WO2002059351A2 - Accessing microbial diversity by ecological methods - Google Patents

Abstract

Methods for enriching a microbial population from a natural environment, such as an oligotrophic environment (e.g., aquatic, marine, extreme or geothermal) are described. Also described are methods for screening for bioactive small molecules in an enriched microbial population. Previously uncultivated microorganisms can be cultured, isolated and their nucleic acids identified by methods described herein. Methods also describe phylogenetic characterization of enriched microbial populations.

Description

ACCESSING MICROBIAL DIVERSITY BY ECOLOGICAL METHODS

This application claims priority from U.S. application No. 09/770,771 filed on 26 January 2001.

BACKGROUND OF THE INVENTION

The growing use of biological catalysts in the chemical synthesis, research reagent, diagnostic reagent and chemical process industries has increased the demand for the discovery and development of new enzymes. Until now all commercially available enzymes have been derived from already cultivated bacteria or fungi. The realization that less than 1% of naturally occurring microorganisms can be isolated and grown in pure culture has created great interest in developing methods to get access to uncultivated microbes in order to exploit a larger fraction of the microbial diversity than has been possible with the presently available technology.

Methods for producing good quality genomic DNA that can be used for constructing gene libraries from cultivated microbes is well known and proven in practice. Cultivated microbes are therefore preferred as the most convenient sources to screen directly for bioactive small molecules and for isolation of genes that code for potentially new valuable enzymes and bioactive molecules. Considerable efforts have therefore in the past been put into developing methods that allow cultivation or isolation of the largest possible fraction of the viable cells in the environment.

Both direct microscopic count and diversity studies based on various molecular methods, such as analysis of 16S rRNA genes, have shown that both the number and types of the microbes present in environmental samples, exceed by several orders of magnitude, the viable cell count done on plates or with most-probable-number techniques (Amann, R. I. et al, Microbial. Reviews 59: 143-169 (1995); Skirnisdόttir, S. et al, Appl Environ. Microbiol. 66: 2835-2841 (2000)). It is generally accepted that the majority of microscopically visualized cells are viable but do not form colonies on plates or grow in laboratory media that only select for certain organisms (Roszak, D. B. and R. R. Colwell, Microbiol. Rev. 51: 365- 379 (1987); Staley, J. T., and A. Konopka, Annu. Rev. Microbiol. 39: 321-346 (1985)).

Isolation and growth of new organisms has usually been done by employing various forms of the traditional laboratory enrichment methods (Alexander, M., Extreme environments. Mechanisms of microbial adaption. Heinrich M. R. eds., (New York Academic Press.), 3-25 (1976)). The main reason for the low ratio of presently cultivated microbial species is that for obvious practical reasons their isolation takes place under both space and time limited laboratory conditions. Other factors that explain the low ratio of cultivated species include unknown chemical components supplied to the species in their natural environment and missing in laboratory media and requirements for interdependent co- cultivation of two or more different species. To obtain sufficient biomass for DNA extraction from small volumes they must therefore be grown fast and to high densities. Rich laboratory medium is not a natural medium for environmental bacteria, copiotrophic organisms therefore gain a competitive edge and out compete oligotrophic species, although they may be more abundant in the habitat. Furthermore, rich medium may be growth inhibiting for oligotrophic species. Therefore, mainly copiotropic species that can grow fast under high-nutrient conditions have been isolated in the laboratory.

If natural communities exist at equilibrium, competitive exclusion ought to be the rule and the community would be dominated by a few species, the best competitors. However, if population growth rates are low for all members of the community, the competitive equilibrium is approached so slowly that it is never reached (Connell, Science, 199: 1302-1310 (1978)). Thus, species diversity can be maintained by periodic disturbance or by environmental fluctuations (i.e., nutrients, pH and temperature) (Buckling et al, Nature 408:961-964 (2000)).

The conditions that allow microbes to grow to high density in short time are "unnatural" for most organisms that normally grow slowly, at very low and steady-state concentrations of nutrients (Fry, J. C, "Oligotrophs" In Microbiology of Extreme Environments, C. Edward Ed., Milton Keynes, (Open University Press), 93-116 (1990)). Recreation of "nature-like" or natural, low-nutrient or oligotrophic conditions have been attempted in a few cases but can only be done on a small scale and with great efforts (Huber, R., W. et al, Appl Environ. Microbiol. 64: 3576-3583 (1998)). Growing many oligotrophs in the laboratory on a large enough scale for good DNA yield, would be practically impossible. Different modifications of the enrichment concept have been developed in order to obtain more novel organisms in culture. These include dilutions or pretreatment of the sample. The purpose is to kill or dilute out numerically abundant or fast growing organisms in the sample before inoculating the enrichment medium (GroBkopf, R. et al, Appl. Environ. Microbiol. 64:960-969 (1998); Santegoeds, C. M. et al, Appl. Environ. Microbiol. 62:3922- 3928 (1996)).

Still another attempt towards "nature-like" enrichments is the technique of in situ enrichment or substrate colonization that has been used in several environments. The in situ enrichment is based on the principle of introducing one or few new factors into an existing "natural" environment. Techniques of in situ enrichments have been of interest to microbiologists ever since bacteria were found to colonize microscope slides submerged in aquatic environments (ZoBell, C. E. et al., J. Bacteriol. 46:39-56 (1943); ZoBell, C. E., and D. 0. Anderson, Biol. Bull. 71: 324-342. (1936)). Such techniques have been used in hot springs to obtain specific groups of microorganism, by using specific substrates such as cellulose (Stainthorpe, A. C, and R. A. D Williams, Int. J. System. Bacteriol. 38:119-121 (1988)) and acetate (Konradsdottir, M., M. et al, System. Appl Microbiol. 74:190-195 (1991)). In situ enrichment for heterotrophic bacteria has also been performed at deep-sea vent environments (Marteinsson, V. T. et al, Can. J. Microbiol. 43: 694-697 (1997)).

Even with all of the cultivation and enrichment techniques mentioned above, only a very small proportion of the existing natural microbes have been accessed. In order to obtain novel protein-based catalysts from a larger proportion of this natural diversity, substantial efforts have been put into developing techniques to isolate DNA directly from environmental samples and to prepare gene expression libraries that can be screened for biological activities (U.S. Patent No. 6,001,574). There are however, three main problems associated with this approach. i) DNA isolated directly from environmental samples tends to be fragmented into small size and contain enzyme inhibitors that results in difficulties in cloning and expression of the desired genes from such DNA. Furthermore, the unavoidable fragmentation of DNA prevents the construction of bacterial artificial chromosome gene libraries with average DNA insert size larger that 20-40 kb (Rondon, M.R. et al, Appl. Environ. Microbial. 66:25A\-25 1 (2000)). Fragments in the range of 150 to 300 kb are preferable for ensuring more optimal genomic coverage and for managing metagenomic analysis and sequencing. ii) Environmental samples are not homogenous but composed of many microniches, where for example in hot springs large temperature gradients can cover only few mm. Large parts of the isolated DNA can therefore be from a non-target organism (i.e., mesophiles when one is targeting thermophiles). iii) Gene libraries constructed from DNA extracted directly from environmental samples represent the microbial genomes in approximately the same abundance as they occur in the natural population. Microbial species differ vastly in abundance in natural ecosystems and species inequality is particularly pronounced in extreme environments such as in hot springs. Typically 99% of a gene library prepared from hot spring samples represents fewer than 10 species (Reysenbach, A. L. et al, Extremophiles, 4:61-67 (2000); Skirnisdόttir, S. et al, Appl Environ. Microbiol 66: 2835-2841 (2000); Reysenach, A. L. et al, Appl. Environ. Microbiol. 60: 2113-2119 (1994)). Since a given sample may contain as many as 10,000 species, most of the other species will be very rare and their genes therefore practically unreachable with the above approach.

Attempts have been made to circumvent this problem of species inequality in natural populations by developing chemical and physical methods to fractionate the isolated DNA and thereby "normalizing" the abundance of the genomes present within the environmental sample (U.S. Patent No. 6,001,574). The applicability of those normalization methods have however, only been demonstrated to work on artificial mixtures of few cultivated microbes and not on natural genome mixtures extracted from real environmental samples (U.S. Patent No. 6,001,574).

Novel methods for efficient cultivation of a large part of the presently uncultivated microbes present in the environment are needed and therefore highly desirable.

