, SEQ ID No. 30 to SEQ ID N ° 32, and SEQ ID N ° 115 to SEQ ID N ° 120.
• a polyketide produced by the expression of at least one nucleotide sequence above is preferably the product of the activity of several coding sequences included within a functional operon whose translation products are the different enzymes necessary for the synthesis of a polyketide, one of the above sequences being understood and expressed in said operon.
• Such an operon comprising a nucleic acid sequence according to the invention coding for a polyketide synthase can be constructed for example according to the teaching of Borchert et al. (1992).
• the invention also relates to a pharmaceutical composition
• a pharmaceutical composition comprising a pharmacologically active amount of a polyketide according to the invention, where appropriate in combination with a pharmaceutically compatible vehicle.
• Such pharmaceutical compositions will advantageously be suitable for the administration, for example parenterally, of an amount of a polyketide synthesized by a polyketide synthase of type I according to the invention ranging from 1 ⁇ g / kg per day to 10 mg / kg per day, preferably at least 0.01 mg / kg per day and most preferably between 0.01 and 1 mg / kg per day.
• compositions according to the invention can be administered either orally, rectally, parenterally, intravenously, subcutaneously or even intradermally.
• the invention also relates to the use of a polyketide obtained by the expression of a polyketide synthase type I according to the invention for the manufacture of a medicament, in particular of a medicament with antibiotic activity.
• FIG. 1 illustrates the diagram of the different lysis steps carried out according to protocols 1, 2, 3n 4a, 4b, 5a, and 5b described in Example 1.
• Figure 2 illustrates one. 0.8% agarose gel electrophoresis of DNA extracted from 300 mg of soil no. 3 (Côte St André) after various lysis treatments (protocols 1 to 5, see Fig. 1).
• M lambda phage molecular weight marker
• Figure 3 illustrates the proportion of different genera of actinomycetes cultivated following treatments 1 to 5 (see Fig. 1).
• the number of cfu colony forming unit
• Figure 4 illustrates the. recovery of lambda phage DNA digested with Hind ⁇ added in soils at different concentrations before (G) or after (G * ) grinding.
• T treatments thermal shock
• S sonication
• the quantification was carried out by phospho-imager analysis after dot-blot hybridization. A sample of each soil was used for each concentration of lambda phage added. The soil characteristics are reproduced in Table 1. The samples corresponding to 10 and 15 ⁇ g of added DNA were not treated.
• Figure 5 illustrates the PCR amplification of DNAs extracted from soil No. 3 according to protocols 1, 2, 3, 5a and 5b.
• Primers FGPS 122 and FGPS 350 (Table 2) were used to target Streptosporangium spp. native. The DNA extracts were used undiluted or diluted 1/10 th and 1/100 th .
• M 123 bp molecular weight marker (Gibco BRL),
• C amplification control without DNA.
• Figure 6 illustrates the quantities of DNA extracted after inoculation of spores (a) or mycelium (b) of S. lividans OS48.3 inoculated in soils at different concentrations.
• the amount of mycelium added to the soil corresponds to the number of spores inoculated into the germination medium. About 50% of the spores have germinated, the number of cells or genomes contained in the hyphae of the germinated spores has not been determined. The quantities of spores and mycelium inoculated are therefore not directly comparable.
• the extraction protocol was carried out according to protocol 6 (cf. equipment and methods section).
• the symbol (') indicates that RNA has been included in the extraction buffer.
• the target DNA was amplified by PCR with the primers FGPS 516 and FGPS 517, the quantification was carried out by phosphoimager after hybridization in dot blot using the probe FGPS 518. A sample of each sol was used for each concentration of hyphae or spores. The soil characteristics are described in Table 1.
• FIG. 7 represents the phylogenetic tree obtained by the Neighbor Joining algorithm, positioning the 16S rDNA sequences contained in the soil DNA bank, with respect to cultured reference bacteria.
• gray the sequences from the clone pools of the bank.
• the bootstrap values are indicated at the node level, after resampling 100 repetitions.
• the scale bar indicates the number of substitutions per site.
• the access number of the sequences in the Genbank database is indicated in brackets.
• FIG. 8 represents a diagram of the vector pOSint 1.
• FIG. 9 represents a diagram of the vector pWED1.
• FIG. 10 represents a diagram of the vector pWE15 (ATCC No. 37503).
• FIG. 11 represents a diagram of the vector pOS 700I.
• FIG. 12 represents a diagram of the vector pOSV010.
• FIG. 13 represents the fragment containing a "cos" site inserted into the plasmid pOSV010 during the construction of the vector pOSV 303.
• FIG. 14 represents a diagram of the vector pOSV 303.
• FIG. 15 represents a diagram of the vector pE116.
• FIG. 16 represents a diagram of the vector pOS 700 R.
• FIG. 17 represents a diagram of the vector pOSV 001.
• FIG. 18 represents the diagram of the vector pOSV 002.
• FIG. 19 represents a diagram of the vector pOSV 014.
• FIG. 20 represents a diagram of the vector pBAC 11.
• FIG. 21 represents a diagram of the vector pOSV 403.
• FIG. 22 represents the DNA electrophoresis gels of the library after digestion with the enzymes BamHI and Dral of the positive clones of the library screened with the oligonucleotides PKS-I.
• FIG. 23 illustrates the production of puromycin by the recombinants of S. lividans compared to the production of the wild strain S. alboniger.
• Figure 24 illustrates Alignment of soil PKSs with conserved active sites of other PKSs.
• the references for each peptide are indicated.
• the beta-ketoacyl synthase domains were aligned using the PILEUP program from GCG (Wisconsin Package Version 9.1, Genetics Computer Group, Madison, Wisc).
• Figure 25 illustrates the construction of a conjugative integrative cosmid.
• Figure 26 illustrates the construction of an integrative conjugative BAC.
• FIG. 27 illustrates the construction diagram of the vector pOSV 308.
• FIG. 28 illustrates the construction diagram of the vector POSV306.
• FIG. 29 illustrates the construction diagram of the vector pOSV307.
• FIG. 30 illustrates the construction diagram of the vector PMBD-1.
• FIG. 31 presents a detailed map of the plasmid pMBD-2 as well as a diagram of construction of the vector pMBD-3.
• Figure 32 illustrates a detailed map of plasmid pMBD-4.
• FIG. 33 illustrates the scheme of construction of the plasmid pMBD-5 from the plasmid pMBD-1.
• Figure 34 illustrates the detailed map of the pBTP-3 vector.
• Figure 35 illustrates the construction scheme of the vector pMBD-6 from the vector pMBD-1.
• FIG. 36 illustrates the map of cosmid a26G1, the DNA insert of which contains open reading frames coding for several polyketide synthase.
• FIG. 37 is a diagram representing the DNA insert (strand +) of the cosmid a26G1, on which the different reading frames coding for several polyketide synthase are positioned.
• EXAMPLE 1 Method for preparing a collection of nucleic acids from a soil sample containing organisms, comprising a step of direct extraction of DNA from the soil sample.
• the content of clay and organic matter ranges respectively from 9 to 47% and from 1.7 to 4.7%, the pH varying from 4.3 to 5.8.
• Soil samples were collected from the surface layer 5-10 cm deep. All visible roots have was removed and the soils were stored at 4 ° C for a few days if necessary, after which they were dried for 24 hours at room temperature and sieved (average mesh size 2 mm) before being kept for several months at 4 ° C.
• the avirulent strain of Bacillus anthracis was used as an inoculum of bacterial cells. Bacillus anthracis was multiplied on a culture broth of the "trypticase soy broth” (TSB) type (Biomérieux, Lyon, France) for approximately 6 hours, checking that the DO ⁇ oo was kept below 0.6. These conditions allow the development of vegetative cells without spore formation (Patra et al., (1996), FEMS Immunol. Médical Microbiology, vol.15: 223-231.). The spores of Streptomyces lividans OS48.3 (CLERC-BARDIN et al.
• Streptosporangium fragile AC1296 (Institute Pushino, Moscow) were cultivated according to techniques described by HICKEY and TRESNER (1952).
• the DNA of the spores and hyphae of S. Lividans was extracted from the pure cultures according to the lysis protocol 6 described below (except that no grinding was carried out), while the spores of S. hygroscopicus and S. fragile were extracted by chemical / enzymatic lysis (Hintermann et al., 1981).
• a TENP buffer 50 mM Tris, 20 mM EDTA, 100 mM NaCl, 1% w / v of polyvinylpolypyrrolidone developed by PICARD (1992) was used. Similar buffers were later used by d '' other authors (CLEGG et al., 1997; KUSKE et al., 1998; ZHOU et al., 1996).
• Tris and EDTA protect DNA from nuclease activity
• NaCI provides a dispersing effect
• PVPP absorbs humic acids and other phenolic compounds
• protocol 6 This protocol was used to quantify DNA from native Actinomycetes and DNA from Gram-positive bacteria inoculated in selected soils. In all cases, the soil samples were dried and sieved as described above. After grinding, 0.5 ml of TENP buffer was added to 200 mg dry weight of soil (except for protocol 1 in which the buffer was added to unground ground).
