# Patent

## Title
Metabolic primers for the detection of perchlorate-reducing bacteria and methods of use thereof

## Abstract

The present invention is directed to metabolic primers for the detection of perchlorate-reducing bacteria and methods and compositions for use of the same in environmental bioremediation.

## Background
This application is a U.S. Utility Application that claims the benefit of U.S. Provisional Application No. 60/833,825, which was filed Jul. 27, 2006. The entire disclosure of the foregoing application is incorporated herein by reference.


Certain aspects of the studies described herein were Federally-funded with the support of grant # DACA72-00-C-0016 from the Department of Defense.


BACKGROUND
1. Field of the Invention
This invention relates to bioremediation of contaminants in the environmental samples, including for example, contamination in particulates such as soil and also in fluids such as groundwater. More particularly, the present invention is directed to methods and compositions for the detection of perchlorate-reducing bacteria at target decontamination site.
2. Background of the Related Art
Chemical contamination of the environment, particularly of soil and groundwater, is a widespread problem throughout the industrialized world. Industrial pollution has contaminated millions of acres of soil and associated aquifers. Often, cleanup of the contamination is hindered because the cost of remediation is significant. Moreover, many of the remediation techniques create additional problems which cause the land to remain unused or abandoned.
Recently, widespread perchlorate contamination of drinking water wells throughout the United States and especially throughout the southwest has become a significant cause for concern. Perchlorate contamination of ground and surface waters originates from, and is a direct effect of, unregulated ammonium perchlorate disposal practices from 1950 to 1997 (Renner et al., Environ. Sci. Technol. News 32:210A, 1998). Ammonium perchlorate is an oxidant that is widely used in the aerospace, munitions, and fireworks industries. Widespread contamination has been documented in the waterways of California, and at least 19 other states in the United States. Similar contaminations have been reported in other countries that have aerospace, munitions and fireworks industries.
Perchlorate has been linked to a number of problems in human health. Excessive intake of perchlorate blocks iodine uptake and inhibits thyroid function and production of thyroid hormones, in addition, gastrointestinal irritation and skin rash, and hematological effects including agranulocytosis and lymphadenopathy have also been observed. In addition, it has been established that there is neurodevelopmental toxicity associated with perchlorate ingestion. As a result of these significant health concerns, drinking water utilities have begun monitoring and reporting perchlorate levels to the State agencies. In some states the Health Services Departments have set maximum limits on the amount of perchlorate in drinking water; this figure is typically in the order of 18 parts per billion (ppb) in order to minimize the risks to human health. The Environmental Protection Agency has established a provisional reference dose (“RFD”) of 14 mg of perchlorate per kg of body weight per day. Practical and efficient methods to treat water contaminated by perchlorate are needed to insure a safe drinking water supply in many communities.
Current methods of perchlorate remediation rely on the use of ion exchange resins to sequester perchlorate ions. (see e.g., U.S. Pat. No. 6,059,975). Conventional perchlorate-removal ion exchange resins have low selectivity coefficients and as such these resins are capable of loading only a few kilograms of perchlorate per cubic meter of removal resin. This produces a waste mass of loaded resin that must be disposed through, e.g., incineration. Disposal costs for these resins are therefore prohibitive because of the bulk volume of loaded to be disposed relative to the amount of perchlorate removed from the environmental target site. Other methods of perchlorate removal are actively being pursued, with bioremediation technologies emerging as a cost-effective and less-invasive alternative to physical or chemical practices (Urbansky et al., Biorem. J.: 81-95, 1998).
Natural attenuation of perchlorate is a cost-effective alternative to current methods of perchlorate remediation. Such natural attenuation systems have been used in bioremediation of other contaminants and include the use of microbial populations to accelerate the breakdown of solids and the various contaminants associated with waste water. Such microbes are permitted to act upon the waste water or contaminated soils and they act to remove the pollutants faster than if nothing were used, and do so without the hazards and difficulties associated with chemical treatment.
The success of natural perchlorate remediation is dependent on the presence and activity of dissimilatory (per)chlorate-reducing bacteria (DPRB) within the target site that is undergoing remediation. Within the last 7 years, more than 40 different strains of dissimilatory (per)chlorate-reducing bacteria (DPRB) have been isolated from a diverse range of environments (Bruce et al., Environ. Microbiol. 1:319-329, 1999; Coates et al., Appl. Environ. Microbiol. 65:5234-5241., 1999; Kim et al., Water Res. 35:3071-3076., 2001; Logan et al., Appl. Environ. Microbiol. 67:2499-2506, 2001, 18, Rikken et al., Appl. Microbiol. Biotechnol., 45:420-426, 1996; Wallace et al., J. Ind. Microbiol. 16:68-72, 1996). Because of the metabolic capability and ubiquity of DPRB (Coates et al., Appl. Environ. Microbiol. 65:5234-5241., 1999), natural attenuation of perchlorate is garnering more and more interest. While studies by various groups have shown the ability of microbes to remediate perchlorate under environmental conditions (Hunter, Curr. Microbiol. 45:287-292., 2002; Kim et al., Water Res. 35:3071-3076., 2001; Tipton et al., J. Environ. Qual. 32:40-46, 2003), a quick, reliable method for detecting the presence and effectiveness of DPRB is needed to determine the natural attenuation candidacy of a contaminated site as well as for monitoring active degradation.
Traditionally, contaminant site evaluation for the presence of DPRB has been performed using labor-intensive enumeration and isolation techniques. However, it is well known that cultivation techniques are time-consuming and often prove unsuccessful in isolating the target bacteria due to both media selectivity and organism culturability (Dunbar et al., Appl. Environ. Microbiol. 63:1326-1331, 1997; Kaeberlein et al., Science, 296:1127-1129, 2002). To alleviate the limitations of cultivation-based methods, molecular techniques using the 16S rRNA gene have been employed to examine bacterial diversity in the environment (Aman et al., Microbiol. Rev. 59:143-169, 1995, Olsen et al., Annu. Rev. Microbiol. 40:337-365, 1986), and numerous primer sets have been developed for the 16S rRNA gene that target specific groups of bacteria. However, due to the fact that there is significant phylogenetic diversity of DPRB and because of their close phylogenetic relationships to non-(per)chlorate-reducing relatives, detection of DPRB using 16S ribosomal DNA (rDNA) primers is not recommended (Achenbach et al., Int. J. Syst. Evol. Microbiol. 51:527-533, 2001). As such, there remains a need to identify a more inclusive approach to the detection of DPRB that would allow an efficient molecular identification technique that will facilitate the rapid identification of the presence of DPRB at a given target site and/or allow prediction of whether a given target site is capable of undergoing perchlorate remediation.
SUMMARY OF THE INVENTION
The present invention is directed to methods and compositions for use in bioremediation-related applications. Specific detection of chlorate reducing bacteria is facilitated by the present invention because it involves determining the presence of perchlorate reductase in a given sample. Perchlorate reductase is only present in perchlorate reducing bacteria as opposed to chlorite dismutase, which is present in both chlorate reducing bacteria and perchlorate reducing bacteria.
A specific aspect of the present invention, which in one aspect provides a composition comprising a first primer and a second primer, wherein the first primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:1 or SEQ ID NO:11 and the second primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:2 or SEQ ID NO:12, wherein the first and second primers are capable of hybridizing to a perchlorate reductase gene, more particularly, capable of hybridizing to pcrA gene. In preferred embodiments, the pcrA gene has the sequence of SEQ ID NO: 27 (from Genbank Accession No. AY180108).
In additional embodiments, the composition may further comprise a third primer and a fourth primer, wherein the third primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:3 and the fourth primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:4, wherein the third and fourth primers are capable of hybridizing to a perchlorate reductase gene.
In an alternative embodiment, the composition is one in which there is third primer and a fourth primer, wherein the third primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:5 or SEQ ID NO:13 and the fourth primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:6 or SEQ ID NO:14, wherein the third and fourth primers are capable of hybridizing to a perchlorate reductase gene.
In yet another alternative embodiment, the composition is one in which there is a third primer and a fourth primer wherein the third primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:7 and the fourth primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:8 wherein the third and fourth primers are capable of hybridizing to a perchlorate reductase gene.
In other specific embodiments, there is a composition that comprises a first primer and a second primer, wherein the first primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:3 and the second primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:4, wherein the first and second primers are capable of hybridizing to a perchlorate reductase gene. In other embodiments, such a composition may further comprise a third primer and a fourth primer, wherein the third primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:5 or SEQ ID NO:13 and the fourth primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:6 or SEQ ID NO:14, wherein the third and fourth primers are capable of hybridizing to a perchlorate reductase gene. In still other embodiments, the composition is one in which the third primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:7 and the fourth primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:8 wherein the third and fourth primers are capable of hybridizing to a perchlorate reductase gene.
Still other compositions of the invention comprise a first primer and a second primer, wherein the first primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:5 or SEQ ID NO:13 and the second primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:6 or SEQ ID NO:14, wherein the first and second primers are capable of hybridizing to a perchlorate reductase gene. In exemplary embodiments, such compositions may further comprise a third primer and a fourth primer, wherein the third primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:7 and the fourth primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:8 wherein the third and fourth primers are capable of hybridizing to a perchlorate reductase gene.
Additional aspects contemplate compositions that further comprise a fifth primer and a sixth primer, such that in addition to SEQ ID NO:1 or 11, SEQ ID NO:2 or 12, SEQ ID NO:3, SEQ ID NO:4, the composition further comprises a fifth primer and a sixth primer, wherein the fifth primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:5 or SEQ ID NO:13 and the sixth primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:6 or SEQ ID NO:14, wherein the fifth and sixth primers are capable of hybridizing to a perchlorate reductase gene. In still other alternatives, the composition is one in which the fifth primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:7 and the sixth primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:8, wherein the fifth and sixth primers are capable of hybridizing to a perchlorate reductase gene.
Other preferred compositions are those in which further comprise a seventh primer and an eighth primer, wherein the seventh primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:7 and the eighth primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:8, wherein the seventh and eighth primers are capable of hybridizing to a perchlorate reductase gene.
Preferably, each of the primers each independently comprise between 20 and 30 nucleotide bases in length. Other preferred embodiments contemplate that the primers each independently comprise between 20 and 40 nucleotide bases in length. Still additional embodiments contemplate that each of the primers each independently comprise between 20 and 50 nucleotide bases in length.