SUMMARY OF THE INVENTION

The present invention provides methods for producing fresh cultures or biomass of rare and previously uncultivated microorganisms for screening of bioactive molecules and for isolation of high quality genomic DNA suitable for sequencing of genes. More particularly, methods are described for enriching different fractions of microbial populations from natural environments with variable diversity depending on substrate and physicochemical conditions. Methods are described for enriching a microbial population from a natural environment by obtaining a sample containing microorganisms from an environment, maintaining the sample under conditions substantially similar to the environment from which the sample was obtained for expanding the microbial population and allowing a sufficient quantity of a microbial population to expand; whereby the population has been enriched. This method can further comprise enriching the environmental conditions with a chemical additive to select for a microbial population that is influenced by the chemical additive. A method for collecting large amounts of hot geothermal fluid from sub-surface hydrothermal vents for retrieving biomass and for media preparations is also described. Also described are methods for screening for bioactive small molecules by enriching a microbial population; expanding the population; combining the population with a bioactive small molecule detector; and detecting bioactivity of sample versus control to determine presence of a bioactive small molecule in the enriched population. The present invention provides efficient nature-mimicking techniques including fluctuating physicochemical conditions that affect the microbial diversity to cultivate simultaneously many uncultivated microbes to sufficient densities so that they can be screened directly for small bioactive molecules, such as secondary metabolites.

In another embodiment, genes from microorganisms that can be grown under oligotrophic or in situ, nature-mimicking conditions can be isolated (e.g., by extraction) from an enriched population.

In yet another embodiment, methods are described for producing a normalized representation of genomes from enriched microbial populations from a natural environment is described by obtaining samples from a natural environment containing microbial populations; maintaining the samples under conditions substantially similar to the natural environment from which the microbial populations were obtained to expand microbial populations contained therein; monitoring cell density of each microbial population to obtain a sufficient quantity of microbial population for genome normalization; and combining the samples of the expanded microbial populations such that the combination represents a normalized genome. The DNA can be extracted and isolated from the normalized genome for making high quality gene libraries and for sequenced based screening for genes of interest, such as PCR amplification and DNA hybridization.

A method of screening for microbial modulators is also described as well as methods for determining phylogenetic characterization of an enriched microbial population. The present invention describes a method of screening for microbial modulators from an enriched microbial population by obtaining spent fluid or cells from an enriched microbial population; preparing extracts from the fluid or cells; combining the extract with a microbial biological entity; and determining the biological activity of the fluid extract or cell extract and comparing the biological activity of fluid extract or cell extract with the biological activity of biological entity in the absence of the extract; wherein the difference of activity is indicative of the presence of a modulator of the biological entity. The microbial modulator may be an inhibitor.

Also described are methods of assessing the nucleic acids from an enriched population for unknown family sequences of a known gene family, isolation, cloning and expression or detection of a target gene. The nucleic acids from enriched populations can be used as templates for sequence base screening, such as amplification and hybridizations methods. Sequences derived by amplification or cloning can be used as PCR primers or hybridization probes for detection of target genes in the same or other DNA sources. The methods described herein offer the ability to recover high diversity of active cells that have been growing under known and controlled physiological states during enrichments. Another advantage is that nucleic acid samples are more easily isolated and purified with previously described culture techniques than, from "dirty" environmental samples. Furthermore, large amounts of un-fragmented DNA may be obtained which is free from enzyme inhibitors, there is less risk of PCR artifacts and whole genes, gene clusters, promotor and intergenic sequences, large chromosomal fragments or gene operons will therefore be more easily accessible for DNA sequencing, PCR amplifications and cloning. Moreover, cloning of large fragments of DNA may provide access to metagenomic DNA for genomic sequencing of non-cultivated species (Rondon, M.R. et al, Appl. Environ. Bacteriol. 66: 2541-2547 (2000)).

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention.

Fig. 1 shows phylogenetic relationships of bacterial 16S rRNA sequences as determined by neighbor-joining analysis. The tree demonstrates results obtained by extracting DNA directly from environmental biomass (SRI clones) and by oligotrophic in situ enrichments (OLI clones).

Fig. 2 shows a phylogenetic tree constructed according to the amino acid alignment of the new sequences with sequences of selected amylolytic enzymes from thermophilic bacteria. The tree, constructed with the neighbor-joining method (Saitou, N., and M. Nei, Mol. Biol Evol 4: 406-425 (1987)), demonstrates varied nature of the amylolytic enzymes in the in situ enrichment cultures.

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

SAMPLING AND OLIGOTROPHIC ENRICHMENTS

The present invention provides special enrichment techniques to obtain biomass from rare target organisms from a variety of sources and obtain from the organisms nucleic acids of high quality and in sufficient quantity for use in biochemical and molecular biology research.

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hot spring) at various temperatures and depth or they may be incubated at specific conditions such as with programmed fluctuating in the laboratory. The containers may be filled with natural liquid and different gases (e.g., nitrogen, hydrogen) in various volumes as headspace of the enrichments. Various substrates in low concentration, from complex nutrients (e.g., yeast extract) to monomers (e.g., amino acids) may be added to the culture containers as well as other vital increments at will. In order to induce growth of microbes that contain genes coding for desired enzymes such as amylases and that may be active at certain temperature range, a container may be placed in a hot spring with in situ geothermal fluid and starch or other appropriate substrate, nutrients or inhibitors. Also, a probe for continuous monitoring of the temperature or pH may be put inside the containers. The additions can also include carbohydrates (e. g., cyclic sugars, monosaccharides, disaccharides, oligosaccharides, polysaccharides, glycoproteins, lectines and phosphate esters of carbohydrates), proteins (e.g., peptides, polypeptides, polypeptone, keratins, collagen, elastin etc.), fatty acids (e.g., propionate, butyrate, succinate, long chain fatty acids etc.), nucleic acids (e.g., nucleosides, nucleotides, deoxyribonucleic acids, ribonucleic acid etc.), lipids (e.g. triacylglycerols, phosphoglycerides etc.), or various other organic compounds such as alcohols, oils, cell extracts, dietary fibers, etc. Also, other modulating compounds like inhibitors (e.g., heavy metals, organic solvents or detergents) and anti-microbial agents (e.g. drugs, antibiotics, and preservatives) may be added. Various modes of energy conservation, other than organic substrates may also be used, such as hydrogen or sulfur compounds as electron donors and carbon dioxide, oxygen, nitrate or sulfur compounds as electron acceptors. A small sample of natural biomass typically millilitres of liquid, milligrams of solids or any dilution thereof may be used as additional inoculants.

The containers may be placed for incubation at the same location where the fluid was taken or it may be incubated at a different place such as a laboratory. Cell growth may be easily monitored by phase-contrast microscopy and the enrichment can be terminated at any time at any cell density. Series of enrichments can be done in different containers containing fluid from the same site with different incremental additions. After monitoring the cultures, the cells can be mixed in different proportions before concentrating the cells by centrifugation, in order to normalize the genome representation before DNA is extracted, followed by isolation of genes by PCR amplification or making of gene libraries. The cultures so obtained can also be screened directly for production of bioactive molecules and secondary metabolites. As used herein, "normalized" refers to making the amount of cells of different species approximately equal in quantity or numbers before DNA extraction of cell mixture in order to obtain a more even representation of their genomes.

The methods described herein offer the ability to recover high diversity of active cells that have been growing under known and controlled physiological states during enrichments. Another advantage is that nucleic acid samples are more easily isolated and purified with previously described culture techniques than, from "dirty" environmental samples. Furthermore, large amounts of un-fragmented DNA may be obtained which is free from enzyme inhibitors and there is less risk of undesirable artificial PCR amplifications. Also, these methods allow complete sequencing of whole genes, of gene operons or clusters of genes that code for enzymes for a particular biosynthetic pathway (e.g., metabolism of (synthesis and/or degradation) amino acids, vitamins, coenzymes or other secondary metabolites such as antibiotics and pigments).

Conditions of the enrichments may be influenced by chemical additions to induce growth and allow selective target groups of microbes to flourish. The target groups of the microbes are influenced by the chemical additive. For example, one may enrich for microorganisms that use starch in their metabolism and contain genes encoding for desired biological catalysts, e.g., amylolytic enzymes that are active at least at 65°C. The fluid in the container is supplemented with starch for inducing growth of such microorganisms which are able to use starch as an energy source. The container containing the microorganisms and inducer is placed at some depth in a hot spring at a desired temperature. After time the culture is collected and the data from the temperature probe is read to record the actual temperature fluctuations during the enrichment period. Allowing the microbes to grow in the presence of starch would enrich for organisms able to induce starch degrading enzymes. DNA may be isolated and the culture screened for microbial diversity and/or diversity of genes encoding amylolitic enzymes. Various substrates in low or high concentration may be added such as but not limited to carbohydrates (e.g., cyclic sugars, monosaccharides, disaccharides, oligosaccharides, polysaccharides, glycoproteins, lectines and phosphate esters of carbohydrates), proteins (e.g., peptides, polypeptides, polypeptone, keratins, collagen, elastin etc.), fatty acids (e.g., propionate, butyrate, succinate, long chain fatty acids etc.), nucleic acids (e.g., nucleosides, nucleotides, deoxyribonucleic acids, ribonucleic acid etc.), lipids (e.g. triacylglycerols, phosphoglycerides etc.), or various other organic compounds such as alcohols, oils, cell extracts, dietary fibers, etc. Also other modulating compounds can be used such as but not limited to inhibitors (e.g., heavy medals, organic solvents or detergents) and anti-microbial agents (e.g. drugs, antibiotics, and preservatives). Various modes of energy conservation other than organic substrates may also be used, such as hydrogen or sulfur compounds as electron donors and carbon dioxide, oxygen or sulfur compounds as electron acceptors.