• the soil suspensions were Vortexed for ten minutes and centrifuged (4000 g for five minutes), after which an aliquot fraction (25 ⁇ l) of the supernatant was analyzed by gel electrophoresis (0.8% agarose).
• Protocol 1 No lysis treatment.
• TENP buffer was added to unground ground, a DNA extraction step was performed as described above.
• the TENP buffer is then added and the DNA is extracted as described above.
• the size distribution of the DNA fragments is similar regardless of the method used.
• Protocol 3 In protocols 3 to 5, the effectiveness of several other treatments of lysis subsequent to the grinding of the soil was tested, either separately or in different combinations. Protocol 3:
• This protocol is identical to protocol 2, except that it includes a homogenization step using an Ultraturrax type mixer (Janker and Kunkel, IKA Labortechnik, Germany) set at half the maximum speed for 5 minutes .
• PROTOCOLS 4a and 4b are PROTOCOLS 4a and 4b:
• Protocol 3 These protocols are identical to protocol 3 except for an additional sonication step.
• sonicator Two types were compared: a titanium microtip sonicator (600W Vibracell Ultrason icator, Bioblock, lllkirch, France) (Protocol 4a) and a Cup Horn type sonicator (protocol 4b).
• the Vibracell microtip producing ultrasound is in direct contact with the soil solution.
• the soil solution is stored in tubes which are placed in a water bath through which the ultrasound passes.
• the best compromise in terms of quantity of DNA extracted and size of fragments, consists of sonication with the titanium microtip and the Cup Horn type sonicator respectively for 7 and 10 minutes, by setting the power to 15 W and with active cycles at 50%.
• Protocol 6 is identical to protocol 5b except that, before sonication, the soil suspensions are subjected to a Vortex treatment and then agitated by rotation on a wheel for two hours before being frozen at - 20 ° C.
• Protocol 6 was used in the experiments in which the soils were seeded with bacterial cells as well as in the experiments in which the native actinomycetes were quantified (see below).
• the bacterial cells were counted using an epifluorescence microscope of the Zeiss Universal type with a 100x objective. For each soil type, three filters were counted, and at least 200 cells were counted on each of the filters.
• Actidione 50 mg / l
• nystatin 50 mg / ml
• Actinomycete colonies were counted after incubation for 15 days at 28 ° C.
• total CFU The total quantity of cultivable bacteria was also determined for each of the lysis protocols 1 to 5.
• the soil suspensions were diluted in series and seeded in triplicate on Bennett agar medium (WAKSMAN et al., 1961) supplemented with nystatin and actidione (each at 50 mg / l).
• Each Petri dish was covered with a cellulose nitrate filter (Millipore) and incubated for three days at 28 ° C. After the colony count on the membranes, the filters were removed and the petri dishes were again incubated for 7 days at 28 ° C and then counted again.
• a cellulose nitrate filter Millipore
• Dilutions corresponding respectively to 0, 2.5, 5, 7.5, 10 and 15 ⁇ g of DNA / g of dry weight of soil were prepared in volumes of 60 ⁇ l. These DNA dilutions were added to batches of 5g of dry soil which were subsequently vigorously mixed by vortexing for 5 minutes before grinding.
• Phage lambda DNA was also added to a soil before grinding at concentrations corresponding to 0, 10 and 15 ⁇ g of DNA / g of dry weight of the soil.
• the extraction buffer is added and the DNA is extracted according to protocol 2 (see above).
• RNA In order to determine whether the saturation of the nucleic acid adsorption sites of soil colloids could increase the recovery rate of DNA, sandy soil (soil no. 4) and the clay soil (soil No. 5) were incubated with an RNA solution before any other treatment.
• Commercial RNA from Saccharomyces cerevisiae
• the DNA was extracted according to protocol No. 2. It was subsequently determined that an identical effect of the addition of RNA on DNA recovery could be achieved by adding the RNA directly to the extraction buffer.
• RNA was then added at a concentration corresponding to 50 mg of RNA / g of dry weight of the soil.
• the hybridization membranes (GeneScreen plus, Life Science Products, Boston, United States of America) were prehybridized for at least 2 hours in 20 ml of a solution containing 6 ml of 20 x SSC, 1 ml of solution of DENHARDT's, 1 ml of 10% SDS and 5 mg of salmon sperm DNA.
• Hybridization was carried out overnight in the same solution in the presence of a probe labeled beforehand with two membrane washes in 2 x SSC buffer for 5 minutes at room temperature, then a third wash in 2 x SSC buffer, 0.1% SDS and a fourth wash in 1 x SSC buffer, 0.1% SDS for 30 minutes at hybridization temperature.
• the hybridization signals were quantified with a BIORAD radioanalytical imaging system (Molecular Analyst Software, BIORAD, Ivry S / Seine, France).
• This probe which hybridizes at positions 1392-1406 of the 16.c rDNA gene of E. coli (Amann et al. (1995)) was labeled at its ends with ATP ⁇ 32 P using a T4 polynucleotide kinase (BOEHRINGER MANNHEIM , Melan, France).
• a calibration curve was prepared from the DNA of E.coli DH5 ⁇ .
• the conversion of stones to bacteria in the soil required simplification, assuming that the average number of copies (rm) is 7, as for E.coli.
• HindIII digested lambda phage DNA was used to quantify the recovery of extracellular DNA. Unamplified extracts from soils, to which lambda phage DNA had been added, were hybridized with HindIII digested lambda phage DNA randomly labeled using the Klenow fragment (Boehringer Mannheim, Melan, France ).
• the amounts of DNA were calculated by interpolation from a calibration curve prepared with the purified DNA.
• Taq DNA polymerase (Appligene Oncor, France) was used according to the manufacturer's instructions.
• the PCR program used for all the amplifications is as follows: initial denaturation for 3 minutes at 95 ° C, then 35 cycles consisting of 1 minute at 95 ° C, 1 minute at 55 ° C and 1 minute at 72 ° C, followed by a final extension at 72 ° C for 3 minutes.
• the DNA isolated and purified from fragile Streptosporangium was used as a control at concentrations ranging from 100 fg to 100 ng.
• the primers FGPS122 and FGPS350 were chosen, complementary to part of the 16S rDNA, after alignment of the 16S rDNA sequences of actynomycetes. Their specificity has been tested on a collection of strains of actynomycetes (Streptomyces, Streptosporangium and other closely related genera).
• PCR products were hybridized with the oligonucleotide probe FGPS643 (Table 2).
• controls of pure DNA from S. fragile were mixed with the soil extracts obtained after treatments according to the lysis protocols 4b and 5b then purified according to protocol D.
• the soil extracts were treated with DNase (one unit of DNase / ml, GIBCO BRL) for 30 minutes at room temperature. DNase was then inactivated by heating at 65 ° C for 10 minutes. Inactivation verification was performed by PCR.
• the humic acid concentrations were measured by spectrophotometry (DO 2 sonm) against a standard curve of commercial humic acids (Sigma). Undiluted, 10x diluted and 10x diluted Dnase sol solutions were mixed from 100. fg to 100ng of S. fragile DNA before PCR amplification.
• increasing concentrations of Streptomyces hygroscopicus DNA from (100 pg to 1 ⁇ g) were added to the DNA of S. fragile in order to simulate the presence of non-target DNA and its influence on the PCR process.
• each of the phases was transferred to another tube, mixed with 100 ⁇ l of the sample and left at 4 ° C overnight to allow separation.
• the lower phase was dialyzed for one hour through a Millipore membrane in the presence of an excess of a 7.5 TE buffer (10 mM Tris, 1 mM EDTA at pH 7.5 and 1 M Mg Cl 2 ) in order to d '' remove excess salts.
• Each protocol is terminated by an ethanol precipitation step, and the DNA is resuspended in 10 ⁇ l of TE 7.5 buffer.
• the efficiency of the purification protocols was verified by PCR amplification of undiluted aliquots of the DNA solutions and of aliquots diluted 10 and x 100 times, using standard protocols (see below).
• the quantities of hyphae of S. lividans were calculated on the basis of the number of spores from which they originate. After adding the bacterial suspensions, the soil samples are mixed vigorously by vortexing for 5 minutes before grinding. The DNA is extracted according to protocol No. 6 (see below).
• DNA extraction was carried out according to lysis protocol No. 6. PCR amplification and hybridization were performed as described above. The primers and probes are targeted to chromosomal regions located outside the 16S region, and are highly specific for the respective organisms, so as to avoid background signals.
• primers R499 and R500 were used (Patra et al. (1996)) and the amplification products were hybridized with the oligonucleotide probe C501 (Table 2).
• the PCR reactions were carried out using the primers FGPS516 and FGPS517, and the amplification products were hybridized with the oligonucleotide probe FGPS518 (Table 2).
• the amplified region is a part of the cassette specifically constructed to obtain the strain OS48.3 (CLERC-BARDIN et al., Unpublished).
• the calibration accounts were in all cases obtained using the purified DNA of the target organism.
• the soil suspensions were precipitated with isopropanol, and aliquots of the resuspended pellets were analyzed by gel electrophoresis, in a first step, in order to estimate the quality and the amount of DNA released.