In specific aspects of the invention, it is contemplated that the perchlorate reductase gene is from dissimilatory perchlorate-reducing bacteria (DPRB) species. DPRB species are well known to those of skill in the art, and simply by way of example, include, but are not limited to a bacterium from the Dechloromonas spp., Azoarcus spp., Dechlorospirillum spp., Dechloromarinus spp., Ideonella spp., Magnetospirillum spp., Pseudomonas spp., Rhodocyclus spp., Rhodospirillum spp., Azospirillum spp., Wolinella spp., Xanthomonas spp. In specific embodiments, the DPRB is selected from the group consisting of Dechloromonas agitate, Dechloromonas aromatica, Azospira suillum, Dechlorospirillum anomalous, Dechloromarinus chlorophilus, Ideonella dechloratans, and Magnetospirillum magnetotacticum. 
In specific embodiments, the primer is detectably labeled.
Also contemplated herein is an oligonucleotide primer pair wherein the first primer of the primer pair comprises a sequence of SEQ ID NO:1 or SEQ ID NO:11 and the second primer of the primer pair comprises a sequence of SEQ ID NO:2 or SEQ ID NO:12. Other preferred oligonucleotide primer pairs are those in which the first primer of the primer pair comprises a sequence of SEQ ID NO:3 and the second primer of the primer pair comprises a sequence of SEQ ID NO:4. Yet further preferred primer pairs include those in which the first primer of the primer pair comprises a sequence of SEQ ID NO:5 or SEQ ID NO:13 and the second primer of the primer pair comprises a sequence of SEQ ID NO:6 or SEQ ID NO:14. In still another exemplary embodiment, the first primer of the primer pair comprises a sequence of SEQ ID NO:7 and the second primer of the primer pair comprises a sequence of SEQ ID NO:8. Preferably, in such compositions, at least one of the primers in a primer pair of the invention is detectably labeled.
Other aspects specifically contemplated an oligonucleotide primer which has the nucleotide sequence defined in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 11, 12, 13, or 14. Such an oligonucleotide may be between 20 and 50 nucleotide bases. Preferably, the oligonucleotide primer is detectably labeled. The label may be any label that is conventionally used to facilitate detection of the primer or the product of the reaction in which the primer is used. For example, the primer is labeled with an epitope, fluorophore, metal particle, enzyme, carbohydrate, polypeptide, radioactive isotope, dye, biotin, or digitonin.
The oligonucleotide primer pairs discussed above may be such that at least on the oligonucleotides in the pair is labeled with an epitope, fluorophore, metal particle, enzyme, carbohydrate, polypeptide, radioactive isotope, dye, biotin, or digitonin.
The present invention particularly contemplates methods of detecting the presence of chlorate reducing bacteria in a sample comprising subjecting DNA of bacterial cells in the sample to a first polymerase chain reaction amplification using a pair of primers of an oligonucleotide primer pair wherein the first primer of the primer pair comprises a sequence of SEQ ID NO:1 or SEQ ID NO:11 and the second primer of the primer pair comprises a sequence of SEQ ID NO:2 or SEQ ID NO:12; or where the first primer of the primer pair comprises a sequence of SEQ ID NO:3 and the second primer of the primer pair comprises a sequence of SEQ ID NO:4; or where the first primer of the primer pair comprises a sequence of SEQ ID NO:5 or SEQ ID NO:13 and the second primer of the primer pair comprises a sequence of SEQ ID NO:6 or SEQ ID NO:14 or where the first primer of the primer pair comprises a sequence of SEQ ID NO:7 and the second primer of the primer pair comprises a sequence of SEQ ID NO:8; and detecting the product or products of the first polymerase chain reaction amplification, thereby identifying the presence of the perchlorate-reducing bacteria in the sample.
In additional embodiments, the method may advantageously further comprise subjecting the DNA to a second polymerase chain reaction amplification using a pair of primers of oligonucleotide primer pair (e.g., wherein the first primer of the primer pair comprises a sequence of SEQ ID NO:1 or SEQ ID NO:11 and the second primer of the primer pair comprises a sequence of SEQ ID NO:2 or SEQ ID NO:12; or where the first primer of the primer pair comprises a sequence of SEQ ID NO:3 and the second primer of the primer pair comprises a sequence of SEQ ID NO:4; or where the first primer of the primer pair comprises a sequence of SEQ ID NO:5 or SEQ ID NO:13 and the second primer of the primer pair comprises a sequence of SEQ ID NO:6 or SEQ ID NO:14 or where the first primer of the primer pair comprises a sequence of SEQ ID NO:7 and the second primer of the primer pair comprises a sequence of SEQ ID NO:8), as long as the primer pair in the second polymerase chain reaction amplification is one that is a different primer set than the primer set that is used in first polymerase chain reaction amplification; and detecting the product or products of the second polymerase chain reaction amplification thereby identifying the presence of the perchlorate-reducing bacteria in the sample.
In specific embodiments, the methods of the invention can be used to distinguish whether there are perchlorate reducing bacteria in a sample. As noted above, perchlorate reductase is present only in perchlorate reducing bacteria and is not present in chlorate reducing bacteria. Chlorite dismutase, the only other enzyme in the perchlorate reduction pathway to have been characterized, is useful for identifying both perchlorate reducing bacteria and chlorate reducing bacteria. Hence, using the methods of the invention it is possible to specifically identify perchlorate reducing bacteria in a sample that contains both chlorate and perchlorate reducing bacteria. Exemplary such methods may utilize the chlorite dismutase detection methods described in e.g., PCT application PCT/US2005/004657 (inventors Achenbach et al., filed Feb. 11, 2005) to identify bacteria that are capable of reducing both perchlorate and chlorate, and then subsequently use the perchlorate reductase-based detection methods discussed herein to further identify only those bacteria that are perchlorate reducers.
Other methods of the invention involve detecting the presence of perchlorate reducing bacteria in a sample comprising subjecting DNA from the sample to a first polymerase chain reaction amplification using a pair of primers of (e.g., oligonucleotide primer pair wherein the first primer of the primer pair comprises a sequence of SEQ ID NO:1 or SEQ ID NO:11 and the second primer of the primer pair comprises a sequence of SEQ ID NO:2 or SEQ ID NO:12; or where the first primer of the primer pair comprises a sequence of SEQ ID NO:3 and the second primer of the primer pair comprises a sequence of SEQ ID NO:4; or where the first primer of the primer pair comprises a sequence of SEQ ID NO:5 or SEQ ID NO:13 and the second primer of the primer pair comprises a sequence of SEQ ID NO:6 or SEQ ID NO:14 or where the first primer of the primer pair comprises a sequence of SEQ ID NO:7 and the second primer of the primer pair comprises a sequence of SEQ ID NO:8); and detecting the product or products of the first polymerase chain reaction amplification, thereby identifying the presence of the perchlorate-reducing bacteria in the sample.
Also contemplated is a method of detecting the presence of perchlorate-reducing bacteria in a sample comprising: (a) subjecting DNA from the sample to a first polymerase chain reaction amplification using a pair of primers (e.g., oligonucleotide primer pair wherein the first primer of the primer pair comprises a sequence of SEQ ID NO:1 or SEQ ID NO:11 and the second primer of the primer pair comprises a sequence of SEQ ID NO:2 or SEQ ID NO:12; or where the first primer of the primer pair comprises a sequence of SEQ ID NO:3 and the second primer of the primer pair comprises a sequence of SEQ ID NO:4; or where the first primer of the primer pair comprises a sequence of SEQ ID NO:5 or SEQ ID NO:13 and the second primer of the primer pair comprises a sequence of SEQ ID NO:6 or SEQ ID NO:14 or where the first primer of the primer pair comprises a sequence of SEQ ID NO:7 and the second primer of the primer pair comprises a sequence of SEQ ID NO:8); (b) isolating the amplification products from step (a); (c) using the amplification products isolated in step (b) as a template for a second polymerase chain reaction amplification using a second primer pair that is different from the primer pair used in the first polymerase chain reaction; and (d) detecting the product or products of the second polymerase chain reaction amplification, thereby identifying the presence of the perchlorate-reducing bacteria in the sample.
Another method of the invention comprises three amplification steps for detecting the presence of perchlorate-reducing bacteria in a sample, wherein the method comprises subjecting DNA from the sample to a first polymerase chain reaction amplification using a pair of primers (e.g., oligonucleotide primer pair wherein the first primer of the primer pair comprises a sequence of SEQ ID NO:1 or SEQ ID NO:11 and the second primer of the primer pair comprises a sequence of SEQ ID NO:2 or SEQ ID NO:12; or where the first primer of the primer pair comprises a sequence of SEQ ID NO:3 and the second primer of the primer pair comprises a sequence of SEQ ID NO:4; or where the first primer of the primer pair comprises a sequence of SEQ ID NO:5 or SEQ ID NO:13 and the second primer of the primer pair comprises a sequence of SEQ ID NO:6 or SEQ ID NO:14 or where the first primer of the primer pair comprises a sequence of SEQ ID NO:7 and the second primer of the primer pair comprises a sequence of SEQ ID NO:8); (b) isolating the amplification products from step (a); (c) using the amplification products isolated in step (b) as a template for a second polymerase chain reaction amplification using a second primer pair that is different from the primer pair used in the first polymerase chain reaction; (d) isolating the amplification products from step (c); (e) using the amplification products isolated in step (d) as a template for a third polymerase chain reaction amplification using a third primer pair that is different from the primer pair used in the first or the second polymerase chain reaction; and (f) detecting the product or products of the third polymerase chain reaction amplification, thereby identifying the presence of the perchlorate-reducing bacteria in the sample.
Still a further method is one which employs four amplification reaction steps for detecting the presence of perchlorate-reducing bacteria in a sample comprising (a) subjecting DNA from the sample to a first polymerase chain reaction amplification using a pair of primers (oligonucleotide primer pair wherein the first primer of the primer pair comprises a sequence of SEQ ID NO:1 or SEQ ID NO:11 and the second primer of the primer pair comprises a sequence of SEQ ID NO:2 or SEQ ID NO:12; or where the first primer of the primer pair comprises a sequence of SEQ ID NO:3 and the second primer of the primer pair comprises a sequence of SEQ ID NO:4; or where the first primer of the primer pair comprises a sequence of SEQ ID NO:5 or SEQ ID NO:13 and the second primer of the primer pair comprises a sequence of SEQ ID NO:6 or SEQ ID NO:14 or where the first primer of the primer pair comprises a sequence of SEQ ID NO:7 and the second primer of the primer pair comprises a sequence of SEQ ID NO:8); (b) isolating the amplification products from step (a); (c) using the amplification products isolated in step (b) as a template for a second polymerase chain reaction amplification using a second primer pair that is different from the primer pair used in the first polymerase chain reaction; (d) isolating the amplification products from step (c); (e) using the amplification products isolated in step (d) as a template for a third polymerase chain reaction amplification using a third primer pair that is different from the primer pair used in the first or the second polymerase chain reaction; (f) isolating the amplification products from step (e); (g) using the amplification products isolated in step (d) as a template for a fourth polymerase chain reaction amplification using a fourth primer pair from claim 20, claim 21, claim 22 or claim 23 that is different from the primer pair used in the first, second or the third polymerase chain reaction; and (h) detecting the product or products of the third polymerase chain reaction amplification, thereby identifying the presence of the perchlorate-reducing bacteria in the sample.