In another aspect of the present invention, hot geothermal fluid from terrestrial wells or from submarine springs that are difficult to access may be pumped in large volumes up to the surface through a flexible tube. Various lengths of flexible tubing can be attached at one end to a stainless steel (titanium, etc.) tube and the other end kept at the surface. The tube can be inserted into a discharge opening at shallow depth by a scuba diver or with a submersible robot or by a man-operated submersible at greater depths. The fluid would be pumped up to the surface and used for oligotrophic enrichments as already described or for media preparation or the fluid may be filtered to harvest the indigenous microbes (or biomass) for diversity analysis. Free-living cells in low concentration or not accessible due to dilution effect may be enriched or concentrated by filtration for example by a cross-flow filtration through sterile hollow fiber cartridge (0. 22 _m). Sufficient amounts of DNA from microorganisms may be obtained by filtration of large quantity of hot geothermal fluid emitted from different locations at various depths on the sea floor or by inoculation into various media for oligotrophic enrichments, as previously described.

The methods described herein can be used for screening for bioactive small molecules in an enriched microbial population. An enriched microbial population from a natural environment is obtained using the techniques set forth herein. The sample is then maintained under conditions substantially similar to the environment from which the sample was obtained for expanding the microbial population thereby allowing a sufficient quantity of a microbial population to expand. The enriched population is then combined with a bioactive small molecule detector under suitable conditions for bioactivity. The extent of detection is determined and compared with a control wherein a difference between the control and sample is indicative of the presence of a bioactive small molecule in the enriched population. The bioactive small molecule may be a macromolecule such as proteins or a secondary metabolite. The bioactive molecules may be small molecule compounds such as anti-microbials, anti-fungals, anti-virals and other therapeutic drugs or molecules with preservation- and detergent activity. The detection can be accomplished by standard methods used in the art such as labeling with a colormetric indicator. DNA ISOLATION

Methods are also described for producing a normalized representation of genomes from multiple enriched microbial environments. Samples containing microorganisms are obtained from multiple natural environments. The samples are then maintained under conditions substantially similar to the natural environments. The enriched microbial populations are combined, DNA extracted, isolated and characterized, thereby producing a normalized representation of the genomes derived from these multiple enriched microbial environments. The enriched microbial population also provides large quantities of cells allowing use of different isolation techniques that ensure little fragmentation of the DNA, such as casting the cells in agar plugs and using mild enzymatic methods of cell lysis and DNA purification in order to obtain sufficiently large fragments for construction of bacteria metagenomic libraries (Rondon, M.R. et al, Appl. Environ. Bactertiol. 66: 2541-2547 (2000)). Such libraries facilitate the genetic screening for whole genes and operons coding for enzymes involved in cooperative synthesis of low weight secondary metabolites.

Also described herein are methods of screening for enriched microbial modulators, inhibitors or enhancers. A modulator as used herein refers to a change or an alteration of activity. A modulator can be an inhibitor or an enhancer. Spent fluid from microbial enrichment is obtained. Extracts from the fluid are prepared and combined with a microbial biological entity that can be whole cells (e.g., bacteria, fungi or protozoa) or isolated molecules (e.g., receptors, enzymes or other proteins) or cell cultures (e.g., plant or mammalian cells or tissues cultures). The biological activity of the extract and biological entity are determined and compared to the biological activity of biological entity in the absence of the extract whereby the difference in activity is indicative of the presence of a modulator of the biological entity. The microbial biological entity may be a whole organism.

Also described are methods of screening for enriched microbial modulators. Cells are obtained from oligotrophic enrichment and extracts prepared from the cells. The extracts are then combined with a microbial biological entity. The bioactive molecules may be small molecule compounds such as anti-microbials, anti-fungals, anti virals and other therapeutic drugs or molecules with preservation- and detergence activity. The biological activity of the extracts is determined and compared with the biological activity of biological entity in the absence of the extract, whereby the reduction of activity is indicative of the presence of a modulator of the biological entity. The biological entity may be a whole organism. The inhibitors inhibit the growth of microbes and may be used to control the growth or certain organisms such as pathogenic organisms. Enhancers augment the growth of microbes. Also described herein are methods of determining phylogenetic characterization of a enriched microbial population. Total DNA of a natural environment microbial population sample is randomly isolated. The SSU rRNA genes are amplified by PCR using specific primers and the amplified genes are cloned creating a SSU rRNA gene library. The SSU rRNA genes clones are then sequenced and assessed for phylogenetic characterization using sequence contigs from selected libraries.

A normalized gene library that is useful for screening may also be prepared by cultivating individual species separately and then mixing them in approximately equal proportions to each other before DNA isolation. The advantages with using cultivated species is that large amounts of un-fragmented DNA which is free from enzyme inhibitors, is more easily isolated and purified from microbes freshly cultivated than from "dirty" environmental samples that adversely affects the quality of the DNA, where the microbes are mostly dormant or in unknown physiological state. Such mixing of fresh cultures can readily be used for species that are present in strain collections or that can be easily isolated with current laboratory techniques. It is apparent that traditional laboratory isolations and cultivation of most uncultivated species would be an impossible task, the solution to this problem is achieved by the enrichment methods described herein.

If the estimate is correct that most microbial ecosystems contain thousands of different species, even if some normalization can be achieved with the described methods (U.S. Patent No. 6,001,574), they will still only allow researchers to access a small proportion of the existing microbes. The use of expression gene libraries for screening for bioactive molecules such as those having pharmaceutical applications is also very limited since few of such molecules like many secondary metabolites, will be correctly synthesized by heterologous hosts. In order to screen larger part of currently uncultivated microbes for bioactive molecules and to benefit in the advantages of more targeted approach, the higher quality DNA obtained from freshly cultivated microbes with closer to "normalized" genome representation, new cultivation methods are needed.

Extraction of DNA is an important step for molecular analysis. DNA can be isolated from samples using various techniques well known in the art and no special procedure is required. However, when extracting DNA directly from an environmental sample, such as hot springs, many physical, chemical and biological factors can interfere with the extraction or with the nucleic acid. DNA isolation is an important and difficult step in the generation of a normalized DNA library from an environmental sample, but no reliable method exist which can deal with all the interfering barriers found in an environment. Preferably, cells may be separated, cultured and harvested from interfering factors in the environment by using the enrichment techniques described herein.

RETRIEVAL OF GENES

A useful embodiment of the invention involves assessing the nucleic acids from the enriched population for unknown homologous family sequences of one or more known genes using polymerase chain reaction.

The term 'homologous' as used herein refers to sequences of shared evolutionary origin, i.e. that have a common genetic ancestor.

In one preferred embodiment of the invention, the isolated nucleic acids comprises a step of amplifying the copy number of genes by the use of primers that are designed on the basis of alignments of sequences from specific protein families after alignments of sequences from gene families. The primers used are designed on the basis of conserved regions in these families and include techniques of using both two degenerate, forward and reverse primers or only a single degenerate primer where the second primer is targeted to an adapter site or one supplied by a cloning vector (Morris, D.D. et al, Appl. Environ. Microbiol. 61:2262-2269 (1995); Shyamala, V. & Ames, G.F., Gene. 84 -S (1989); Timothy, M.R., et al, Nucleic Acids Research 2:1628-1635 (1998)).

Another embodiment of the invention involves making a gene library from the isolated DNA (Woo, S.S. et al., Nucleic Acid Res. 22:2922-4931 (1994); Rondon, M.R., PNAS 96: 6451-6455 (1999)).

Yet, another embodiment involves mixing the cells of a series of oligotrophic enrichments in such a way to create a cell mixture that contains normalized representation of genomes from all enrichments in the series.