• a visual comparison of the intensities of the colored DNA with ultraviolet radiation allowed a semi-quantitative estimate of the effectiveness of the treatments.
• the presence of migration profiles of multiple sizes of DNA fragments (discrete bands) and the disappearance of long fragments indicates that DNA degradation has taken place.
• Hybridization showed that the quantities of extracellular DNA, as determined by extraction without lysis treatment (protocol No. 1), ranged from 4 ⁇ g / g for the acidic soil (No. 6) to 36 ⁇ g / g for soil n ° 3 (table 3).
• Ground grinding increased the amounts of DNA extracted from all soils (e.g. 26 ⁇ g / g soil) for soil 6 and 59 ⁇ g / g soil ( for floor 3) (table 3; figure 2).
• the band intensity of the smallest fragments is very low, indicating that most of the fragments are much larger than 1 kb.
• Protocol No. 3 includes a homogenization step in an Ultraturax-type mixer device after the addition of the buffer extraction to soil samples. This step leads to an increase in the quantities of DNA extracted, as determined by dot blot hybridization for two of the soils (sandy soil No. 3 and acidic soil No. 6), while the two soils rich in organic matter (soil no. 1 and no. 2) led to the production of lower amounts of DNA.
• Protocols No. 4a and No. 4b made it possible to assess the influence of two types of sonication on DNA yields from previously ground and homogenized soils. Sonication did not have a positive effect on
• the number of bacteria per gram of dry weight of the soil ranged from 1.4 x 10 9 (+/- 0.4) in tropical soil No. 5 to 10 x 10 9
• colonies of Streptomyces sp. dominated the viable actinomycete flora when no lysis treatment was applied (protocol 1), and represented 65% of the total number of colonies identified. After grinding, the percentage of colonies of Streptomyces decreased to 51%, while the proportion of colonies belonging to the genus Micromonospora increased from 14% to 41%.
• Organisms belonging to the genera such as Streptosporangium, Actinomadura, Microbispora, Dactilosporangium and Actinoplanes appeared on the plates in small numbers (2-8% of the total number of colonies identified) after grinding, homogenization with the Ultraturrax device, and were sonicated, but were generally absent when these treatments were combined with chemical / enzymatic lysis.
• the total number of cultivable bacteria remaining after each lysis treatment (protocols 2 to 5) was also sought for soil No. 4. The results indicate that the number of cultivable bacteria does not decrease with the intensity of the lysis treatments (approximately 2 ⁇ 10 6 CFU / g of soil in all cases, and also when a treatment is applied, as according to Protocol 1).
• DNA could either be a fraction of extracellular DNA released from already dead organisms, which can persist in the soil for months (WARD et al., 1990), or DNA released from organisms easily lysed during the first stages of treatment.
• lambda phage DNA digested with HindIII was added, in various concentrations, to the soils before and after grinding.
• a combination of other lysis treatments has been tested, including sonication (Cup device
• the 23 kb band was absent in several cases, indicating that the long fragments were preferentially adsorbed to soil particles, or were more sensitive to degradation, compared to the short fragments.
• the hybridization signals obtained from soil suspensions which were treated by thermal shock and sonication were, at most, weak.
• the sample with the highest amount of DNA (15 ⁇ g DNA / g dry weight of soil) was the only one for which the signal obtained was significantly different from the level of background noise. No difference, (or small differences) was observed between the samples treated by thermal shock and those treated by thermal shock and sonication, indicating that thermal shock has a detrimental effect on DNA.
• the best recoveries were observed for soil # 2, which has the highest organic matter content (Table 1), while no DNA was recovered from clay soil # 5.
• the samples were extracted immediately or after a one hour incubation period at 28 ° C, then the DNA extracts were purified and analyzed by gel electrophoresis.
• the intensity of the bands obtained from samples seeded with DNA after grinding increases with the concentration of RNA, indicating that the treatment could have a positive effect.
• hybridization signals for the control samples did not differ from the background noise levels, significant amounts of DNA were released from the RNA-treated samples, and the signals increased with the amount of DNA added as well as with RNA concentration.
• protocol B (see section Materials and Methods) was used for all the experiments in which PCR amplifications and / or hybridizations on task (Dot Blot) were carried out.
• the first step was to determine whether the quantities of PCR product were proportional to the number of target DNA molecules initially present in the reaction tube. Streptosporangium fragile DNA was used as target (see Materials and Methods section).
• the primers used were the primers FGPS122 and FGPS350 (Table 2).
• the gel electrophoresis of the PCR products showed that the band intensity increases with the increase in the concentration of the targets.
• the PCR products were hybridized with the oligonucleotide probe FGPS643 (table 2), and the signals were quantified by phosphorescence imaging (phospho-imaging).
• the various amounts of target S. fragile DNA made it possible to provide the expected amounts of PCR product when, before amplification, the DNA of S. fragile was mixed with DNA of Streptomyces hygroscopicus and added to the PCR mixture of 50 ⁇ l in a range of 100 pg to 1 ⁇ g in order to simulate the non-target DNA released from the soil microflora.
• extraction protocol No. 6, purification method D, and PCR amplification combined with spot hybridization (Dot Blot) and phosphorescence imaging (phospho-imaging) were used to count target DNAs that had been released.
• the extracted DNA can be clearly distinguished from background noise only when the number of added spores exceeds 10 5 for soils 3 and 5 and 10 7 for soil 2 ( Figure 6a).
• the extracted DNA can be detected above an amount corresponding to 10 3 spores / g of soil for soils 2 and 3, and above 10 7 spores / g for soil n ° 5 (figure b).
• the hybridization signal increases with increasing amounts of the inoculated cells.
• a 100-fold increase in the number of seeded cells leads to an almost 100-fold increase in DNA yield. This increase is clearly less when the hyphae are inoculated, particularly in soils 2 and 3 ( Figure 6).
• the results obtained when phage lambda DNA was used as an inoculum the DNA was also recovered from clay-rich soil (# 5) when the bacterial cells were used as an inoculum.
• RNA treatment increased the recovery of Streptomyces DNA from this soil for both the spores and the mycelium ( Figure 6).
• Example 2 Construction of a DNA library of low molecular weight ( ⁇ 10 kb) from a soil contaminated with lindane: cloning and expression of the linA gene
• This example describes the construction of a soil DNA library in E. coli. It demonstrates the cloning and expression of small genes from a non-cultivable microflora. Lindane is an organochlorine pesticide, recalcitrant to degradation and persistent in the environment. Aerobically, its biodegradation is catalyzed by a dehydrochlorinase, coded by the linA gene, making it possible to transform lindane into 1,2,4-trichlorobenzene.
• the linA gene has only been identified among two strains isolated from the soil: Sphingomonas paucimobilis, isolated in Japan (Seeno and Wada 1989, Imai et al 1991, Nagata et al 1993) and Rhodanobacter lindaniclasticus isolated in France (Thomas et al 1996, Nalin et al 1999).
• Dry soils are ground for 10 minutes in a Restch centrifugal mill equipped with 6 tungsten balls. 10 grams of ground ground are suspended in 50 ml of TENP pH 9 buffer (Tris 50 mM, EDTA 20 mM, NaC1 100 mM, polyvinylpolypirrolidone 1% w / v), and homogenized using a vortex for 10 min.
• TENP pH 9 buffer Tris 50 mM, EDTA 20 mM, NaC1 100 mM, polyvinylpolypirrolidone 1% w / v
• the supernatant is precipitated with sodium acetate (3M, pH 5.2) and isopropanol, to be taken up in sterile TE buffer (10 mM Tris, EDTA 1 mM, pH 8.0).
• sterile TE buffer (10 mM Tris, EDTA 1 mM, pH 8.0).
• the extracted DNA is then purified on a S400 molecular sieving column (Pharmacia) and on an Elutip d ion exchange column (Schleicher and Schuell), according to the manufacturers' instructions, then stored in TE.
• the vector pBluescript SK- and the DNA extracted from the soil are each digested by the enzymes Hind ⁇ and BamHI (Roche), at the rate of 10 units of enzymes for 1 ⁇ g of DNA (incubation for 2 hours at 37 ° C. ). DNA is then ligated by the action of T4 DNA ligase (Roche), overnight at 15 ° C, at the rate of one unit of enzyme per 300 ng of DNA (approximately 200 ng of DNA insert and 100 ng of digested vector) .
• ElectroMAX DH10B TM (Gibco BRL) are transformed by the ligation mixture (2 ⁇ l) by electroporation (25 ⁇ F, 200 and 500 ⁇ , 2.5 kV) (Biorad Geneuterer).
• the transformed cells are diluted so as to obtain approximately 100 colonies per dish and then are spread on LB medium (10 g / l Tryptone, 5 g / l yeast extract, 5 g / NaCl ) added with Ampicilin (100 mg / l), ⁇ -HCH (500 mg / l), X-gal 60 mg / l (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside) , and IPTG 40 mg / l (isopropylthio- ⁇ -D-galactoside), and incubated overnight at 37 ° C. Since ⁇ -hexachlorocyclohexane (Merck-Schuchardt) is insoluble in water, a 50 g / l solution is prepared in DMSO (dimethyl sulfoxide) (Sigma).