In such methods, the DNA is preferably isolated from a bacterial lysate from the bioremediation formulation prior to the initial first polymerase chain reaction amplification step. In specific embodiments, the bioremediation formulation is a cocktail of microorganisms that are used to remove contaminants from a sample of soil or water in need of decontamination, wherein the cocktail of microorganisms comprises a mixture of DPRBs. In preferred embodiments, the cocktail of microorganisms further microorganisms that are denitrifiers. Preferably, the cocktail of microorganisms further comprises microorganisms that can degrade toluene, xylene, benzene, petroleum, and creosote.
The soil or water samples have been contaminated with perchlorate as a result of for example, waste disposal from paper mill waste, airbag production, firework manufacture and use, fertilizer manufacture and use.
Also provided are methods of determining whether a bioremediation formulation that comprises bacteria will be effective at decreasing perchlorate contamination, wherein the method comprises subjecting DNA from the bioremediation formulation to a first polymerase chain reaction amplification using a pair of primers (oligonucleotide primer pair wherein the first primer of the primer pair comprises a sequence of SEQ ID NO:1 or SEQ ID NO:11 and the second primer of the primer pair comprises a sequence of SEQ ID NO:2 or SEQ ID NO:12; or where the first primer of the primer pair comprises a sequence of SEQ ID NO:3 and the second primer of the primer pair comprises a sequence of SEQ ID NO:4; or where the first primer of the primer pair comprises a sequence of SEQ ID NO:5 or SEQ ID NO:13 and the second primer of the primer pair comprises a sequence of SEQ ID NO:6 or SEQ ID NO:14 or where the first primer of the primer pair comprises a sequence of SEQ ID NO:7 and the second primer of the primer pair comprises a sequence of SEQ ID NO:8); and detecting the product or products of the first polymerase chain reaction amplification, wherein the presence of the amplification products indicates the presence of perchlorate reducing bacteria in bioremediation formulation thereby indicating that the formulation is effective at reducing perchlorate contamination. The method may further comprise subjecting the DNA to a second, third or fourth polymerase chain reaction amplification using a second, third or fourth pair of primers that is different from the first (and corresponding second, and third where there are three or four amplifications) pair of primers; and detecting the presence of perchlorate-reducing bacteria in the bioremediation formulation by visualizing the product or products of the second polymerase chain reaction amplification, wherein the presence of the amplification products indicates that the formulation is effective at reducing perchlorate contamination.
Also taught is a method of determining whether a bioremediation formulation that comprises bacteria will be effective at decreasing perchlorate contamination comprising
(a) subjecting the DNA from the bioremediation formulation to a first polymerase chain reaction amplification using a pair of primers (e.g., an oligonucleotide primer pair wherein the first primer of the primer pair comprises a sequence of SEQ ID NO:1 or SEQ ID NO:11 and the second primer of the primer pair comprises a sequence of SEQ ID NO:2 or SEQ ID NO:12; or where the first primer of the primer pair comprises a sequence of SEQ ID NO:3 and the second primer of the primer pair comprises a sequence of SEQ ID NO:4; or where the first primer of the primer pair comprises a sequence of SEQ ID NO:5 or SEQ ID NO:13 and the second primer of the primer pair comprises a sequence of SEQ ID NO:6 or SEQ ID NO:14 or where the first primer of the primer pair comprises a sequence of SEQ ID NO:7 and the second primer of the primer pair comprises a sequence of SEQ ID NO:8); (b) isolating the amplification products from step (a); (c) using the amplification products isolated in step (b) as a template for a second polymerase chain reaction amplification using a primer pair that is different from the pair of primers used in the first polymerase chain reaction amplification; and (d) detecting the perchlorate-reducing bacteria by visualizing the product or products of the second polymerase chain reaction amplification.
Still another method is one for determining whether a bioremediation formulation that comprises bacteria will be effective at decreasing perchlorate contamination comprising
(a) subjecting the DNA from the bioremediation formulation to a first polymerase chain reaction amplification using a pair of primers (e.g., oligonucleotide primer pair wherein the first primer of the primer pair comprises a sequence of SEQ ID NO:1 or SEQ ID NO:11 and the second primer of the primer pair comprises a sequence of SEQ ID NO:2 or SEQ ID NO:12; or where the first primer of the primer pair comprises a sequence of SEQ ID NO:3 and the second primer of the primer pair comprises a sequence of SEQ ID NO:4; or where the first primer of the primer pair comprises a sequence of SEQ ID NO:5 or SEQ ID NO:13 and the second primer of the primer pair comprises a sequence of SEQ ID NO:6 or SEQ ID NO:14 or where the first primer of the primer pair comprises a sequence of SEQ ID NO:7 and the second primer of the primer pair comprises a sequence of SEQ ID NO:8); (b) isolating the amplification products from step (a); (c) using the amplification products isolated in step (b) as a template for a second polymerase chain reaction amplification using a primer pair that is different from the pair of primers used in the first polymerase chain reaction amplification; (d) isolating the amplification products from step (c); (e) using the amplification products isolated in step (d) as a template for a third polymerase chain reaction amplification using a primer pair that is different from the pair of primers used in the first and second polymerase chain reaction amplification; and (f) detecting the perchlorate-reducing bacteria by visualizing the product or products of the second polymerase chain reaction amplification.
Still another method for determining whether a bioremediation formulation that comprises bacteria will be effective at decreasing perchlorate contamination comprises (a) subjecting the DNA from the bioremediation formulation to a first polymerase chain reaction amplification using a pair of primers (e.g., oligonucleotide primer pair wherein the first primer of the primer pair comprises a sequence of SEQ ID NO:1 or SEQ ID NO:11 and the second primer of the primer pair comprises a sequence of SEQ ID NO:2 or SEQ ID NO:12; or where the first primer of the primer pair comprises a sequence of SEQ ID NO:3 and the second primer of the primer pair comprises a sequence of SEQ ID NO:4; or where the first primer of the primer pair comprises a sequence of SEQ ID NO:5 or SEQ ID NO:13 and the second primer of the primer pair comprises a sequence of SEQ ID NO:6 or SEQ ID NO:14 or where the first primer of the primer pair comprises a sequence of SEQ ID NO:7 and the second primer of the primer pair comprises a sequence of SEQ ID NO:8); (b) isolating the amplification products from step (a); (c) using the amplification products isolated in step (b) as a template for a second polymerase chain reaction amplification using a primer pair that is different from the pair of primers used in the first polymerase chain reaction amplification; (d) isolating the amplification products from step (c); (e) using the amplification products isolated in step (d) as a template for a third polymerase chain reaction amplification using a primer pair that is different from the pair of primers used in the first and second polymerase chain reaction amplification; (f) isolating the amplification products from step (e); (g) using the amplification products isolated in step (f) as a template for a fourth polymerase chain reaction amplification using a primer pair that is different from the pair of primers used in the first second and third polymerase chain reaction amplification; and (h) detecting the perchlorate-reducing bacteria by visualizing the product or products of the second polymerase chain reaction amplification.
These methods may be supplemented by further determining the presence of chlorite dismutase in the sample prior to determining the presence of perchlorate reductase in the sample. Such further methods advantageously may use polymerase chain reactions using primers selected from the group consisting of SEQ ID NO:15 through SEQ ID NO:24.
Also contemplated by the invention is a method of determining whether a sample contains bacteria that is reducing perchlorate in the sample comprising: isolating nucleic acid from the sample; incubating the nucleic acid with a DNase to isolate RNA; performing a reverse transcription reaction on the RNA using one or more of the primers selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 11, 12, 13 and 14; and detecting the product or products of the reverse transcription reaction that are expressing perchlorate reductase, thereby identifying the presence of bacteria in the sample that are expressing perchlorate reductase for reducing the perchlorate content of the sample.
Another aspect contemplated is a method of determining whether a sample contains bacteria that is reducing perchlorate in the sample comprising: (a) isolating nucleic acid from the sample; (b) incubating the nucleic acid with a DNase to isolate RNA; (c) performing a reverse transcriptase reaction on the RNA using one or more of the primers selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 11, 12, 13 and 14; (d) isolating the reaction products from step (c); (e) using the reaction products isolated in step (d) as a template for a polymerase chain reaction amplification using a primer pair (e.g., oligonucleotide primer pair wherein the first primer of the primer pair comprises a sequence of SEQ ID NO:1 or SEQ ID NO:11 and the second primer of the primer pair comprises a sequence of SEQ ID NO:2 or SEQ ID NO:12; or where the first primer of the primer pair comprises a sequence of SEQ ID NO:3 and the second primer of the primer pair comprises a sequence of SEQ ID NO:4; or where the first primer of the primer pair comprises a sequence of SEQ ID NO:5 or SEQ ID NO:13 and the second primer of the primer pair comprises a sequence of SEQ ID NO:6 or SEQ ID NO:14 or where the first primer of the primer pair comprises a sequence of SEQ ID NO:7 and the second primer of the primer pair comprises a sequence of SEQ ID NO:8); and (f) detecting the product or products of the polymerase chain reaction amplification of step (e), thereby identifying the presence of bacteria in the sample that are expressing perchlorate reductase for reducing the perchlorate content of the sample.
In some embodiments, the method may further comprise determining the presence of chlorite dismutase in the sample prior to determining the presence of perchlorate reductase in the sample, for example, determining the presence of chlorite dismutase in the sample comprises performing polymerase chain reactions using primers selected from the group consisting of SEQ ID NO:15 through SEQ ID NO:24.
Further methods contemplate performing additional rounds of amplifications using primer pairs that are distinct from the first round of amplification.
The invention also is directed to kits for amplifying perchlorate reductase polynucleotide, the kit comprising: composition or an oligonucleotide pair or an oligonucleotide as described herein above, wherein the composition; and instructions for carrying out any one or more of the methods discussed above. Preferably, each of the primers are in separate containers. In specific embodiments, the kits further comprise an oligonucleotide primer or primer pair for amplifying chlorite dismutase, e.g., an oligonucleotide primer selected from the group consisting of SEQ ID NO:15 through SEQ ID NO:24. Preferably, the kits may further comprise enzymes and nucleotide components of a PCR reaction. The kits of the invention also may comprise one or more solid supports. In certain embodiments, the primers described herein are preferably arranged as arrays on a solid support. Preferably, the arrays are addressable and/or detectable such that the skilled individual may readily detect the primer from a signal or from a specific “address” or location on the solid support. Other embodiments contemplated are those in which the primers are provided in separate containers.
Also contemplated herein is a library of primers for the detection of a perchlorate reductase gene from DPRB, the library comprising at least 6 primers derived from the sequences set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 11, 12 13, or 14. Another library of primers is one for the detection of DPRB, the library comprising at least 6 primers derived from the sequences set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 11, 12, 13, or 14 for detecting perchlorate reductase and the sequences set forth in SEQ ID NO:15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 for detecting chlorite dismutase.
Other features and advantages of the invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, because various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.



BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further illustrate aspects of the present invention. The invention may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
FIG. 1. Diagram of the pcrABCD genes of D. agitata and D. aromatica. The N-terminal sequences of PcrA are indicated, and the twin-arginine motif is in boldface type.
FIG. 2. Amino acid alignment of the □-subunit of perchlorate reductases (PcrB) from strain GR-1 (St.GR1) (N-terminal sequence only), D. agitata (D.agit), and D. aromatica (D.arom), selenate reductase B (SerB) from T. selenatis (T.sele), chlorate reductase (ClrB) from I. dechloratans (I.dech), ethylbenzene dehydrogenase B (EbdB) from Azoarcus sp. strain EbN1 (A.EbN1), dimethyl sulfide dehydrogenase B (DdhB) from R. sulfidophilum (R.sulf), and nitrate reductase H (NarH) from H. marismortui (H.mars). Light shading indicates amino acids identical to amino acids in both perchlorate reductases. Dark shading indicates conserved cysteine clusters for Fe—S center binding. The numbers below the cysteine residues indicate the associated Fe—S centers.
FIG. 3. Amino acid alignment of the □-subunit of perchlorate reductase (PcrC) from D. aromatica (D.arom) and cytochrome c554 from N. europaea (N.euro). Shading indicates identical residues, while boldface and underlining indicate residues shown to bind heme in cytochrome c554
FIG. 4. Predicted model for electron transfer during (per)chlorate reduction. Electrons from a quinone pool are transferred from the membrane via a NirT-type cytochrome to the PcrABC reductase. While PcrD is absent from the functional enzyme, this protein is predicted to be involved in enzyme assembly.
FIG. 5. Anaerobic growth of wild-type D. aromatica and pcrA mutant with nitrate (a), perchlorate (b), or chlorate (c) as the sole electron acceptor. F, wild-type growth; E, pcrA mutant growth. The data are averages for duplicate incubations.
FIG. 6. Unrooted neighbor-joining tree indicating the evolutionary distances in the DMSO reductase family of molybdoenzymes. GenBank accession numbers are indicated after the names.
FIG. 7. Slot blot hybridization of genomic DNAs from DPRB and non-perchlorate-reducing close relatives of DPRB using the D. agitata, pcrA probe. A total of 250 ng of genomic DNA was loaded for each strain. Row A (left to right): 1, D. agitata; 2, R. tenuis; 3, D. aromatica; 4, Dechloromonas sp. strain JJ. Row B: 1, D. anomolous strain WD; 2, M. magnetotacticum; 3, Pseudomonas sp. strain PK; 4, P. stutzeri. Row C: 1, D. chlorophilus strain NSS; 2, A. suillum; 3, Dechloromonas sp. strain LT-1; 4, I. dechloratans. D. agitata, D. aromatica, D. anomalous, A. suillum, and Dechloromonas sp. strain LT-1 are capable of perchlorate reduction; Pseudomonas sp. strain PK, D. chlorophilus strain NSS, and I. dechloratans are capable of only chlorate reduction.