DIVERSITY ANALYSIS

Analysis of the complexity of the nucleic acid recovered from the enrichments can be monitored by SSU rRNA analysis (Reysenbach, A. L. et al, Appl. Environ. Microbiol. 58: 3417-3418 (1992); DeLong, E. F., Proc. Natl. Acad. Sci. 89: 5685-5689 (1992); Bams, S. M., et al, Proc. Natl. Acad. Sci. USA. 97:1609-1613 (1994); Skirnisdόttir, S. et al, Appl. Environ. Microbiol. 66: 2835-2841 (2000)). Primers have been described for the specific amplifications of SSU rRNA genes from each of the three described domains of life, Eukarya, Bacteria and Archaea. The SSU rRNA analysis is made mainly of five major steps, e.g., (i) random isolation of the total DNA of the biomass sample; (ii) specific amplification of the SSU rRNA genes through polymerase chain reaction (PCR); (iii) cloning of the amplified fragments and creation of a SSU rRNA genes library; (iv) sequencing of the SSU rRNA genes clones; and (v) phylogenetic characterization of the population members.

GENE MINING

The enrichment methods described herein are useful for obtaining diverse nucleic acids, such as from uncultured organisms. These nucleic acids may be further assessed and utilized in known methods to obtain gene families, investigate polymorphisms, cloning and expression of target genes as well as using the nucleic acids probes as primers for the detection of target genes. Furthermore, to use the nucleic acids as targets in sequence based screening by PCR amplifications using primers derived from known sequences of genes and proteins, or DNA by hybridization using known genes or probes derived from known genes or proteins. In these methods, nucleic acids also include variants which hybridize under high or low stringency hybridization conditions (e.g., for selective hybridization) to a target nucleotide sequence.

A "target gene" is a nucleic acid sequence of interest that may be obtained or detected by the methods described herein. Such nucleic acid molecules can be detected and/or isolated by specific hybridization (e.g., under high or low stringency conditions). Any of those conditions can be varied to increase the possibility of detecting distantly related sequence. "Stringency conditions" for hybridization is a term of art which refers to the incubation and wash conditions, e.g., conditions of temperature and buffer concentration, which permit hybridization of a particular nucleic acid to a second nucleic acid; the first nucleic acid may be perfectly (i.e., 100%) complementary to the second, or the first and second may share some degree of complementarity which is less than perfect (e.g., 70%, 75%, 85%, 95%). For example, certain high stringency conditions can be used which distinguish perfectly complementary nucleic acids from those of less complementarity. "High stringency conditions", "moderate stringency conditions" and "low stringency conditions" for nucleic acid hybridizations are explained on pages 2.10.1-2.10.16 and pages 6.3.1-6.3.6 in Current Protocols in Molecular Biology (Ausubel, F.M. et al., "Current Protocols in Molecular Biology", John Wiley & Sons, (1998), the entire teachings of which are incorporated by reference herein). The exact conditions which determine the stringency of hybridization depend not only on ionic strength (e.g., 0.2XSSC, 0.1XSSC), temperature (e.g., room temperature, 42°C, 68°C) and the concentration of destabilizing agents such as formamide or denaturing agents such as SDS, but also on factors such as the length of the nucleic acid sequence, base composition, percent mismatch between hybridizing sequences and the frequency of occurrence of subsets of that sequence within other non-identical sequences. Thus, equivalent conditions can be determined by varying one or more of these parameters while maintaining a similar degree of identity or similarity between the two nucleic acid molecules. Typically, conditions are used such that sequences at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 95% or more identical to each other remain hybridized to one another. By varying hybridization conditions from a level of stringency at which no hybridization occurs to a level at which hybridization is first observed, conditions which will allow a given sequence to hybridize (e.g., selectively) with the most similar sequences in the sample can be determined.

Exemplary conditions are described in Krause, M.H. and S.A. Aaronson, Methods in Enzymology, 200:546-556 (1991). Also, in, Ausubel, et al, "Current Protocols in Molecular Biology", John Wiley & Sons, (1998), which describes the determination of washing conditions for moderate or low stringency conditions. Washing is the step in which conditions are usually set so as to determine a minimum level of complementarity of the hybrids. Generally, starting from the lowest temperature at which only homologous hybridization occurs, each °C by which the final wash temperature is reduced (holding SSC concentration constant) allows an increase by 1% in the maximum extent of mismatching among the sequences that hybridize. Generally, doubling the concentration of SSC results in an increase in T_m of ~17°C. Using these guidelines, the washing temperature can be determined empirically for high, moderate or low stringency, depending on the level of mismatch sought.

For example, a low stringency wash can comprise washing in a solution containing 0.2XSSC/0.1% SDS for 10 min at room temperature; a moderate stringency wash can comprise washing in a prewarmed solution (42°C) solution containing 0.2XSSC/0.1% SDS for 15 min at 42°C; and a high stringency wash can comprise washing in prewarmed (68°C) solution containing 0.1XSSC/0.1%SDS for 15 min at 68°C. Furthermore, washes can be performed repeatedly or sequentially to obtain a desired result as known in the art. Equivalent conditions can be determined by varying one or more of the parameters given as an example, as known in the art, while maintaining a similar degree of identity or similarity between the target nucleic acid molecule and the primer or probe used.

The percent identity of two nucleotide or amino acid sequences can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence). The nucleotides or amino acids at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences ( . e. , % identity = # of identical positions/total # of positions x 100). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 60%, and even more preferably at least 70%, 80% or 90% of the length of the reference sequence. The actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. A preferred, non-limiting example of such a mathematical algorithm is described in Karlin et al, Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) as described in Altschul et al, Nucleic Acids Res., 25:389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. In one embodiment, parameters for sequence comparison can be set at score=100, wordlength=12, or can be varied {e.g., W=5 or W=20).

Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the CGC sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12 , and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Bioscl, 10:3-5; and FASTA described in Pearson and Lipman (1988) PNAS, 55:2444-8.

Additionally, the percent identity between two amino acid sequences can be accomplished using the GAP program in the CGC software package (available at http://www.cgc.com) using either a Blossom 63 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4. Also, the percent identity between two nucleic acid sequences can be accomplished using the GAP program in the CGC software package (available at http://www.cgc.com), using a gap weight of 50 and a length weight of 3.

The present invention also provides methods of obtaining isolated nucleic acid molecules that contain a fragment or portion that hybridizes under highly stringent conditions to a target nucleotide sequence. The nucleic acid fragments obtained by the methods of the invention described herein can be at least about 15, preferably at least about 18, 20, 23 or 25 nucleotides, and can be 30, 40, 50, 100, 200 or more nucleotides in length. Longer fragments, for example, 30 or more nucleotides in length, which encode antigenic polypeptides described herein are particularly useful, such as for the generation of antibodies.

In a related aspect, target genes in the DNA obtained by the methods of the invention can be identified with the help of probes or primers in assays such as those described herein. "Probes" are oligonucleotides that hybridize in a base-specific manner to a complementary strand of nucleic acid molecules. Such probes include polypeptide nucleic acids, as described in Nielsen et al, Science, 254, 1497-1500 (1991). Typically, a probe comprises a region of nucleotide sequence that hybridizes under highly stringent conditions to at least about 15, typically about 20-25, and more typically about 40, 50 or 75, consecutive nucleotides of a nucleic acid molecule comprising a nucleotide sequence selected from the target gene and the complement of the target gene. More typically, the probe further comprises a label, e.g., radioisotope, fluorescent compound, enzyme, or enzyme co-factor.

As used herein, the term "primer" refers to a single-stranded oligonucleotide which acts as a point of initiation of template-directed DNA synthesis using well-known methods {e.g., PCR, LCR) including, but not limited to those described herein. The appropriate length of the primer depends on the particular use, but typically ranges from about 15 to 30 nucleotides.

The nucleic acid molecules isolated by the methods of the invention such as those described above can be identified and isolated using standard molecular biology techniques. For example, nucleic acid molecules can be amplified and isolated by the polymerase chain reaction (including all types of PCR, e.g., inverse PCR) using one or more synthetic oligonucleotide primers designed based on one or more target sequences and/or the complement of a target sequence. See generally PCR Technology: Principles and Applications for DNA Amplification (ed. H.A. Erlich, Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods and Applications (Eds. Innis, et al, Academic Press, San Diego, CA, 1990); Mattila et al, Nucleic Acids Res., 19:4967 (1991); Eckert et al, PCR Methods and Applications, 7:17 (1991); PCR (eds. McPherson et al, IRL Press, Oxford); and U.S. Patent 4,683,202. The nucleic acid molecules can be amplified using cDNA, mRNA or genomic DNA as a template, cloned into an appropriate vector and characterized by DNA sequence analysis.

Other suitable amplification methods include the ligase chain reaction (LCR) (see Wu and Wallace, Genomics, 4:560 (1989), Landegren et al, Science, 241:1077 (1988), transcription amplification (Kwoh et al, Proc. Natl. Acad. Sci. USA, 86:\ 173 (1989)), and self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 57: 1874 (1990)) and nucleic acid based sequence amplification (NASBA). The latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.