• DMSO dimethyl sulfoxide
• the screening of the library is carried out by visualization of a halo of degradation of lindane around the colony (lindane precipitating in culture media). Out of 10,000 clones screened, 35 thus exhibited a degradation activity of lindane. The presence of the linA gene in these clones could be confirmed by PCR using specific primers, described by Thomas et al (1996). Digests carried out on the inserts as well as on the amplification products showed identical profiles between all the screened clones and the reference control, R. lindaniclasticus. The clones carrying the linA gene also presented an insert of the same size (approximately 4 kb).
• a method of preparing a collection of nucleic acids from a soil sample comprising a step of indirect DNA extraction.
• the cell ring located at the interphase of the aqueous phase and the Nycodenz phase, is removed, washed in 25 ml of sterile water and centrifuged at 10,000 g for 20 minutes. cell is then taken up in a 10 mM Tris solution, EDTA 100 mMn pH 8.O.
• a step of enriching the soil in a solution of yeast extract can be included in order to allow in particular the germination of the bacterial spores of the soil.
• the cells are lysed in a 10 mM Tris solution, 100 mM EDTA, pH 8.0 containing 5 mg.ml '1 of lysozyme and 0.5 mg.ml "1 of achromopeptidase for 1 hour at 37 ° C.
• a solution of lauryl sarcosyl ( 1% final) and proteinase K (2 mg.ml "1 ) is then added and incubated at 37 ° C for 30 minutes.
• the DNA solution is then purified on a cesium chloride density gradient by centrifugation at 35,000 rpm for 36 hours on a Kontron 65.13 rotor.
• the cesium chloride gradient used is a gradient at 1 g / ml of CsCI, having a refractive index of 1.3860 (Sambrook et al., 1989).
• the cells are mixed with an equal volume of agarose at 1.5% (weight / volume) Seaplaque (Agarose Seaplaque FMC Products. TEBU, Le Perray en Yvelines, France), at low melting point and poured into a 100 ⁇ l block.
• the blocks are then incubated in a lysis solution: EDTA 250 mM, sucrose 10.3%, lysozyme 5 mg.ml "1 and achromopeptidase 0.5 mg.ml " 1 at 37 ° C for 3 hours.
• the blocks are then washed in a 10 mM Tris - 500 mM EDTA solution and incubated overnight at 37 ° C. in 500 mM EDTA containing 1 mg.ml '1 of proteinase K and 1% lauryl sarcosyl. After several washes in Tris-EDTA, the blocks are stored in 500 mM EDTA.
• the quality of the DNA thus extracted is checked by electrophoresis in pulsed fields.
• the amount of DNA extracted was evaluated on an electrophoresis gel against a standard range of calf thymus DNA.
• the DNAs extracted from the soil are characterized by PCR hybridization, a method which consists in first amplifying the DNAs using primers located on universally conserved regions of the rR16S gene, then in hybridizing the amplified DNAs with different oligonucleotide probes of known specificity (Table 4), for the purpose quantify the intensity of the hybridization signal with respect to an external standard range of genomic DNA.
• the DNAs extracted from the soil as well as the genomic DNAs extracted from pure cultures are amplified with the primers FGPS 612-669 (Table 1) under the standard conditions of amplification by PCR.
• the amplification products are then denatured by an equal volume of 1N NaOH, deposited on a nylon membrane (GeneScreen Plus, Life Science Products) and hybridized with an oligonucleotide probe marked at its end with g 32 P ATP by action of the T4 polynucleotide kinase.
• One of the decisive steps for the extraction of soil cells is the dispersion of the soil sample in order to dissociate the cells adhering to the surface or inside the aggregates of soil particles.
• Three successive grinding cycles of one minute each make it possible to obtain a better extraction efficiency of the cells as well as a larger quantity of DNA recovered, compared to a single grinding cycle of one minute 30.
• Table 5 reports the extraction efficiencies obtained after centrifugation on a Nycodenz gradient, on the total viable microflora (counted by microscopy after staining with acridine orange), on the total cultivable microflora (counted on solid medium Trypticase-Soy 10% ), and on the microflora of actinomycetes cultivable on HV agar medium (after incubation at 40 ° C in a solution of yeast extract 6% -SDS 0.05% in order to cause sprout germination).
• the extracted DNA was quantified either after a lysis of the cells in a liquid medium (without purification on a cesium chloride gradient) or after a lysis of the cells included in an agarose block (after digestion of the agarose with a b-agarase).
• the quantity of DNA extracted from the cells is 330 ng per gram of dry soil.
• Electrophoresis in pulsed fields showed that the soil DNA extracted after Nycodenz and CsCI gradient could reach a size of 150 kb and that the agarose block lysis made it possible to extract fragments greater than 600kb.
• the aim of the molecular characterization of the DNA extracted from the soil is to obtain profiles representing the proportions of the different bacterial taxa present in the DNA extract. It also involved knowing the extraction biases induced by the prior separation of the cell reaction from the soil, in comparison with a direct extraction method due to the lack of direct visualization of the microbial diversity present in the soil. Indeed, little information has been gathered on the extraction of cells on a Nycodenz gradient according to their morphological structure (cell diameter, filamentous or sporulated forms).
• the method implemented according to the present invention consists in universally amplifying a 700 bp fragment inside the 16S rDNA sequence, in hybridizing this amplifier with a probe.
• oligonucleotide of variable specificity in terms of reign, order, subclass or genus
• the amplification prior to hybridization makes it possible to quantify genera or species of microorganisms which are not abundant.
• amplification with universal primers makes it possible, during hybridization, to use a large series of oligonucleotide probes. It allows them to compare different lysis modes (direct or indirect extraction) on well-defined taxonomic groups. The results are collated in Table 6.
• This stage causes the germination of the spores, and on the one hand certainly allows better recovery of this type of cells and on the other hand greater efficiency of lysis on germinating cells.
• the tipA gene codes for a 19 KD protein, the transcription of which is induced by the antibiotic thiostrepton or nosiheptide.
• the tipA promoter is well regulated: induction in exponential phase and in stationary phase (200X) Murakami T, Holt TG, Thompson CJ. J. Bacteriol
• the resistance gene codes for a phosphotransferase (hph)
• the gene used comes from a cassette constructed by Blondelet et al in which the hyg gene is under the control of its own promoter and the promoter plac inducible by IPTG Blondelet-Rouault et al; .
• the pSAM2 element is integrated into the chromosome by a site-specific integration mechanism. Recombination takes place between two identical 58 bp sequences present on the plasmid (attP) and on the chromosome (atfB).
• the int gene located near the aftP site, is involved in the site-specific integration of pSAM2, and its product has similarities to the integrases of temperate bacteriophage enterobacteria. It has been shown that a fragment of pSAM2 containing only the attP attachment site as well as the int gene was able to integrate in the same way as the whole element. See French patent n ° 88 06638 dated 05/18/1988, as well as Raynal A et al. Mol Microbiol 1998 28: 333-42).
• Step 1 / The TipA promoter was isolated from the plasmid pPM927 (Smokvina et al. Gene 1990; 94: 53-9) on a 700 base pair Hindlll-BamHI fragment cloned into the vector pUC18 (Yannish-Perron et al. , 1985) digested with Hindlll / BamHI
• Step 2 This HindIII-BamHI fragment was subsequently transferred from pUC18 to pUC19 (Yannish-Perron et al., 1985).
• Step 3 A BamHI-BamHI insert of 1500 base pairs carrying the int gene and the attP site of pSAM2 was isolated from the plasmid pOSintl, represented in FIG. 8, (Raynal A et al. Mol Microbiol 1998 28: 333-42 ) and cloned at the BamHI site of the preceding vector (pUC19 / TipA), in the orientation making it possible to put the int gene under the control of the TipA promoter.
• Step 4 The BamHI site located 5 ′ of the int gene was deleted by partial BamHI digestion then treatment with the Klenow enzyme. A Hindlll-BamHI fragment carrying TipA-int-attP was thus isolated from pUC19 and transferred to pBR322 Hindlll / BamHI.
• Step 5 The Hygromycin cassette isolated from pHP45 ⁇ hyg (Blondelet-Rouault et al., 1997) on a Hindlll-Hindlll fragment was cloned at the Hindlll site located upstream of the TipA promoter.
• Step 6 The Hindlll site located between the ⁇ Hyg cassette and the TipA promoter was removed by Klenow treatment after partial Hindlll digestion.
• Step 7 The plasmid obtained at the end of the previous step makes it possible to isolate a single Hindlll-BamHI fragment, carrying all the ⁇ Hyg / TipA / int attP elements, which was cloned after Klenow treatment at the EcoRV site of the cosmid pWED Le cosmid pWED1, shown in Figure 9, is derived from cosmid pWE15, shown in Figure 10 (Wahl GM, et al. Proc Natl Acad Sci USA 1987 84: 2160-4) by deletion of a Hpal-Hpal fragment carrying the Neomycin gene and SV40 origin.