DESCRIPTION OF THE PREFERRED EMBODIMENTS
There are significant environmental problems associated with the presence of perchlorate in the ground water and soil compositions of areas where there has previously been significant activities from the aerospace, munitions, and fireworks industries. These industries have produced significant quantities of ammonium perchlorate and the unchecked disposal of this compound over the last 5 decades has resulted in the contamination the ground and surface waters throughout the United States and other countries (Renner et al., Environ. Sci. Technol. News 32:210A, 1998). Remediation of soils and water to remove the perchlorate contaminants has typically involved resin-based ion exchange chromatography, which is an inefficient method because of the bulk of resin required and the fact that the perchlorate-loaded resin ultimately must be disposed.
Biological methods for remediation or clean-up of the environment are compelling in that they employ natural organisms. Microorganisms have been used to clean-up oil spills, sewage effluence, chlorinated solvents, pesticides and the like. The ability of microorganisms to remove the contaminants from a contaminated site is nature's way of cleaning-up the environment. In the present application, there are provided methods and compositions for determining the presence and efficacy of one such set of microorganisms.
DPRB are excellent microbial bioremediators in that they are able to live in a diverse range of environments and are able to metabolize (per)chlorate. As such, natural attenuation of (per)chlorate contaminants is of significant economic and environmental interest. The present invention specifically is directed to identification of the presence of these bacteria and compositions that may be used in the field to determine whether a given target site will undergo bioremediation naturally or whether additional intervention is required. As noted above and described in PCT application PCT/US2005/004657 (inventors Achenbach et al., filed Feb. 11, 2005, which is incorporated herein by reference in its entirety), chlorite dismutase is an enzyme useful for the identification of both chlorate and perchlorate reducing bacteria. Perchlorate reductase, and the compositions and methods described herein is useful for further identifying the bacteria that can be used in perchlorate bioremediative processes in that the methods and compositions of the present invention are suitable for specifically identifying perchlorate reducing bacteria to such a specificity level that they can be distinguished from chlorate reducing bacteria.
Metabolic primer sets have been applied to a variety of bioremediative studies for the detection of specific bacteria. For example, since many denitrifiers are able to degrade toluene and xylene, Braker and colleagues developed primer sets targeting two nitrate reductase genes that allowed for the qualitative detection of denitrifiers in the environment (Braker et al., Appl. Environ. Microbiol., 64:3769-3775, 1998). And while primers for the catechol 2,3-dioxygenase were used to detect bacteria capable of aerobically degrading benzene, toluene, and xylene, they were also used in quantitative PCR to show an increase in gene copy number after soil samples were amended with petroleum (Mesearch et al., Appl. Environ. Microbiol. 66:678-683, 2000). In specific embodiments, the invention is directed to primers that are able to target a gene that is essential to the metabolic pathway of (per)chlorate reducing bacteria but do not target their close phylogenetic non-(per)chlorate-reducing relatives. These primers allow for the rapid, sensitive and inexpensive identification of presence of DPRB. Certain aspects of the invention are described in further detail herein below.
To date, chlorite dismutase and perchlorate reductase are the only enzymes in the perchlorate reduction pathway that have been isolated and characterized. The use of primers for the detection of chlorite dismutase have been described in PCT application PCT/US2005/004657 (inventors Achenbach et al., filed Feb. 11, 2005, which is incorporated herein by reference). Together with the chlorite dismutase gene, the gene for perchlorate reductase, and more particularly the pcrA gene (GenBank Accession No. AY180108), is a good candidate for the environmental detection of DPRB and can be used to particularly identify perchlorate reducing activity in a sample.
Just as studies have shown that chlorite dismutation is essential to the (per)chlorate reduction pathway (Bruce et al., Environ. Microbiol. 1:319-329., 1999; Coates et al., Appl. Environ. Microbiol. 65:5234-5241., 1999; van Ginkel et al., Arch. Microbiol. 166:321-326, 1996), the first step in microbial perchlorate reduction is the reduction of perchlorate (ClO4−) to chlorite (ClO2−) by the perchlorate reductase enzyme.
In the present invention there is provided a metabolic primer set targeting the perchlorate reductase gene (and more particularly the pcrA gene (GenBank Accession No. AY180108)). Such a primer set would be useful for the molecular detection of DPRB in the environment. However, the efficacy of this metabolic primer set is dependent upon regions of sequence conservation within the perchlorate reductase gene, information which is currently unavailable due to the paucity of gene sequences in the publicly available databases. The primers of the present invention are designed to universally detect this gene. Hence, this gene is the target nucleic acid for the methods of the present invention. As used herein, the term “target nucleic acid” or “nucleic acid target” refers to a particular nucleic acid sequence of interest. The “target” can exist in the presence of other nucleic acid molecules or within a larger nucleic acid molecule.
In the present invention, target nucleic acid is a sequence that is a perchlorate reductase gene and more particularly the pcrA gene, from any DPRB. The DPRB may be present in any sample, including soil samples, groundwater samples, isolated populations of DPRBs, cocktails of bacterial populations used in bioremediation processes and the like. The inventors have developed the primers of the present invention such that they hybridize to the pcrA gene from DPRB. The results and discussion of the studies leading to the identification of these primers is provided in the Examples.
It is particularly contemplated that the primers designed to identify the pcrA gene may be provided in kits or compositions which are designed to detect the chlorite dismutase gene, a gene that is unique to and required by all DPRB.
A. PRIMERS, OLIGONUCLEOTIDES AND PRODUCTION THEREOF
The following section provides a discussion of preferred primer and oligonucleotide compositions of the invention. The term “nucleic acid” as used herein refers to a linear sequence of nucleotides (bases) linked to one another by a phosphodiester bond between 3′-position of a pentose of one nucleotide and 5′-position of a pentose of another nucleotide. The term “polynucleotide” refers to a nucleic acid including a sequence of nucleotides more than about 100 bases. The term “oligonucleotide” refers to a short polynucleotide or a portion of polynucleotide including about 2-100 bases.
As used herein a “primer” refers to an oligonucleotide, which is capable of acting as a point of initiation of nucleic acid synthesis when the primer is placed under conditions in which synthesis of a primer extension product that is complementary to a nucleic acid strand is induced, i.e. in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH. The primer may be a naturally occurring oligonucleotide or may be a purified restriction digest or produced synthetically. The oligonucleotide primers may be used, for example, in a PCR method as a primer for polymerization, in specific embodiments, the same primer may be in reverse transcription reaction in which the enzyme catalyzing the polymerization is a reverse transcriptase. Herein, oligonucleotides and primers of the invention may contain some modified linkages such as a phosphorothioate bond. The primers may also comprise a degenerate base, such as N base. Alternatively, one or more of the bases may be a universal base, such as e.g., hypoxanthine, as its ribo- or 2′-deoxyribonucleoside which is known for its ability to form base pairs with the other natural DNA/RNA bases. Nucleotide analog can be incorporated into the primers by methods well known in the art. The only requirement is that the incorporated nucleotide analog must serve to base pair with target polynucleotide sequences. For example, certain guanine nucleotides can be substituted with hypoxanthine, which base pairs with cytosine residues. Alternatively, adenine nucleotides can be substituted with 2,6-diaminopurine, which can form stronger base pairs than those between adenine and thymidine.
In exemplary embodiments of the present invention, primer sets targeting the perchlorate reductase gene were designed based on areas of amino acid and nucleotide sequence conservation in the pcrA gene.
The primers of SEQ ID NO:1 through 8 were developed based on the amino acid conservation of the pcrA sequence of the perchlorate reductase operon from Dechloromonas agitata, and Dechloromonas aromatica. Primers were synthesized by Integrated DNA Technologies, Coralville, Iowa. The primers include the following:








DPR.F SEQ ID NO:1:



GCNCCNCCNGTNGCNTT(T/C)ATG


 


DPR.R SEQ ID NO:2


(G/A)TC(G/A)TTNAC(T/C)TCNGC(G/A)TG(A/G/T)AT


 


DPR1.F SEQ ID NO:3


ACAGCGA(G/C/T)(G/C/T)TGCT(A/G/T)(A/G/T)TGCG


 


DPR1.R SEQ ID NO:4


(A/G/C)GCCGAAGG(A/G/C)A(A/G/T)AACCA(G/C/T)(A/G/C)


 


UPR.F SEQ ID NO:5


GA(T/C)CCNGCNCTNGA(G/A)GGNAA(G/A)


 


UPR.R SEQ ID NO:6


CCA(A/G/T)ATNGTNGT(T/C)TG(G/A)CA


 


UPR1.F SEQ ID NO:7


AGACCAC(G/C/T)ATCTGGACCTATGTC


 


UPR1.R SEQ ID NO:8


GG(A/G/C)A(A/G/T)AACCA(G/C/T)(A/G/C)TC(A/G/C)GAATA


 


(T/C)A






Each of the above primers is a degenerate primer. Degenerate primers are useful for pulling out one part of a gene sequence when the gene sequence in related organisms is unknown. Degenerate primers are thus designed to match an amino acid sequence. Typically, one gathers sequences from a large range of organisms and translates them to amino acid sequence and aligns them. Based on these amino acid alignments, regions of the sequence that are highly conserved at the amino acid level are readily identified. These conserved regions become possible locations for degenerate primers. Preferably, at least 2 blocks of conserved amino acids should be present to enable the design of PCR primers. A further alignment can be done at nucleotide level if desired.
Typically, the conserved regions chosen as the basis for the primer design should be at least 5 amino acids in length, more preferably 6, 7, 8, 9, 10 or more amino acids in length. As such, the primers should be 20-30 mer in length and it is preferred that the minimum size is 20 bases in length (i.e., a 20 mer). In certain embodiments, the efficiency of the degenerate primers may be increased by adding an oligonucleotide tail to the degenerate primers on the 5′ ends. This helps to increase the PCR efficiencies of these primers by increasing primer length and hence allows an increase in the annealing temperature. Although the tails do not help in the first few rounds of PCR when only the genomic template is being amplified, the tails do match in subsequent PCR cycles when the short PCR products containing the primers at each end are being amplified. Tails from commonly used restriction sites are particularly useful. For example a tail from an EcoRI site e.g., GCGCGGAATTC (SEQ ID NO:9) can be added to the 5′ end of the degenerate primer. Another useful tail, GCGCGCAAGCTT (SEQ ID NO:10), from the HindIII restriction site could be added to the 5′ end of a primer of the present invention.
Degeneracy of the primers depends on a multitude of factors including the template. 1000-10,000 fold degeneracy have been done. However the degeneracy can be lowered with the use of inosines for substituting 4 base wobbles instead of using all 4 base substitutions Thus, one or more of the “N” sequences in the above primers may be replaced with an inosine residue. To obtain the degeneracy of the primers, all the degeneracy values incorporated into the primer sequence are multiplied. Thus, other specific primers of the present invention include:








SEQ ID NO:11:



GC(I/N)CC(I/N)CC(I/N)GT(I/N)GC(I/N)TT(T/C)ATG


 


SEQ ID NO:12


(G/A)TC(G/A)TT(I/N)AC(T/C)TC(I/N)GC(G/A)TG(A/G/T)


 


AT


 


SEQ ID NO:13


GA(T/C)CC(I/N)GC(I/N)CT(I/N)GA(G/A)GG(I/N)AA(G/A)


 


SEQ ID NO:14


CCA(A/G/T)AT(I/N)GT(I/N)GT(T/C)TG(G/A)CA






The above specific primers for the detection of perchlorate reductase may be provided in remediation detection kits which comprise primers for the detection of chlorite dismutase. Exemplary primers for detection of chlorite dismutase include:








SEQ ID NO:15:



[5′-GA(A/G)CGCAA(A/G)(A/G)GNGCNGCNG(A/C)NGA(A/G)


 


GT-3′]


 


SEQ ID NO:16:


[5′-TC(A/G)AA(A/G)TANGT(A/T/G)AT(A/G)AA(A/G)TC-3′]


 


SEQ ID NO:17


[5′-T(C/T)GA(A/C/G)AA(A/G)CA(C/T)AAGGA(A/T/C)AA


 


(A/C/G)GT-3′]


 


SEQ ID NO:18:


[5′-GAGTGGTA(A/C/G)A(A/G)(C/T)TT(A/C/G)CG(C/T)


 


TT-3′]


 


SEQ ID NO:19


[5′-GANCGNAANNGNGCNGCNGNNGANGT-3′]


 


SEQ ID NO:20


[5′- TCNAANTANGTNATNAANTC-3′]






Exemplary chlorite dismutase primers where one or more of the “N” residues are replaced with inosine include for example:








SEQ ID NO:21:



[5′-GA(A/G)CGCAA(A/G)(A/G)G(I/N)GCIGC(I/N)G(A/C)


 


(I/N)GA(A/G)GT-3′]


 


SEQ ID NO:22:


[5′-TC(A/G)AA(A/G)TA(I/N)GT(A/T/G)AT(A/G)AA(A/G)


 


TC-3′]


 


SEQ ID NO:23:


[5′-GA(I/N)CG(I/N)AA(I/N)(I/N)G(I/N)GC(I/N)GC(I/N)


 


G(I/N)(I/N)GA(I/N)GT-3′]


 


SEQ ID NO:24:


[5′-TC(I/N)AA(I/N)TA(I/N)GT(I/N)AT(I/N)AA(I/N)


 


TC-3′]