The amplified DNA can be radiolabelled or non radioactively labeled and used as a probe for screening a gene library derived from the DNA obtained by the methods of the invention, in a suitable vector. Corresponding clones can be isolated, DNA can be obtained following in vivo excision, and the cloned insert can be sequenced in either or both orientations by art recognized methods to identify the correct reading frame encoding a polypeptide of the appropriate molecular weight. For example, the direct analysis of the nucleotide sequence of nucleic acid molecules of the present invention can be accomplished using well-known methods that are commercially available. See, for example, Sambrook et al, Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York 1989); Zyskind et al, Recombinant DNA Laboratory Manual, (Acad. Press, 1988)). Using these or similar methods, the polypeptide and the DNA encoding the polypeptide can be isolated, sequenced and further characterized.

Antisense nucleic acid molecules of the invention can be designed using the nucleotide sequences of and/or the complement of, and/or a portion of or the complement of, and constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid molecule {e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Alternatively, the antisense nucleic acid molecule can be produced biologically using an expression vector into which a nucleic acid molecule has been subcloned in an antisense orientation {i.e., RNA transcribed from the inserted nucleic acid molecule will be of an antisense orientation to a target nucleic acid of interest).

In general, the isolated nucleic acid sequences of the invention can be used as molecular weight markers on Southern gels, and as chromosome markers which are labeled to map related gene positions. The nucleic acid sequences can also be used as probes, such as to hybridize and discover related DNA sequences or to subtract out known sequences from a sample. The nucleic acid sequences can further be used to raise anti-polypeptide antibodies using DNA immunization techniques. Portions or fragments of the nucleotide sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to map their respective genes on a chromosome; and, thus, locate gene regions in the corresponding species. Additionally, the nucleotide sequences of the invention can be used to identify and express recombinant polypeptides for analysis, characterization and therapeutic use.

Another aspect of the invention pertains to nucleic acid constructs containing a nucleic acid molecule and the complement of the nucleic acid molecule (or a portion thereof). The constructs comprise a vector (e.g., an expression vector) into which a sequence or open reading frame obtained by the invention has been inserted in a sense or antisense orientation. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced {e.g., bacterial vectors having a bacterial origin of replication). Other vectors {e.g., bacterial transposon vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors, expression vectors, are capable of directing the expression of genes to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., bacterial phages, plant and animal viruses) that serve equivalent functions.

Preferred recombinant expression vectors of the invention comprise a nucleic acid molecule of the invention in a form suitable for expression of the nucleic acid molecule in a host cell. This means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably or operatively linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence {e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements {e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed and the level of expression of polypeptide desired. The expression vectors of the invention can be introduced into host cells to thereby produce polypeptides, including fusion polypeptides or genetically modified polypeptides, encoded by nucleic acid molecules obtained as described herein.

The recombinant expression vectors of the invention can be designed for expression of a polypeptide of the invention in prokaryotic or eukaryotic cells, e.g., bacterial cells such as E. coli, or using expression vectors, fungal cells, yeast cells or plant cells. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, a nucleic acid molecule of the invention can be expressed in bacterial cells {e.g., E. coli), fungal cells, yeast or plant cells. Other suitable host cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing a foreign nucleic acid molecule {e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al {supra), and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker {e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Nucleic acid molecules encoding a selectable marker can be introduced into a host cell on the same vector as the nucleic acid molecule of the invention or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid molecule can be identified by drug selection {e.g. , cells that have incorporated the selectable marker gene will survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce {i.e., express) a polypeptide obtained by the invention. Accordingly, the invention further provides methods for producing a polypeptide using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a polypeptide of the invention has been introduced) in a suitable medium such that the polypeptide is produced. In another embodiment, the method further comprises isolating the polypeptide from the medium or the host cell.

The host cells of the invention can also be used to produce recombinant or transgenic organisms. For example, in one embodiment, a host cell of the invention is a prokaryote, fungal, yeast or plant cell molecule of the invention has been introduced. Such host cells can then be used to create recombinant or transgenic organisms in which exogenous nucleotide sequences have been introduced into the genome. Such organisms are useful for studying or changing the function and/or activity of the nucleotide sequence and polypeptide encoded by the sequence and for identifying and/or evaluating modulators of their activity. As used herein, a "recombinant organism", is an organism preferably a prokaryote such as Escherichia coli, in which one or more of the cells of the organism includes a transgene or a recombinant gene. Other examples of transgenic organisms include fungi, yeast and plants. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic organisms develops and which remains in the genome of the mature organism, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic organisms. Methods for generating recombinant prokaryotes by homologous recombination are described in Hamilton et al, J. Bacteriol. 171:4617-4622 (1989) and in Fridjonsson et al, Extremophiles 4:23-33 (2000).

The invention will now be illustrated by the following Examples which are not intended to be limiting in any way. All references cited herein are incorporated by reference in their entirety. EXAMPLE 1

OLIGOTROPHIC ENRICHMENT WITH HOT SPRING WATER IN LABORATORY

Samples were collected in a sulfide rich hot spring in Hveragerdi (Grensdalur), Iceland. About thirty liters of hot spring water were collected in a sterile container. Sulfur- mat or filaments were collected at 65°to 75°C and the biomass sample was stored in a sterile flask at 4°C. All media and inoculations were prepared on the day of sampling. Three series of media with different concentration of additional supplements were prepared with 500 ml spring water as aqueous base solutions, in Erlenmeyer flasks for aerobic cultivation and in closed bottles for anaerobic processes. The following stock solutions, which had been sterilized by autoclavation were added later: 1% starch (w/v), 25% (w/v) (NH₄)₂S0₄, 12.5% NaCl (w/v) and 10%) (w/v) Yeast Extract (Difco). The natural hot spring water was not autoclaved before inoculation. The biomass sample was homogenized by shaking and diluted in series with spring water down to a 10^" -fold. Each series of media (1 to 10) was inoculated with 5 ml of a specific dilution of the biomass mix. The series inoculated with 10^"2 dilution was designated as Rl to R10, the series inoculated with 10^"4 was designated as Gl to G10 and the series inoculated with 10^" as <j>l to φlO. The inoculum for the series R was specifically treated with 50 % ethanol (vol/vol) for 10 min. before inoculation. Series 2 to 6 were supplemented with 0. 1% starch and 1. 0% (NH₄)₂Sθ₄ final concentration. Series 8 to 10 with 0. 002% starch and 0. 02% (NH₄)₂S0_4. Series 7 with 0. 02% starch and 1. 0% (NH₄)₂S0 _. All series were cultivated aerobically except for series 3 and 7. Anaerobiosis was achieved by applying a vacuum to the media and saturating it with nitrogen gas (N₂). Finally, the media were reduced by adding a sterile solution of Na S . 9H₂0 (final concentration, 0. 025% [wt/vol]). Nothing was added to series 1. The pH was adjusted to 9. 5 with NaOH (1 N) in series 4 and 8, and to pH 4. 0 with HCl (1 N) in series 6 and 9. In series 5 and 10, 0. 5% (w/v) NaCl was added as final concentration. Media, inoculated with 10^"7 dilution were prepared and supplemented with final concentration of 0. 5% starch, 0. 1 % and 0. 01% yeast extract in spring water and designated as S, YE.l and YE.01, respectively. All cultures were incubated at 65°C without shaking in a incubation oven (Gallencamp).

Cells were observed with a Leica DM LB light microscope equipped with a phase- contrast oil immersion objective (magnification, xlOO) and were counted by using a Petroff- Hausser chamber (depth, 0. 02 mm [Hausser Scientific Partnership, Horsham, PA, USA]). Each culture was stopped when the cell concentration had reached to about 10⁷ cells/mL. Before pelleting, a 20 ml sample of each culture was removed and stored either aerobically or anaerobically at 4°C.

Results from oligotrophic enrichments in three series of natural hot spring media with different concentration of additional supplements are presented in Table 1. No growth was observed in enrichments containing 0. 001%) Y. E. or lower after 16 days. When 0.005% Y.E. was added after 16 days of cultivation, cell numbers in series R, G, and φ reached 10⁵ - 10⁸ cell/ml within 2 to 42 days.

DNA was extracted from all enrichments showing positive growth and stored at -20°C. All cultures contained Bacterial 16S rRNA genes but no Archaea 16S rRNA genes. A total of 13 enrichments were selected for creating 16S rRNA genes libraries for SSU gene sequencing (R2, R3, R6, R10, G2, G3, G5, G7, φ2, φ7, φlO and S).

All clones were sequenced with R805 reverse primer and all sequences could be aligned to each other and to sequences in the Ribosomal database. Only sequences with reliable nucleotide sequence were edited and aligned with reference strains. Table 2, shows the closest database matches for the sequence in contigs after BLAST searches.