• Example 5 Construction of several conjugative and integrative cosmids in Streptomyces, the vector pOSV 303. POSV306 and POSV307
• Step 1 the pOSVOOl vector
• the vector map pOSV 001 is shown in Figure 17.
• Step 2 the vector pOSV002
• Hygromycin marker ( ⁇ hyg cassette), and selectable from Streptomyces, so that the gene conferring resistance to hygromycin is transferred last, which ensures complete transfer of the BAC with the DNA insert from the ground.
• the PstI and Hindlll ends are made compatible after treatment with the Klenow fragment of the DNA polymerase making it possible to generate "blunt ends".
• the orientation of the ⁇ hyg fragment is determined at the end of construction.
• the vector map pOSV002 is shown in Figure 18.
• Step 3 the vector pOSV010
• the Xbal-Hindlll fragment isolated from the plasmid pOSV002 and containing the hygromycin resistance marker and the origin of transfer is cloned into the plasmid pOSintl digested with Xbal and Hindlll.
• the orientation of the sites is such that the hygromycin marker will always be transferred last.
• the plasmid pOSintl represented in FIG. 8, was described in the article by Raynal et al. (Raynal A et al. Mol Microbiol 1998 28: 333-42).
• the principle is to insert a "cos" site in the plasmid pOSV010 allowing the packaging in the plasmid pOSV010, represented in FIG. 12.
• This fragment is obtained by PCR. From a fragment carrying the cohesive ends (cos) of ⁇ (lambda bacteriophage or cosmid pHC79), amplification by PCR is carried out using oligonucleotides corresponding to the sequences -50 / + 130 relative to the cos site. These oligonucleotides also contain the Nsil, PstI compatible, Xhol, Sali compatible, EcoRV, "blunt end" cloning sites.
• the PCR fragment is bounded by a PstI site at the 5 'end and by a Hincll site at the 3' end, allowing cloning into the vector pOSV010 ( Figure 12) previously digested with the enzymes Nsil and EcoRV, causing the deletion of the laclq repressor.
• the vector map pOSV303 is shown in Figure 14.
• the vector pOSV303 contains cloning sites such as the Nsil site,. PstI compatible, the Xhol site, Sali compatible or the EcoRV site for obtaining "free ends”.
• Step 1 Construction of the vector POSV308.
• the vector pOSV308 was constructed according to the method illustrated in FIG. 27. A 643 bp fragment containing the cos region was amplified using the pair of primers with sequences SEQ ID No. 107 and
• SEQ ID No. 108 from the cosmid vector pHc79 described by HOHM B and COLLINS (1980). This amplified nucleotide fragment was cloned directly into the vector pGEMT-easy sold by the company PROMEGA, as illustrated in FIG. 27 in order to produce the vector pOSV308.
• Step 2 Construction of the vector pOSV306.
• the vector pOSV010 was constructed as described in step 3 of construction of the vector pOSV303, as described in paragraph 5.1 of this example.
• the vector pOSVIO was digested with the enzymes EcoRV and Nsil in order to excise a fragment of 7874 bp which was subsequently purified, as illustrated in FIG. 28.
• the vector pOSV308 obtained in step 1) above was subjected to digestion with the enzymes EcORV and PstI in order to excise a fragment of 617 bp, which was subsequently purified.
• the 617 bp cos fragment obtained from the vector pOSV308 was ligated into the vector pOSVI O, in order to obtain the vector pOSV306, as illustrated in FIG. 28.
• the cosmid pOSV307 still contains the Laclq gene, in order to improve the stability of the cosmid in Streptomyces, for example in the strain S17-1 of Streptomyces.
• the vector pOSV010 was subjected to digestion with the enzyme Pvull, to obtain a fragment of 8761 bp which was purified, then dephosphorylated.
• the vector pOSV308, as obtained as described in step 1) of paragraph 5.2 above, was digested with the enzyme EcoRI in order to obtain a fragment of 663 bp, which was then purified and treated by Klenow's enzyme.
• Example 6 Construction of the replicative cosmid shuttle E. coli-Streptomvces pOS700R.
• the transformation efficiency is approximately 3000 transformants per ⁇ g of DNA.
• the small fragments are very preferably integrated.
• Equipped with a screen to select the plasmids carrying an insert This screen makes it possible, by eliminating the vectors which are closed on themselves and not digested, to work with a higher ratio between vector and DNA to be inserted, which allows better cloning efficiency to constitute banks.
• Step 1 the pOSVOOl vector
• the vector map pOSV 001 is shown in Figure 17.
• Step 2 the vector pOSV002
• the vector map pOSV002 is shown in Figure 18.
• Step 3 the pOSVOIQ vector
• the Xbal-Hindlll fragment isolated from the plasmid pOSV002 and containing the hygromycin resistance marker and the origin of transfer is cloned into the plasmid pOSintl digested with Xbal and Hindlll.
• the orientation of the sites is such that the hygromycin marker will always be transferred last.
• the plasmid pOSintl represented in FIG. 8, was described in the article by Raynal et al. (Raynal A et al. Mol Microbiol 1998 28: 333-42). This construction allows the expression of integrase in E. coli and in Streptomyces.
• Step 4 the vector POSV014
• This "cassette” carries the gene coding for the repressor C1 of phage ⁇ and the gene conferring resistance to tetracycline.
• This gene carries in its 5 'non-coding region the target sequence of the repressor. insertion of DNA in the HindIII site located in the coding sequence of C1 leads to the non-production of the repressor and therefore to the expression of resistance to tetracycline.
• the vector map pOSV014 is shown in Figure 19.
• Step 5 the vector pOSV 403, integrative and conjugative BAC vector
• the Pstl-Pstl fragment of the vector pOSV014 carrying all of the elements and functions described above is cloned into the pasmide pBAC11 (pBeloBACH) digested with NotI. The ends are made compatible by treatment with the Klenow enzyme.
• the vector map pOSV403 is shown in Figure 21. The diagram in Figure 21 shows the orientation selected.
• the vector pOSV403 contains the Hindlll and Nsil sites.
• the Nsil site is quite rare in Streptomyces and has the advantage of being compatible with PstI.
• the PstI site is frequent at
• Streptomyces and can be used to perform partial digestions.
• Recombinant clones carrying an insert cloned into the repressor C1, and therefore inactivating this repressor become resistant to tetracycline. Since the BACs are only present at the rate of one copy per cell, it is necessary to select the recombinant clones with a lower dose of tetracycline than the usual dose of 20 ⁇ g / ml, for example with a dose of 5 ⁇ g / ml. Under these conditions there is no background noise. It is also possible to use a system developed and marketed by the company InVitrogen, in which the insertion of DNA into the vector inactivates a gyrase inhibitor whose expression is toxic for E. coli. The fragment is preferably isolated from the vector pZErO-2 (http://www.invitrogen.com/).
• Example 9 Construction of a S. alboniger bang in the 2 integrative (pOS700l) and replicative .pOS700R_ cosmids
• the puromycin biosynthesis pathway from Streptomyces alboniger was cloned in the two cosmid shuttles pOS700l and pOS700R.
• the genes of the puromycin biosynthetic pathway are carried by a BamHI DNA fragment of approximately 15 Kb.
• DNA has a molecular weight between 20 and 150 Kb, determined by pulsed field electrophoresis.
• the two cosmids were digested with the enzyme BamHI (single cloning site).
• the BamHI partial digestion conditions for genomic DNA were determined (50 ⁇ g of DNA and 12 units of enzyme, 5 minutes of digestion). After checking the size by agarose gel electrophoresis, the partially digested DNA was introduced into the vectors. In the ligation, 15 ⁇ g of genomic DNA + 2 ⁇ g of the integrative vector or 5 ⁇ g of the replicative vector were used.
• Each ligation mixture was used for the in vitro packaging of DNA in the heads of lambda bacteriophage.
• the cosmids were used to transfect E. coli and thus generate two libraries of approximately 25,000 ampicillin-resistant clones.
• the DNA of all of these clones was isolated and quantified. To test the libraries, several clones were chosen, the DNA purified and was digested with BamHI, in order to verify the presence and the size of the inserts. The clones tested contain between 20 and 35 Kb of S. alboniger insert.
• the clones capable of containing the complete puromycin biosynthesis pathway were identified by hybridization with a probe corresponding to the puromycin resistance gene, the 1.1 kb pac gene. (Lacalle et al. Gene 1989; 79, 375-80)
• Three resistant clones were selected to verify the production of puromycin. They correspond to the recombinants of S. lividans containing an insert in the integrative vector pOS700l (G 20) or an insert in the replicative vector (G21 and G22).
• the strains are seeded in a culture medium, the composition of which is as follows: Bacteriological peptone Organotechnics 5 g / l of final medium
• 50 ml of liquid culture medium distributed in 250 ml Erlenmeyer flasks, are inoculated with 2 ml of aqueous suspension of spores and mycelium of each of the strains.
• the cultures are incubated for 4 days at 28 ° C. with shaking at 220 rpm.
• 50 ml of production media distributed in 250 ml Erlenmeyer flasks, are then seeded with 2 ml of these precultures.
• the production medium used is an industrial medium optimized for the production of pristinamycin (RPR 201 medium).
• the cultures are incubated at 28 ° C., with shaking at 220 rpm.
• Comparative HPLC analyzes from the cultures of the different strains show the production of puromycin in the culture of the wild strain from 24 h of incubation. A production, although lower, is also clearly detected from 48 h on in the culture of clone G20 containing the cosmid pOS700l (FIG. 23). Puromycin was also detected as a trace in clone G23 containing the full operon coding for the compound in the plasmid pRCPH. However, no production was observed in the cultures of clones G21 and G22 containing the cosmid pOS700R. The results are shown in Figure 23. c) Conclusions
• EXAMPLE 10 DNA CLONING OF SOLDANS OF VECTORS 1) - Preparation of DNA from the soil to be cloned
• the size of the molecules must be between 30 and 40kb.
• the DNA extracted from the soil is heterogeneous in size and includes molecules reaching 200 or 300kb.
• the DNA is broken mechanically by passing the solution through a 0.4mm diameter needle. Fragments of a size close to 30kb are not affected by these repeated passes through a needle and it is therefore not necessary to make a separation by size especially since the packaging in the particles automatically eliminates the short inserts.
• the soil DNA is separated by pulsed field electrophoresis (CHEF type) under conditions such that the fragments between
• 100 and 300kb are concentrated in a band of about 5mm. This is obtained by carrying out the migration in a gel with 0.7% normal agarose or 1% low melting agarose with a pulse time of 100 seconds for 20 hours and at a temperature of 10 ° C.
• the porosity of a 0.7% agarose gel allows the DNA to be removed by electroelution provided that there is a total absence of ethidium bromide.
• This DNA is then manipulated with pipetting instruments with an enlarged and hydrophobic orifice to avoid mechanical fragmentation of the molecules. - Between 100 and 300 kb
• the strip containing the fragments of a size between 100 and 300kb is cut.
• a 1% agarose gel with a low melting point is used for migration. This property makes it possible to melt the gel at a tolerable temperature for DNA of 65 ° C and to then digest it with agarase (Agarase marketed by the company Boehringer) at a temperature of 45 ° C according to the supplier's prescriptions.
• agarase Agarase marketed by the company Boehringer
• a cosmid vector, open to any cloning site, is modified at the 3 'ends by adding a monotonic polynucleotide.
• the DNA to be cloned is modified at the 3 'ends by adding a monotonic polynucleotide which can pair with the previous one.
• the vector-fragment association to be cloned is made by these polynucleotides and the cos sequence of the vector allows the in vitro packaging of DNA in capsids of phage Lamda.
• the vector used is a self-replicating vector in E. coli and integrative in Streptomyces.
• E. coli For E. coli, the selection is made on resistance to ampicillin and for Streptomyces, it is made on resistance to hygromycin.
• the cosmid is open at one of the 2 possible sites (BamHI or Hindlll) and the 3 ′ ends are lengthened by polyA with the terminal transferase under the conditions where the supplier of the enzyme provides for the addition of 50 to 100 nucleotides.
• the 3 ′ ends of the DNA are lengthened by polyT with terminal transferase under the conditions providing an elongation comparable to that of the vector. Under the experimental conditions described by the manufacturer, the polyA polyT tails are 30 to 70 bases long. Molecule assembly and in vitro packaging.
• a vector molecule is mixed for an inserted DNA molecule.
• the mass concentration of DNA is 500 ⁇ g.ml "1 .
• the mixture is packaged and the transfection efficiency depends on the strain used as receptor and on the inserted DNA: zero with the test DNA and the strain DH5 ⁇ , the efficiency is comparable for the strains SURE and DH10B; on extraction, the DNA yield is however higher with the strain DH10B.
• Soil DNA is blunt-ended by eliminating outgoing 3 'sequences and filling outgoing 5' sequences. This is done with: Klenow enzyme, T4 polymerase, the 4 nucleotide triphosphates.
• the cosmid vector is digested with BamHI, then treated with the Klenow enzyme to make it blunt and then dephosphorylated to prevent it from closing on itself. After ligation, the mixture is packaged and transfected as previously described.
• the conjugative and integrative pBAC plasmid has Hindlll and Nsil sites as cloning sites.
• the insertion of a DNA sequence at these sites inactivates the repressor C1 of the phage Lambda which controls the expression of the gene for resistance to tetracycline. Inactivation of the repressor therefore makes the cell resistant to this antibiotic ( ⁇ g.ml "1 ). Cloning at these sites is facilitated by the modification of the vector and the preparation of the DNA to be cloned. Vector preparation. Hindlll example
• the Hind III site is modified: the first base (A) is replaced to form an outgoing 5 'sequence, which cannot pair with its similars.
• the operation is carried out by the Klenow enzyme in the presence of dATP.
• the success of the operation is verified by carrying out a ligation of the vector on itself before and after treatment with the Klenow enzyme. With the same amount of DNA tested, 3000 clones are obtained before treatment and 60 after treatment.
• DNA preparation (size between 100 and 300kb). DNA blunt ends.
• the DNA is blunt-ended by eliminating the outgoing 3 'sequences and filling in the outgoing 5' sequences. This operation is done with: Klenow enzyme, T4 polymerase, the 4 nucleotide triphosphates.
• oligonucleotides recognizing the modified HindIII sequence of the vector. They contain rare restriction sites to allow subsequent cloning (Swal; Notl). This technique is derived from that of: Elledge SJ, Mulligan JT, Ramer SW, Spottswood M, Davis RW. Proc Natl Acad Sci U S A 1991 Mar 1; 88 (5): 1731-5 Two complementary oligonucleotides are used: Oligo 1: 5'-GC ⁇ ATTTAAATATTAATGCGGCCGCCCGGG-3 '
• Oligo 2 5'-CCCGGGCGGCCGCATTAATATTTAAATA-3 '(SEQ ID N ° 26) They are phosphorylated 5 ′ by the T4 polynucleotide kinase in the presence of ATP, after their hybridization. This phosphorylation step can be eliminated using the already phosphorylated oligonucleotides.
• the ligation of this double-stranded adapter with the DNA to be inserted into a vector is made by the T4 ligase in the presence of a very large excess of adapter (1000 molecules of adapter for a DNA molecule to be inserted), in 15 hours at 14 ° C. The excess adapter is removed by electrophoresis on an agarose gel and the molecules of interest are recovered from the gel by hydrolysis of the latter with agarase or by electroelution.
• Ligation is carried out at 14 ° C. over 15 hours with 10 vector molecules for one insert molecule.
• the receptor strain is strain DH10B.
• the transformation is done by electroporation. To express resistance to tetracycline, the transformants are incubated at 37 ° C. for 1 hour in an environment without antibiotics. The selection of the clones is done by culture overnight, on LB agar medium supplemented with tetracycline at ⁇ g.ml * 1 .
• Example 12 Construction of a cosmid bong in E. coli and Streptomyces lividans: Cloning of soil DNA
• the objective is the construction of a large DNA library from the environment, without prior stage of culture of microorganisms, in order to access the metabolic genes of bacteria (or any other organism) that we do not know how to cultivate under standard laboratory conditions.
• the procedure described was used to generate a DNA library in Escherichia coli using the shuttle cosmid E. coli-S. lividans pOS700l and DNA extracted and purified from the bacterial fraction of a soil. This latter method makes it possible to obtain DNA of high purity and an average size of 40 kb.
• the DNA and the vector are assembled by mixing a vector molecule and an inserted DNA molecule.
• the mixture is then packaged in the heads of lambda bacteriophages (Amersham kit) which are used to transfect E. coli DH10B.
• the transfected cells are then seeded on LB agar medium in the presence of ampicillin for selection of the recombinants resistant to this antibiotic.
• a bank of around 5000 clones of E. ampicillin resistant coli was obtained. Each clone was seeded in LB or TB medium + ampicillin in a microplate well (96 wells) and stored at -80 ° C.
• the sequence at the sites of insertion of the soil fragments into the vector, pOS7001, generated during the construction of the library was analyzed. To do this, 17 cosmids from the library were purified and sequenced with a primer, seq. 5 'CCGCGAATTCTCATGTTTGACCG 3', which hybridizes between the BamHI sites and the HindIII cloning site present in the vector.
• the sequences obtained made it possible to estimate that the length of the homopolymeric tails at the junction points is very variable, between 13 and 60 poly-dA / dT. Beyond the tails, the sequences of the soil fragments thus generated have a G + C percentage between 53 and 70%. Such high percentages were unexpected, but similar results have already been reported on crude DNA preparations from soil (Chatzinotas A. et al., 1998).
• a “pooling” strategy of 48 or 96 clones was used for the analysis of microbial and metabolic wealth.
• the cosmid DNA extracted from these "clone pools” was then used to carry out PCR or hybridization experiments.
• Example 13 Diversity of 16S ribosomal DNA within cloned DNA.