The results show closest matches to cultivated species that belong to seven genera {Bacillus, Thermits, Meiothermus, Caloramator, Thermoterrabacterium, Chloroflexus and Moorella), one potential new genus and five non-cultivated bacterial OTUs. One belongs to unidentified green non-sulfur bacterium clone OPB34, another to unidentified Cytophagales clone OPB88, two to new candidates for new bacterial divisions, OP9 and OP12 (Hugenholtz, P., C. et al, "Novel division level bacterial diversity in a Yellowstone hot spring," J Bacteriol 180: 366-376 (1998)), and the last one to unidentified Thermits clone SRI248 (Skirnisdόttir et l, Appl. Environ. Microbiol 66:2835-2841 (2000)).

Sequence contigs from ten libraries out of thirteen selected enrichments were used for the construction of the phylogenetic tree (Figure 1). Sequences in libraries from enrichments R2, G2 and φ2 were not used to prevent redundancy. The libraries revealed eighteen phylogenetic distinct clusters (that represent at least twelve new species in eleven genera). The oligotrophic enrichment clones were designated OLI. In enrichments G (dilution 10^"4) six new species grew that were gathered to five genera. OLI-16G3 and OLI-15G7 belonged to the genus Thermoterrabacterium, although the last one was distantly related to the reference sequence. OLI-3G7 and OLI-9G7 were related to candidate division OP 12 and OP9 respectively (Hugenholtz et al, J. Bacteriol. 180:366-376 (1998)). OLI-10G5 is closely related to Bacillus flavothermus and OLI-14G7 to unidentified green non sulfur bacterium OPB34 (Hugenholtz et al, J. Bacteriol. 180:366-376 (1998)). In enrichments R (dilution 10^"2) two new species grew that were gathered in two genera. OLI-12R3 was closely related to Caloramator indicus and OLI-12R6 to Thermits SRI248 (Skirnisdottir et al, Appl. Environ. Microbiol. 66:2835-2841 (2000)). Enrichment S (dilution 10^"7) gave species belonging to five genera. Clone OLI-6S was closely related to Chloroflexus aurantiacus and clone OLI-16S to Meiothermus ruber. OLI-22S and OLI-12S belonged to Thermits ZFI A.2 and Thermits SRI96 respectively (Skirnisdottir et al, Appl. Environ. Microbiol. 66:2835-2841 (2000)). OLI-5S was only distantly related to unidentified Cytophagales OPB88 (Hugenholtz et al, J. Bacteriol. 180:366-376 (1998)). Finally, in φ enrichments (dilution 10^"8, clones designated F) five species were detected. OLI-11F3, OLI-10F7 and OLI-4F10 were closely related to Caloramator fervidus, Moorella glycerini and Thermits oshimai, respectively. Clone OLI-12F10 was distantly related to M. glycerini and OLI-15F3 showed very low homology to the genus Caloramator and might be a representative to a potential new genus.

The phylogenetic tree in Figure 1 shows alignment of 16S rRNA sequences obtained with oligotrophic in situ culture method and by extracting DNA direct from environmental biomass (Skirnisdottir et al, Appl. Environ. Microbiol. 66:2835-2841 (2000)). Samples were taken from the same spot. Different kind of species and genera were detected with each method. The oligotrophic method obtained much more diversity in the hot spring than the culture-independent method (Skirnisdottir et al, Appl. Environ. Microbiol. 66:2835-2841 (2000)). The following known bacterial genera: Morrella, Thermoterrabacterium, Caloramator, Bacillus, Chloroflexus, Meiothermus and Thermits were detected. Other bacterial sequences belonged to non-cultivated and unidentified microorganisms, like unidentified green non-sulfur bacterium OPB34, candidate division OP12 (clone OPB54), candidate division OP9 (clone OPB47), and to unidentified Cytophagales (clone OPB88). Only Thermus was also detected with the culture-independent method.

EXAMPLE 2

OLIGOTROPHIC ENRICHMENT IN CULTURE CONTAINERS IN HOT SPRING

Spring water from a hot spring with surface about 6 m² and 0. 3 to 1. 5 m deep was poured into two sterile 950 ml polyethylene containers. One of them was inoculated with 0. 005% (w/v) Yeast Extract (Difco) and designated "BrusiY", while the other one contained 0. 25% (w/v) starch and designated "BrusiS". Both BrusiY and S contained 1% (w/v) NH₄C1 (final concentration). The two containers were filled up with the spring water and then closed and placed at 1 m depth at 65°C for 21 days. A temperature probe was used to measure the temperature inside the container with 5 minutes interval during the enrichment. Over the incubation period the temperature fluctuated between 57°C and 72°C. The initial temperature was about 67°C, 65°C on the second day, up again to 72 on the forth day, and down to 59°C on the fifth day. After the fifth day, the temperature was fluctuating between 59°C and 66°C for 16 days. The fluctuations were close to being periodical with 1 or 2 days between peaks.

Both in situ oligotrophic enrichments were positive for growth. Microscopic observation showed that both contained mixed population of rod-forming and coccoid cells.

Large amounts of good quality DNA were extracted from both enrichments. Bacterial 16S rRNA genes could be amplified in both samples but no Archaea 16S rRNA genes. All clones were sequenced with R805 reverse primers and all sequences could be aligned to each other and to sequences in the ribosomal database. Only sequences with reliable nucleotide sequences were edited and aligned with reference strains. At least four genera could be detected, Thermits, Bacillus, Clostridium and Thermoanaerobacterium and at least one non- cultivated genus (Table 3).

EXAMPLE 3

COLLECTING GEOTHERMAL FLUID FROM HYDROTHERMAL VENTS.

A large quantity of hot geothermal fluid was collected from submarine hot springs, located 1. 8 km offshore in the north-eastern part of the fjord Eyjafjordur, Iceland. The vents occur on the east-slope, which rises from 100-m depth from the center of the fjord. At about 65 m in depth, three giant silicate cone structures, have grown at the site to heights of 33, 25 and 45 m above the sea bottom. A scuba diver was sent down with a rubber hose attached to stainless steel tube (0. 4 m _10 mm). The steel tube was placed inside in a discharge opening at 27. 5 m depth. Two successive 12 V booster pumps were mounted inside the tubing, few meters below the sea surface. The other end of the tube was attached to a rubber dingy. The whole system (40 m long) was rinsed with the hot fluid (around 2 liters min^"1) for 30 min before sampling hot fluid for chemical and microbial analysis. The vent fluid was collected or concentrated directly by cross-flow filtration through sterile hollow fibre cartridges (0. 22-μm filter, Amicon). The cells retained inside the cartridge (600 ml) were concentrated further in the laboratory by centrifugation. About 240 liters of 71. 6°C hot vent fluid, from a vent at 27. 5 m depth was pumped and concentrated to 600 ml by filtration and pellated in an eppendorf tube.

The hydrothermal fluid had only about 0. 1 % contamination by seawater and was also used for oligotrophic enrichments as described in Example 1. Microscopic evaluation after 14 days in oligotrophic enrichments at 65 to 80°C revealed complex community of cells. DNA was successfully extracted from the concentrated biomass. Sequencing of environmental clones revealed both Bacteria (45 clones) and Korarchaea (10 clones) sequences (Table 5). The thermophilic taxonomic divisions of Bacteria represented by the clones, included mostly the order Aquificales and one unidentifed Nitrospira clone. Three clones were closest to the mesophilic divisions of Proteobacteria and Firmicutes.

EXAMPLE 4 DNA ISOLATION

Cell pellets were obtained from each culture by centrifugation for 30 minutes at 8. 000 rpm (Sorval) and 4°C.

Cells were disrupted with a sterile mortar (or homogenizer) and incubated for 1 hour at 37°C in lysis TNE buffer (Tris-NaCl-EDTA, (100 mM, 100 mM, 50 mM), pH 8. 0 and 1 mg/ml (final concentration) Lysozyme (Sigma), and for 2 hours at 50°C with 1% SDS, 1% Sarcocyl and 1 mg/ml Proteinase K (Sigma, final concentrations). Gently mixed by inversion. The protein fraction was removed with several extractions with Phenol :Chloroform:Isoamyl alcohol (Sigma, 25:24:1), pH 8. 0. Nucleic acids were ethanol-precipitated and dried during 10 minutes of vacuum centrifugation (SpeedVac). DNA was finally resuspended in 100 μl of TE solution (Tris-EDTA, (100 mM, 50 mM)), pH 8. 0 and its quality analyzed on a 0. 8% TAE-agarose gel electrophoresis. DNA was stored at -20°C.