• cosmids of the bank are extracted from clone pools by alkaline lysis and are then purified on a cesium chloride gradient, in order to take the cosmid DNA band in supercoiled form and with the aim of eliminating any Escherichia coli chromosomal DNA that may interfere with the study. After linearization of the cosmids by the action of nuclease S1
• the 16S rDNA sequences contained in the clone pools are amplified under standard amplification conditions, from the universal primers 63f (5'- CAGGCCTAACACATGCAAGTC-3 ') and 1387r (5'- GGGCGGWGTGTACAAGGC-3 ') defined by MARCHESI et al. (1998).
• the amplification products of approximately 1.5 kilobases are purified from the Qiaquik gel extraction kit (Qiagen) and then directly cloned into the vector pCR II (Invitrogen) in Escherichia coli TOP10, according to the manufacturer's instructions.
• the insert is then amplified using primers M13 Forward and M13 reverse specific to the cloning site of the vector.
• pCR II The amplification products of expected size (approximately 1.7 kb) are analyzed by RFLP (Restriction Fragment Length Polymorphism) using the enzymes Cfol, Mspl and BstUI (0.1 units) in order to select the clones to be sequenced.
• the restriction profiles obtained are separated on 2.5% metaphor agarose gel (FMC Products) containing 0.4 mg of ethidium bromide per ml.
• the 16S rDNA sequences are then determined directly using the PCR products purified by the "Qiaquick gel extraction" kit using the sequencing primers defined by Normand (1995).
• Phylogenetic analyzes are obtained by comparing the sequences with the prokaryotic 16S rDNA sequences gathered in the Ribosomal Database Project (RDP) database, version 7.0 MAIDAK et al. (1999) thanks to the SIMILARITY MATCH program, allowing obtain the similarity values with respect to the sequences in the database.
• RDP Ribosomal Database Project
• proteobacteria 18 sequences with a percentage of similarity between 89 and 99%.
• a second group of sequences is represented by the subclass g of proteobacteria, comprising 9 sequences whose percentages of similarity vary between 84 and 99%).
• the groups of b-proteobacteria, d-proteobacteria, firmicutes with low G + C% and high G + C% respectively comprise 1, 4, 3 and 5 sequences.
• the phylogenetic tree represented in FIG. 7 was produced from the alignment of the sequences by the MASE software (Faulner and Jurak, 1988) and corrected by the method of the 2 parameters of Kimura (1980), and using the Neighbor Joining algorithm (Saitou and Nei 1987).
• Phylogenetic analysis made it possible to compare the 16S rDNA sequences cloned in the soil DNA library, with the prokaryotic 16S rDNA sequences gathered in the Ribosomal databases Database Project (RDP), (version 7.0, SIMILARITY-MATCH program, Maidak et al 1999), and in the GenBank database thanks to BLAST 2.0 software (Atschul et al, 1997).
• S. coelicolor ATCC101478, S. ambofaciens NRRL2420, S. lactamandurans ATCC27382, S. rimosus ATCC109610, B. Subtilis ATCC6633 and B. licheniformis THE1856 (RPR collection) were used as DNA sources for the PCR experiments.
• S. lividans TK24 is the host strain used for the cosmid shuttle POSI700.
• Escherichia coli Top10 (INVITROGEN) was used as host for the cloning of PCR products and E. coli Sure (STRATAGENE) was used as host for the shuttle cosmid pOS700l.
• the culture conditions for E. coli, the preparation of plasmids, the digestion of DNA, the electrophoresis on agarose gel were carried out according to standard procedures (Sambroock et al., 1996).
• pairs of primers a1-a2 and b1-b2 were defined by the team of N. Bamas-Jacques and their use was optimized for the screening of DNA from pure strains and the soil bank for genes encoding PKSI)
• Table 8 PCR primers homologous to the PKSI genes used for screening the library.
• the amplification mixture contained: in a final volume of 50 ⁇ l, between 50 and 150 ng of genomic DNA, 200 ⁇ M of dNTP, 5 mM of MgCl 2 final, 7% DMSO, 1x Appligene buffer, 0.4 ⁇ M of each primer and 2.5U of Taq Polymerase Appligene.
• the amplification conditions used are: denaturation at 95 ° C for 2 minutes, hybridization at 65 ° C for 1 minute, elongation at 72 ° C for 1 minute, for the first cycle, followed by 30 cycles where the temperature is lowered up to 'at 58 ° C as described in K. Seow et al., 1997.
• the final extension step is carried out at 72 ° C for 10 minutes.
• the PCR conditions are the same as above for the a1-a2 pair using between 100 and 500 ng of cosmid extracted from pools of 48 clones.
• the amplification mixture contained 200 ⁇ M dNTP, 2.5mM final MgCI 2 , 7% DMSO, 1x Quiagen buffer, 0.4 ⁇ M of each primer and 2.5U of Taq polymerase Hot-start (Qiagen).
• the amplification conditions used are: denaturation 15 'at 95 ° C followed by 30 cycles: the denaturation at 95 ° C + the hybridization at 65 ° C for the first cycle and 62 ° C for the other cycles , elongation at 72 ° C, final extension step from 10 'to 72 ° C.
• the identification of positive clones from pools of 48 or 96 clones is carried out using replicates of the corresponding mother microplates on solid medium or any other standard replication method.
• the fragments are purified on agarose gel (Gel Extraction Kit (Qiagen)) and cloned in E.coli TOP 10 (Invitrogen) using the TOPO TA cloning kit (Invitrogen).
• the plasmid DNA of subclones is extracted by alkaline lysis on a Biorobot (Qiagen) and dialysis for 2 h on VS membrane 0.025 ⁇ m (Millipore).
• the samples are sequenced with the primers M13 "Universal” and "Reverse” on the sequencer ABI 377 96 (PERKIN ELMER).
• the sequencing of the PCR products obtained with the pair of primers a1-a2 made it possible to identify, from the strain S. ambofaciens, the sequence of a KS gene already described (European Patent Application No. EP0791656) as belonging to the biosynthesis pathway for plantenolide, macrolidic precursor of spiramycin, and two sequences never described, Stramb 9 and Stramb12, (see sequence list).
• the screening method allowed the identification of a KS sequence (sacen / 17) identical to that of the KS of module 1 already published in Genebank (Access number M63677), coding for synthetase 1 (DEBS1) of 6-deoxyerythronolide B.
• KS sequence sequence identical to that of the KS of module 1 already published in Genebank (Access number M63677)
• DEBS1 synthetase 1
• Another sequence not correlated to the erythromycin biosynthetic pathway has been identified and it is the sequence SEQ ID No. 32.
• the screening was carried out under the conditions described in the Materials and Methods section using the pairs of primers validated from producing strains.
• the intensity of the bands obtained was variable, but only one amplification band was present for each target DNA pool. Under these conditions, 8 groups of target DNA were detected, corresponding to 9 positive clones after dereplication. The screening carried out with the second pair of primers, b1-b2, gave less specific amplification results since numerous satellite bands were observed alongside the 700 bp band. However, 9 groups of target DNA were detected, corresponding to 14 positive clones after dereplication from these positive clones, the DNA was extracted for the sequencing and transformation steps of .S. lividans. Cosmid analysis
• Streptomyces lividans by transformation of protoplasts in the presence of PEG 1000.
• the transformation efficiency varies between 30 and 1000 transformants per ⁇ g of cosmid DNA used.
• the PCR method developed on pure strains was used as described on the bank's cosmids and 24 clones were thus identified.
• FIG. 24 The alignment of the protein sequences deduced from PKSs I of the soil with other PKSs I present in different microorganisms (FIG. 24) shows the presence of a very conserved region which corresponds to the consensus region of the active site of b-ketoacyl. synthetase.
• Analysis of the sequences obtained with the "Codonpreference" method revealed the presence of a strong bias in the use of codons rich in G + C in a single reading phase.
• the proteins deduced according to this reading phase show a strong similarity with known type I KSs (Blast program). In particular, the similarity between the sequences of soil KSs and KSs of the erythromycin cluster is approximately 53%.
• the sequence of the PCR product obtained from this clone is identical to that of the pool, which confirms the reliability of the method used.
• G + C in soil sequences suggests that they can be derived from genomes with codon usage similar to that of actinomycetes.
• PKSI ranges from a few kb for penicillin to around 120 kb for rapamycin.
• the size of the cosmid inserts may therefore not be sufficient for the expression of the most complex clusters.
• the inventors identified a cosmid clone containing an insert of 34071 bp containing several open reading frames coding for polypeptides of the polyketide synthase type.
• the cosmid thus identified by screening the library contains six open reading frames coding for polyketide synthase polypeptides or for strongly related polypeptides, non-ribosomal peptides synthase.
• a detailed map of this cosmid is shown in Figure 36.
• the complete nucleotide sequence of the cosmid constitutes the sequence SEQ ID No. 113 of the sequence listing.
• the DNA insert contained in the sequence SEQ ID No. 113 constitutes the complementary nucleotide sequence (strand -) of the nucleotide sequence coding for the various polyketide synthases.