EXAMPLE 5 DIVERSITY ANALYSIS

Bacterial and Archaeal 16S ribosomal RNA genes were specifically amplified with universal oligonucleotide primer sets. The following Bacterial {Escherichia coli) primers were used:

Forward primer (F9) 5'-GAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. : 1) Forward primer (F515) 5'-GTCCCAGCAGCCGCGGTAAATAC-3' (SEQ ID NO. : 2) Reverse primer (R805) 5'-GACTACCGGGTATCTAATCC-3' (SEQ ID NO. : 3) Reverse primer (Rl 544) 5'-AGAAAGGAGGTGATCCA-3' (SEQ ID NO. : 4)

The Archaea specific primer set used was 23 FPL and 1391R (Bams, S. M. et al, Proc. Natl. Acad. Sci. USA. 97:1609-1613 (1994)). Forward primer (23 FPL) 5'-

GCGGATCCGCGGCCGCTGCAGAYCTGGTYGATYCTGCC-'3 (SEQ ID NO. : 5); Y indicates pyrimidine substitution.

Reverse primer (1391R) 5'-GACGGGCGGTGTGTRCA-3' (SEQ ID NO. : 6); R indicates purine substitution.

The PCR solutions were prepared as follows: 4 μl of lOx Buffer (from kit), 4 μl of dNTPs (10 mM), 1 μl of primer (20mM) forward and reverse, 1 μl of template DNA (series of dilutions), 0. 5 μl of DNA polymerase and 28. 5 μl of sterile water (final volume of mix 40 μl). The PCR amplifications of Bacterial and Archaea SSU genes were performed by using DyNAzyme polymerase (Finnzyme) and with Υaq DNA polymerase (QIAGEN) respectively, according to the manufactures instruction. Two protocols were used for amplification of the SSU genes (Skirnisdottir et al , Appl. Environ. Microbiol. 66:2835-2841 (2000)). Bacterial 16S rRNA genes amplification reactions were performed with an initial denaturation step at 95°C for 5 min and 85°C for 1 min, followed by 25 amplification cycles of 95°C for 40 sec, 42°C for 60 sec and 72°C for 3 min, extension was at 72°C for 7 min. Amplifications for Archaeal SSU genes were performed with an initial denaturation step at 94°C for 5 min, then followed by 40 cycles of 94°C for 90 sec, 55°C for 90 sec and 72°C for 2 min and extension at 72°C for 7 min. These protocols were optimized experimentally by modifying number of cycles, annealing temperature, concentration of DNA and concentration of primers to obtain pure PCR product. PCR products were analyzed on a 0. 8% TAE-agarose gel electrophoresis and kept at 4°C until cloning. The amplification reactions were performed on a GeneAmp PCR System 9700 thermal cycler (PE Applied Biosystems). Libraries of fresh PCR products were constructed in E. coli cells by using the Cloning Kit (Invitrogen), according to the manufacturer. PCR products from different primer sets within enrichments were pooled before cloning.

Plasmid DNA's from single colonies were isolated with an automatic plasmid isolation apparatus (AutoGen 740 robot). The DNA was sequenced with an ABI 377 DNA sequencer by using the BigDye Terminator Cycle Sequencing kit (PE Applied Biosystems) according to the manufacturer. The SSU rRNA genes were sequenced with the reverse primer R805, 5'- GACTACCGGGTATCTAATCC-3' (SEQ ID NO. : 3)

Sequences were analyzed with the Sequencing analysis software (ABI), and sequence contigs were built up on maximum likelihood within all sequences by the software. After BLAST searches (http://www. ncbi. nih. nlm. gov/BLAST), the sequences (about 300-400 bases long) were manually aligned with closely related sequences obtained from the Ribosomal Database Project (RDP; http//rrna. uia. ac. be/rma/ssu/forms/index) using ClustalX 1. 8 software (Thompson et al, Nucleic Acids Res. 22: 4673-4680 (1994), and DCSE V3. 4 software (Dedicated Comparative Sequence Editor, De Rijk et al, Department of Biochemistry, University of Antwerp). SeqPupO. 6 (D. C, Gilbert, Biology Dpt, Indiana University, Bloomington) was used as a file translator. Distance trees were constructed by the neighbor joining algorithms with the ARB software (Strunk et al, Lehrstuhl fuer Mikrobiologie, Technical University of Munich).

EXAMPLE 6

PCR-AMPLIFICATION OF UNKNOWN AMYLASE GENE SEQUENCES FROM

ENRICHMENTS

Primers were designed according to the CODEHOP strategy by using the CODEHOP program (Rose, T. M. et al, Nucleic Acids Research, 26: 1628-1635 (1998)). The primers were degenerate at the 3' core region of length 11-12 bp across four codons of highly conserved amino acids. In contrast they were non-degenerate at the 5' region (consensus clamp region) of 18-25 bp with the most probable nucleotide predicted for each position. Reducing the length of the 3' core to a minimum decreases the total number of individual primers in the degenerate primer pool. The 5' non-degenerate consensus clamp stabilizes hybridization of the 3' degenerate core with the target template.

For the primer construction, amino acid sequences of various amylolytic enzymes were retrieved from protein database (Bateman, A. et al, Nucleic Acids Research 27: 260-262 (1999)) and aligned by using CLUSTALX version 1. 8. (Thompson et al. , Nucleic Acids Res. 22: 4673-4680 (1994). Furthermore, blocks of multiply aligned amino acid sequences, established with the program Blockmaker (Henikoff, S., et al, Gene 163: 17-26 (1995) were used as input for the CODEHOP program. Subsequently, a set of forward and reverse primers were constructed, aimed to hybridize to the DNA coding sequences of the conserved A- and B- regions, of amylolytic enzymes, respectively (Takehiko, Y., "Enzyme chemistry and molecular biology of amylases and related enzymes," The amylase research society of Japan, CRC Press, pp. 81-100 (1994)).

Nucleic acids were extracted from harvested cells obtained from oligotrophic enrichments cultures in containers located in a hot spring as previously described (EXAMPLE 2). Each forward primer was tested against each reverse primer in a matrix of PCR-reactions. The PCR amplifications were performed with 0. 5 U of DyNAzyme DNA polymerase (Finnzyme), 1-10 ng of template DNA, a 0. 1 μM concentration of each synthetic primer, a 0. 2 mM concentration of each deoxynucleoside triphosphate and 1. 5 mM MgCl₂ in the buffer recommended by the manufacturer. A total of 30 cycles were performed; each cycle consisted of denaturing at 94°C for 50 s, annealing at 50°C for 50 s, and extension at 72°C for 60 s.

Cloning and sequencing of the PCR products was carried out as previously described for the SSU rRNA genes except that Ml 3 forward and reverse primers were used for the sequencing of the cloned PCR products. All data base searches were run with the program BLASTX on server from the National Center for Biotechnology Information, Bethesda, Maryland, USA (Altschul, S. F. et al, J. Mol. Biol. 215: 403-410. (1990)). The alignment of the derived amino acid sequences and construction of phylogenetic trees was as described for the SSU rRNA genes.

To determine the nature and extent of amylolytic enzymes within enrichment cultures, we designed primers to detect unknown amylase-family gene sequences. The amino acid sequences of 199 amylolytic enzymes were multiply aligned and classified according to the alignment. Two sequence regions (A and B) (Takehiko, Y., The Amylase Research Society of Japan, CRC Press, pp. 81-100 (1994)) separated by ~80-200 amino acids were chosen as primer target sites. Sixteen different forward primers with region A as a target site and seven different reverse primers with region B as a target site were constructed according to the classification. The degeneracy of the primer pools ranged from 16-fold to 64-fold and they were 29-32 bp in length.

Electrophoretic analysis revealed bands of expected sizes (-250 - 600 bp) in amplification reactions with certain primer combinations. The corresponding fragments were cloned and 8-12 clones from each band were sequenced. Of 35 cloned fragments, five different corresponded to amylolytic enzyme gene sequences. The results are summarized in Table 4 and Figure 3. No sequence was observed in both types of enrichment cultures. The "BrusiY" amylase sequences revealed similarity to Thermits sequences in accordance to the rRNA sequence analysis, which detected Thermits bacteria only in BrusiY. Table 1. Results of oligotrophic enrichments done in natural fluid base. Yeast extract (0.005 % final concentration) was added to all cultures after 16 days of incubation.

Inoculum Enrichment Starch

Head pH NaCl Cultiv. Microscopic Ceils/ml dilution code (w/v) (w/v) space (%) time observation (days)

JO^"2 Rl - - - 18 Rods 10⁶- 10⁷

R2 0.1% 1.0% air - 21 Rods ιo⁶ - ιo⁷

R3 0.1% 1.0% N₂ - 22 Long & thin rods N.D.

R4 0.1% 1.0% air 9.5 18 Rods 10⁵ - 10⁶

R5 0.1% 1.0% air - 0.5 18 Rods N.D.

R6 0.1% 1.0% air 4 18 Rods 10^δ

R7 0.002% 1.0% N₂ - 22 Cocci, long & thin rods 10⁶ - 10⁷

R8 0.002% 0.02% air 9.5 18 Small rods 10⁶ - 10⁷

R9 0.002% 0.02% air 4 18 Rods of all sizes 10⁶

RIO 0.002% 0.02% air - 0.5 60 Rods & spores 10⁶ - 10⁷

10-¹ 01 - - air - 21 Rods of all size, 10^s - 10⁶ filaments

G2 0.1% 1.0% air - 18 Very thin & small rods 10 - 10⁷

G3 0.1% 1.0% N₂ - 22 Small & thin rods 10⁵ - 10⁶

G4 0.1% 1.0% air 9.5 18 Thin & small rods >10⁷

G5 0.2% 1.0% air - 0 5 18 Rods 10⁶ - 10⁷

G6 0.1% 1.0% air 4 74 No biomass N.D.

G7 0.002% 1.0% N₂ - 50 Cocci & spores, rods 10⁶ - 10⁷

Table 1 cont.

Inoculu Enrichment Starch (NR,)₂S0₄ Head PH NaCl Cultiv. Microscopic Cells/ml m code (w/v) (w/v) space (%) time observation dilution (days)

Φ8 0.002% 0.02% air 10 18 Very small & thin rods 10⁷ - 10*

Φ9 0.002% 0.02% air 4 21 Very small & thin rods 10⁶

Φ10 0.002% 0.02% air - 0.5 60 Rods & spores 10⁷ - 10⁸

10-⁷ YE.l - air - 0.1 7 Rods of all size 10^δ - I0⁷

YE.01 - air - 0.01 1 1 Rods of all size 10' - 10⁷

S 0.5% - air - 12 Rods 10⁵ - 1 o⁷

Table 2. Identification of cloned 16S rRNA sequences (320 clones from 13 enrichments) from oligiotrophic enrichments based on Ribosomal Database BLAST searches.

Table 2 Continued.

Table 3. Identification of SSU rRNA sequences derived from Bacterial libraries obtained from In situ oligiotrophic enrichments BrusiY and BrusiS placed in the hot spring.

In situ oligiotrophic enrichment In situ oligiotrophic enrichment BrusiY BrusiS

Table 4. Amylases and related enzymes from in situ oligotrophic enrichment cultures.

Clone Amylase origin PCR primers (f/r) Homologous enzyme code signature

Enzyme Bacteria Amino acid sequence identity

2.26 ami BrusiS 15.Equ-FNH-f Cyclomaltodextrinase

26.Equ-GWR-r Alicyclobacillus acidocaldarhis 86%

2.27 am2 BrusiS 5. Bac-VNH-f -amylase

31. Equ-AKH-r Alicyclobacillus acidocaldarius 91%

14.1 am3 BrusiY 15.Equ-FNH-f glycosyl hydrolase

26.Equ-GWR-re Deinococcus radiodurans

59%

14.2 am4 BrusiY 15.Equ-FNH-f glycosyl hydrolase

26.Equ-GWR-r Deinococcus radiodurans

57%

1.7 am5 BrusiY 16.Equ-YNH-f α-glucosidase

25.Equ-GFR-r Thermits aquatic

81% Table 5. Molecular diversity analysis of environmental DNA in geothermal fluid from hydrothermal vent.

Type No. of Bacterial division Closest database match (%) sequence clones

OTU

Bacteria library

ST22 1 Nitrospira group Unidentified (OPB67A 97%)

ST56 15 Aquificales Hydrogenobacter thermophilus TK-6 (90%)

ST10 26 Aquificales EM 17 (97%)

ST43 1 Firmicutes Propionobacterium acnes (96%)

ST12 1 -Proteobacteria Caulobacter crescentus (99%)

ST50 1 β-Proteobacteria Alcaligenes sp. (99%)

Archaea library

ST89 10 Korarchaeota Clone pJP78 (99%)

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

A method for enriching a microbial population from a natural environment, comprising:

(a) obtaining a sample containing microorganisms from an environment in which they naturally occur;

(b) maintaining the sample under conditions substantially similar to the environment from which the sample was obtained to thereby expand the microbial population; and

(c) allowing a sufficient quantity of a microbial population to expand; whereby the population has been enriched.

A method as in Claim 1, wherein the microbial population comprises at least one organism selected from the group consisting of: viruses, prokaryotic microorganisms, lower eukaryotic microorganisms and combinations thereof.

A method as in Claim 1, wherein the natural environment is oligotrophic.

A method as in Claim 1, wherein the natural environment is an extreme environment.

A method as in Claim 4, wherein the natural environment is a terrestrial geothermal environment.

A method as in Claim 4, wherein the natural environment is a marine geothermal environment.

A method as in Claim 1, further comprising enriching the environmental conditions with a chemical additive to select for a microbial population that is influenced by the chemical additive.

A method as in Claim 7, wherein the chemical additives are enzyme substrates for enzymatic induction. A method as in Claim 1, where in the sample of step (a) is concentrated by filtration or centrifugation.

A method as in Claim 1, wherein the sample is liquid.

A method as in Claim 1 , where in the sample is solid.

A method for screening for bioactive small molecules in an enriched microbial population, comprising:

(a) obtaining an enriched microbial population from a natural environment;

(b) maintaining the sample under conditions substantially similar to the environment from which the sample was obtained to thereby expand the microbial population;

(c) allowing a sufficient quantity of a microbial population to expand;

(d) combining the enriched population with a bioactive small molecule detector under suitable conditions for bioactivity;

(e) determining the extent of detection; and

(f) comparing the extent of detection with a control, whereby a difference between the control and sample is indicative of the presence of a bioactive small molecule in the enriched population.

A method as in Claim 12, wherein the bioactive small molecule is a secondary metabolite.

A method for obtaining nucleic acids from an enriched microbial population of Claim 1 , comprising:

(a) extracting nucleic acids from the enriched microbial population; and

(b) isolating the nucleic acids therefrom.

A method as in Claim 14, wherein the extracted nucleic acid from the enriched microbial population is biologically normalized by combining different enriched microbial populations prior to extracting the nucleic acids. A method for producing a normalized representation of genomes from enriched microbial populations from a natural environment, comprising:

(a) obtaining samples from a natural environment containing microbial populations;

(b) maintaining the samples under conditions substantially similar to the natural environment from which the microbial populations were obtained to expand microbial populations contained therein;

(c) monitoring cell density of each microbial population to obtain a sufficient quantity of microbial population for genome normalization; and

(d) combining the samples of the expanded microbial populations such that the combination represents a normalized genome.

A method of screening for microbial modulators from an enriched microbial population, comprising:

(a) obtaining spent fluid from an enriched microbial population;

(b) preparing extracts from the fluid;

(c) combining the extract with a microbial biological entity; and

(d) determining the biological activity of (c) and comparing the biological activity of (c) with the biological activity of biological entity in the absence of the extract; wherein the difference of activity is indicative of the presence of a modulator of the biological entity.

A method as in Claim 17, wherein the microbial biological entity is selected from the group consisting of: isolated biochemical molecule, whole cell and cell culture.

A method of screening for enriched microbial inhibitors, comprising:

(a) obtaining cells from oligotrophic enrichment;

(b) preparing extracts from the cells;

(c) combining the extract with microbial biological entity; and

(d) determining the biological activity of (c) and comparing the biological activity of (c) with the biological activity of biological entity in the absence of the extracts; wherein the reduction of activity is indicative of the presence of an inhibitor of the biological entity.

A method as in Claim 19, wherein the microbial biological entity is selected from the group consisting of: isolated biochemical molecule, whole cell and cell culture.

A method of Claim 1 , wherein step (b) is in the natural environment.

A method of Claim 1 , wherein the microbial population to be enriched is previously uncultured.

A method of Claim 14, wherein the nucleic acids from the enriched population are further assessed for unknown homologous family sequences of a known gene using polymerase chain reaction.

A method of Claim 14, wherein the nucleic acids from the enriched population are used as templates for sequence based screening.

A method of Claim 14, wherein the nucleic acid from the enriched population are used as polymerase chain reaction primers for the detection of a target genes in the same or other DNA sources.

A method of Claim 14, wherein the nucleic acid from the enriched population are used as hybridization probes for the detection of a target genes in the same or other DNA sources

A method of Claim 14, wherein the nucleic acid from the enriched population is isolated, cloned into a suitable vector and introduced into a suitable host cell.

A method of Claim 14, wherein the nucleic acid from the enriched population is isolated, cloned into a suitable vector and expressed in a suitable host cell. A method of Claim 28, wherein the nucleic acid encodes a polypeptide which is subsequently purified from the host cell.

A method of Claim 28, wherein the nucleic acid is genetically modified.