• the nucleotide sequence of the DNA insert contained in the cosmid of FIG. 36 which includes the open reading frames coding for the polyketide synthase (strand +) polypeptides is shown diagrammatically in FIG. 37 and constitutes the sequence SEQ ID No. 114 sequence listing.
• the orfl sequence comprises an open partial reading frame with a length of 4615 nucleotides.
• This sequence constitutes the sequence SEQ ID No. 115, which begins at the nucleotide in position 1 and ends at the nucleotide in position 4615 of the sequence SEQ ID No. 114.
• sequence SEQ ID No. 1 15 codes for the ORF1 polypeptide of 1537 amino acids, this polypeptide constituting the sequence SEQ ID No. 121.
• the polypeptide of sequence SEQ ID No. 121 is related to the non-ribosomal peptide synthases. This polypeptide has a degree of amino acid identity of 37% with the peptide synthase of Anabaena sp.90 referenced under the access number "emb CACO1604.1" in the Genbank database.
• the nucleotide sequence orf2 is 8301 nucleotides in length and constitutes the sequence SEQ ID No 116 which begins at the nucleotide at position 4633 and ends at the nucleotide at position 12933 of the sequence SEQ ID No 114.
• the ORF2 sequence codes for the ORF2 peptide with a length of 2766 amino acids, this polypeptide constituting the sequence SEQ ID No. 122.
• the polypeptide of sequence SEQ ID No. 122 has an amino acid sequence identity of 41% with the MtaD sequence of Stigmatella aurantiaca referenced under the access number "gb AAF 19812.1" from the GENBANK database.
• the ORF2 polypeptide constitutes a polyketide synthase.
• the nucleotide sequence orf3 has a length of 5292 nucleotides and constitutes the sequence SEQ ID No. 117.
• the sequence SEQ ID No. 117 corresponds to the sequence which begins at the nucleotide in position 12936 and which ends at the nucleotide in position 18227 of the sequence SEQ ID No. 114.
• the nucleotide sequence SEQ ID No. 117 codes for the polyketide synthase ORF3 polypeptide of 1763 amino acids, this polypeptide constituting the sequence SEQ ID No. 123 according to the invention.
• the ORF3 polypeptide of sequence SEQ ID No. 123 has an identity of 42% in amino acids with the MtaB sequence of Stigmatella aurantiaca referenced under the access number “gb AAF 19810.1” from the GENBANK database.
• the nucleotide sequence orf4 has a length of 6462 nucleotides and constitutes the sequence SEQ ID No. 118 according to the invention.
• the nucleotide sequence SEQ ID No. 118 corresponds to the sequence starting at the nucleotide at position 18224 and ending at the nucleotide at position 24685 of the nucleotide sequence SEQ ID No. 114.
• the nucleotide sequence SEQ ID No. 118 codes for the polyketide synthase ORF4 polypeptide of 2153 amino acids, this polypeptide constituting the sequence SEQ ID No. 124 according to the invention.
• the ORF4 polypeptide of sequence SEQ ID No. 124 has an amino acid sequence identity of 46% with the epoD sequence of Sorangium cellulosum referenced under the access number “gb AAF62883.1 from the GENBANK database.
• the orf ⁇ nucleotide sequence has a length of 5088 nucleotides and constitutes the sequence SEQ ID No. 119 according to the invention.
• sequence SEQ ID No. 119 corresponds to the sequence starting at the nucleotide at position 24682 and ending at the nucleotide at position 29769 of the nucleotide sequence SEQ ID No. 114.
• the nucleotide sequence SEQ ID No 119 codes for the polyketide synthase ORF5 polypeptide of 1695 amino acids, this polypeptide constituting the sequence SEQ ID No 125 according to the invention.
• polyketide synthase ORF5 polypeptide of sequence SEQ ID No. 125 has an amino acid identity of 43% with the epod sequence of Sorangium cellulosium referenced under the access number "gb AAF 62883.1" from the GENBANK database.
• the nucleotide sequence ifif has a length of 4306 nucleotides, and constitutes the sequence SEQ ID No. 120 according to the invention.
• the nucleotide sequence SEQ ID No. 120 corresponds to the sequence starting at the nucleotide at position 29766 and ending at the nucleotide at position 34071 of the sequence SEQ ID ID No. 1 14.
• sequence SEQ ID No. 120 contains an open partial reading frame coding for the ORF6 polypeptide of 1434 amino acids of the polyketide synthase type, this polypeptide constituting the sequence SEQ ID No. 126 according to the invention.
• the polypeptide of sequence SEQ ID No. 126 has an amino acid identity of 43% with the epoD sequence of Sorangium cellulosum referenced under the access number "gb AAF 62883.1" from the GENBANK database.
• EXAMPLE 15 Construction of integrative BAC shuttle vectors at Streptomyces
• the BAC vector pMBD-1 was obtained according to the following steps: Step 1: The vector pOSVO10 was subjected to digestion with the enzymes PsTI and BstZ171 in order to obtain a nucleotide fragment of 6.3 kb.
• Step 2 The vector pDNR-1 was digested with the enzymes PstI and Pvull in order to obtain a nucleotide fragment of 4.145 kb.
• Step 3 The 6.3 kb nucleotide fragment originating from the vector pOSV017 was ligated to the 4.15 kb fragment originating from the vector pDNR-1, in order to produce the vector pMBD-1, as illustrated in the figure. 30.
• the vector pMBD-2 is a vector of the BAC type containing an integrative box " ⁇ c31 int- ⁇ hyg".
• ⁇ c31 is a temperate phage with a broad host spectrum whose attachment site (attP) is well located.
• the ⁇ c31 int fragment is the minimal fragment of the ⁇ c31 actinophagus capable of inducing the integration of a plasmid into the chromosome of Streptomyces Lividans.
• ⁇ hyg is a derivative of the interposon ⁇ capable of conferring resistance to hygromicine in E. coli and S. Lividans.
• BAC vectors containing the integration system ⁇ c31 are described by SOSIO et al. (2000) and in PCT application No. 99 6734 published on December 29, 1999.
• the BAC pmBD-2 vector was constructed according to the following steps:
• Step 1 Construction of an integrative box intc31 int ⁇ hyg in a multicopy plasmid of E.coli.
• the ⁇ c31 int fragment was first amplified from the plasmid pOJ436 using the following pair of primers:
• the primer EV ⁇ c31 l (SEQ ID No. 109) (which makes it possible to introduce an EcoRV site at the 5 ′ end of the sequence ⁇ c31) and the primer Bll ⁇ c31 F (SEQ ID No. 110) (which allows 'introduction of a BgLII site at the 3' end of the sequence ⁇ c31).
• the ⁇ hyg fragment was obtained by digestion using the enzyme BamHI from the plasmid pHP45 ⁇ hyg described by BLONDELET-ROUAULT (1997).
• Step 2 Construction of the vector pMBD-2.
• the bacterial artificial chromosome pBAce3.6 described by FRENGEN et al. (1999) was digested with the enzyme Nhel and then treated with the enzyme Eco polymerase.
• the vector pMCS5 ⁇ c31 int- ⁇ hyg was digested with the enzymes SnaBI and EcoRV in order to recover the integrative box.
• the vector pMBD-3 is an integrative ( ⁇ c31 int) and conjugative (OriT) vector of the BAC type, which comprises the selection marker ⁇ hyg.
• the vector pMBD-3 was obtained by amplifying the OriT gene from the plasmid pOJ436 using the pair of primers with sequences SEQ ID No. 111 and SEQ ID No. 112 which contain pacl restriction sites.
• the nucleotide fragment amplified using the primers SEQ ID No. 111 and SEQ ID No. 112 was cloned into the vector pMBD2 previously digested with the enzyme Pac1.
• the construction diagram of the vector pMBD-3 is illustrated in FIG. 31. 15.4 Construction of the DMBD-4 vector
• the detailed map of the vector pMBD-4 is represented in FIG. 32.
• the vector pMBD4 was obtained by cloning the integrative box ⁇ c31 int- ⁇ hyg into the vector pCYTAC2.
• the construction diagram of the vector pMBD-5 is illustrated in FIG. 33.
• the vector pMBD-5 was constructed by recombination of the nucleotide fragment comprised between the two loxP sites of the vector pMBD-1 illustrated in FIG. 33 with the loxp site contained in the BAC vector designated pBTP3, a detailed map of the plasmid pBTP3 being represented at Figure 34.
• the vector pMBD-6 was constructed by recombining the nucleotide fragment comprised between the two loxP sites of the vector pMBD-1 at the level of the loxP site of the BAC vector pBeloBad 1, as shown in FIG. 35.
• n 3; standard deviation in parentheses.
• d The quantity of DNA extracted was evaluated on an electrophoresis gel relative to a standard range of calf thymus DNA.
• e The quantification was carried out after digestion of the agarose by the action of a b-agarase
• Table 6 Characterization of the DNAs extracted according to their proportion in a, b, and g subclasses of Proteobacteria, in Gram + with low GC% and in Actinomycetes; the hybridization signal with the prokaryotic probe serving as a 100% reference.
• Blondelet-Rouault MH Weiser J, Lebrihi A, Branny P, Pernodet JL.

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