In the primers described herein, according to standard nomenclature “N” refers to any one of the bases A, C, G, or T and it is presented at the third “wobble” position of the nucleic acid codons. Those of skill in the art could modify these primers by fixing one or more of the “N” residues in any of the primers with a specific nucleotide. It is specifically contemplated that the individual sequences derived from the degenerate primers in which the “N” is a set base are specifically part of the present invention. Simply by way of example, SEQ ID NO:2, which is (G/A)TC(G/A)TTNAC(T/C)TCNGC(G/A)TG(A/G/T)AT, a 21 nucleotide-base primer may yield the primer: GTCGTTNACTTCNGCGTGAAT (SEQ ID NO:25) derived by fixing the “G/A” choice at position 1 of the oligonucleotide as “G,” fixing the “G/A” choice at position 4 of the oligonucleotide as “G”, fixing the “T/C” choice at position 10 of the oligonucleotide as “T”, fixing the “G/A” choice at position 16 of the oligonucleotide as “G”, fixing the “AIGIT” choice at position 19 of the oligonucleotide as “A”. In like manner, the other permutations of the above degenerate primers also can be readily determined. Each of these permutations is particularly part of this invention and the specific sequences have not been written out as individual primer sequence, simply for the purposes of clarity and not because they are excluded from the written description.
One embodiment of the invention contemplates a library of primers that are generated from any one or more of the primers of SEQ ID NO:1 through SEQ ID NO:8 and SEQ ID NO:12 through SEQ ID NO:16, including permutations of each of these primers where one or other of the “N,” “Y,” “R,” “B,” “D,” “V” residues in SEQ ID NO:1 through 8 is fixed as a specific nucleotide. In certain exemplary embodiments, it may be desirable to array such a library of primers on a sequencing chip or other nucleic acid microarray. The above library of primers is one which is for detection of perchlorate reductase. The library also may contain primers for the detection of chlorite dismutase, including all permutations of any one or more of the primers of SEQ ID NO:15 through SEQ ID NO:24.
Sequences of about 17 bases long should occur only once in the genome and, therefore, suffice to specify a unique target sequence. The primers of the present invention are typically of this size. As used herein, an oligonucleotide that “specifically hybridizes” to a given target nucleic acid (e.g., DNA; RNA) means that hybridization under suitably (e.g., high) stringent conditions allows discrimination of that target nucleic acid from other genes. Although shorter oligomers are easier to make, numerous other factors are involved in determining the specificity of hybridization. Both binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length. It is contemplated that exemplary oligonucleotides of 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more base pairs can be prepared. In preferred aspects of the invention the oligonucleotide primers are between about 17 to about 50 bases in length and serve as primers in amplification reactions to amplify the sequence of any perchlorate reductase gene sequence from a DPRB sample.
The oligonucleotides may include the degenerate primers (which may be anywhere from 17 to 50 bases in length) but may also include 5′ or 3′ flanking regions or tails to facilitate amplification of the products. In particular, as discussed above, 5′ tails on the degenerate primers are useful for increasing amplification efficiency. Thus, as used herein, the “primer” sequence is the degenerate primer discussed above, an oligonucleotide sequence may be the primer sequence alone, or it may be the primer sequence in addition to other nucleic acids.
The term “complementary” is used when defining a pair of nucleotide sequences, for example, a base pair of A/T or C/G, that match each other according to the base pairing rules. For example, a sequence of 5′-A-G-T-3′ is complementary to a sequence of 3′-T-C-A-5′. Nucleotide sequences may be “partially” or “perfectly” complementary to one another so that they form partially matching base pairs or perfectly matching base pairs.
Methods for the production of primers are well known to those of skill in the art as routine synthesis techniques. For example, oligonucleotides can be synthesized chemically according to the solid phase phosphoramidite triester method described by Beaucage and Caruthers (1981), Tetrahedron Letts., 22(20):1859-1862, e.g., using a commercially available automated synthesizer, e.g., as described in Needham-VanDevanter et al. (1984) Nucleic Acids Res., 12:6159-6168. Synthesis of modified oligonucleotides (e.g., oligonucleotides comprising 2′-O-methyl nucleotides and/or phosphorothioate, methylphosphonate, or boranophosphate linkages, e.g., for use as nuclease resistant primers) are described in e.g., Oligonucleotides and Analogs (1991), IRL Press, New York; Shaw et al. (1993), Methods Mol. Biol. 20:225-243; Nielsen et al. (1991), Science 254:1497-1500; and Shaw et al. (2000) Methods Enzymol. 313:226-257. Detailed procedures for the phospho-triester and hydrogen phosphonate methods of oligonucleotide synthesis are described in the U.S. Pat. No. 4,458,066.
Oligonucleotides, including modified oligonucleotides (e.g., oligonucleotides comprising fluorophores and quenchers, 2′-O-methyl nucleotides, and/or phosphorothioate, methylphosphonate, or boranophosphate linkages) can also be ordered from a variety of commercial sources known to persons of skill. There are many commercial providers of oligo synthesis services, and thus, this is a broadly accessible technology. Companies such as The Midland Certified Reagent Company (www.mcrc.com), The Great American Gene Company (www.genco.com), ExpressGen Inc. (www.expressgen.com), QIAGEN (http://oligos.qiagen.com) and many others provide a readily available commercial service for the synthesis of oligonucleotide and as such the primers and oligonucleotides of the present invention may be commercially synthesized once the identity of the oligonucleotides is provided herein. The probes used herein were prepared by Integrated DNA Technologies, (Coralville, Iowa).
The present invention requires the use of a probe or primer pairs that are specific for perchlorate reductase and/or chlorite dismutase from a DPRB. To develop these probes or primers, one must first determine what genetic sequences are conserved between the many strains of DPRB. If one were to use a sequence derived from only a few strains, one would risk not detecting bacterial strain that had mutated slightly from this group.
The inventors compared the perA sequence from D. agitata, and D. aromatica, with other like sequences and looked for highly conserved nucleotide sequences. As discussed above, the design of degenerate primers typically employs an amino acid sequence of at least 5 amino acids, which would produce a 15-mer, however, it is preferred that the primers should be greater than or equal to 20 nucleotides in length. FIG. 1 describes the amino acid sequences identities of proteins that were compared.
A probe of the invention suitable to hybridize with a perchlorate reductase gene sequence will be at least 20 nucleotides in length and may be chosen from the entire length of the perchlorate reductase sequence. The probe should preferably have a GC content of approximately 50%. Similar considerations, of course are applicable to for example compositions for the detection of chlorite dismutase.
To derive primers from the perchlorate reducatase sequences, one must first choose sequences that when amplified would produce a DNA segment of sufficient length. An exemplary product length is a DNA segment of at least 100 nucleotides. If one wishes to visualize a PCR fragment on an electrophoretic gel, a smaller fragment would suffice. However, for optimum PCR amplification, a fragment of 100 nucleotides is still preferred. Preferably in both cases, the fragment should exceed 150 nucleotides.
The primer should be chosen so that the two primers are not complementary at the 3′ ends. This situation would lead to a hybridization reaction between the primers before the primers hybridize to the substrate material. A complementary region of equal to or greater than 2 nucleotides will cause an unwanted primer hybridization. Preferably, there will be no complementary region at the 3′ end. Also preferred are primers that do not have internal complementary segments that allow formation of hairpins.
B. AMPLIFICATION METHODS AND DETECTION OF PRODUCTS
The primers and oligonucleotide probes of the invention are used in methods of detecting the presence of (per)chlorate reducing bacteria. The presence of such bacteria is detected by determining the presence of the perchlorate reductase gene to which the degenerate primers of the present invention hybridize. The detection of the hybridized nucleotides is effected through the use of a polymerization chain reaction, a technique that is widely known in the field (U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,800,159).
Nucleic acid amplification by template-directed, enzyme-dependent extension of primers is well known in the art. For example, amplification by the polymerase chain reaction (PCR) has been described. Details regarding various PCR methods, including, e.g., asymmetric PCR, reverse transcription-PCR, in situ PCR, quantitative PCR, real time PCR, and multiplex PCR, are well described in the literature. Details regarding PCR methods and applications thereof are found, e.g., in Sambrook et al., Molecular Cloning—A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (2000); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2002); Innis et al. (eds.), PCR Protocols: A Guide to Methods and Applications, Academic Press Inc., San Diego, Calif. (1990); J. P. V. Heuvel, PCR Protocols in Molecular Toxicology, CRC Press (1997); H. G. and A. Griffin, PCR Technology: Current Innovations, CRC Press (1994); Bagasra et al., (1997) In Situ PCR Techniques, Jossey-Bass; Bustin (2000) “Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays” Journal of Molecular Endocrinology 25:169-193; Poddar (2000) “Symmetric vs. asymmetric PCR and molecular beacon probe in the detection of a target gene of adenovirus” Molecular and Cellular Probes 14: 25-32; and Mackay et al. (2002) “Real-time PCR in virology” Nucleic Acids Res. 30:1292-1305, and references therein, among many other references.
Additional details regarding PCR methods, including asymmetric PCR methods, are found in the patent literature, e.g., U.S. Pat. No. 6,391,544 (May 21, 2002) to Salituro et al. entitled “Method for using unequal primer concentrations for generating nucleic acid amplification products”; U.S. Pat. No. 5,066,584 (Nov. 19, 1991) to Gyllensten et al. entitled “Methods for generating single stranded DNA by the polymerase chain reaction”; U.S. Pat. No. 5,691,146 (Nov. 25, 1997) to Mayrand entitled “Methods for combined PCR amplification and hybridization probing using doubly labeled fluorescent probes”; and U.S. patent application Ser. No. 10/281,054 (filed Oct. 24, 2002) by Beckman et al. entitled “Asymmetric PCR with nuclease-free polymerase or nuclease-resistant molecular beacons.”
The PCR reaction is achieved by repeated cycles of denaturation, annealing for hybridizing a target sequence of a sample with a complementary primer, and polymerization using a thermally stable DNA polymerase to extend a DNA double helix from the hybridized primer. If no nucleotide primer hybridizes to the target nucleic acid, there is no PCR product. The PCR primer acts as a hybridization probe.
In brief, PCR typically uses at least one pair of primers (typically synthetic oligonucleotides). Each primer hybridizes to a strand of a double-stranded nucleic acid target that is amplified (the original template may be either single-stranded or double-stranded). A pair of primers typically flanks a nucleic acid target that is amplified. Template-dependent extension of the primers is catalyzed by a DNA polymerase, in the presence of deoxyribonucleoside triphosphates (typically dATP, dCTP, dGTP, and dTTP, although these can be replaced and/or supplemented with other dNTPs, e.g., a dNTP comprising a base analog that Watson-Crick base pairs like one of the conventional bases, e.g., uracil, inosine, or 7-deazaguanine), an aqueous buffer, and appropriate salts and metal cations (e.g., Mg2+). The PCR process typically involves cycles of three steps: denaturation (e.g., of double-stranded template and/or extension product), annealing (e.g., of one or more primers to template), and extension (e.g., of one or more primers to form double-stranded extension products). The PCR process can instead, e.g., involve cycles of two steps: denaturation (e.g., of double-stranded template and/or extension product) and annealing/extension (e.g., of one or more primers to template and of one or more primers to form double-stranded extension products). The cycles are typically thermal cycles; for example, cycles of denaturation at temperatures greater than about 90° C., annealing at 50-75° C., and extension at 60-78° C. A thermostable enzyme is thus preferred.
Other suitable hybridization conditions for the PCR reaction will be well known to those of skill in the art. In certain applications, it is appreciated that lower stringency conditions may be required. Under these conditions, hybridization may occur even though the sequences of probe and target strand are not perfectly complementary, but are mismatched at one or more positions. Conditions may be rendered less stringent by increasing salt concentration and decreasing temperature. For example, a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37° C. to about 55° C., while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20° C. to about 55° C. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results. Those of skill in the art will understand the salt concentrations and temperature parameters can be varied without departing from the nature of the invention.
In other embodiments, hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, at temperatures between approximately 20° C. to about 37° C. Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, at temperatures ranging from approximately 40° C. to about 72° C. Formamide and SDS also may be used to alter the hybridization conditions.
In specific preferred embodiments the annealing temperatures may range from 42° to 55° C., the MgCl2 concentrations may be varied ranging from 1.0 to 3.0 mM, the primer amounts ranging from 15 to 60 μmol. In addition, the PCR methods were carried out in the presence of PCR additives, such as 0.25 mg of bovine serum albumin (BSA)/ml, 5% (vol/vol) dimethyl sulfoxide, and 1 M betaine. In the exemplary reaction mixtures employed in the present application, the reaction mixtures contained 1× Mg-free buffer, 1.0 to 3.0 mM MgCl2, 200 μM of (each) deoxynucleoside triphosphates, 2.5 U of Taq polymerase (Sigma, St. Louis, Mo.), 1 μl of gDNA or environmental DNA, and nuclease-free double-distilled H2O to a final volume of 50 μl. All components were purchased from Promega (Madison, Wis.) except for the polymerase.
In a particularly preferred PCR reaction, the following PCR conditions produced amplicons of the desired size using the forward and reverse primer set: 44° C. annealing temperature, 60 pmol (each) primer, 1.5 mM MgCl2, and 0.25 mg of BSA/ml. In another exemplary reaction with a different primer set PCR conditions were 50° C. annealing temperature, 40 pmol (each) primer, 1.5 mM MgCl2, and 0.25 mg of BSA/ml. Of course, the skilled artisan could vary these conditions and still achieve appropriate results in connection with the detection techniques of the present invention.
In the exemplary embodiments, the cycling parameters were as follows: reactions were initially heated to 94° C. for 2 min, followed by 30 cycles of 94° C. (1 min), annealing temperature (1 min), 72° C. (1 min), with a final 10-min 72° C. extension period. For touchdown cycling, the parameters consisted of a denaturation step at 94° C. for 1 min, a primer-annealing step for 1 min, and an extension step at 72° C. for 1 min. After 38 cycles, a final 10-min incubation was performed at 72° C. During the first 18 cycles, the annealing temperature was decreased by 1.0° C. every two cycles, starting at 59° C., until reaching a touchdown temperature of 50° C.
Automated thermal cyclers, including integrated systems for real time detection of product, are commercially available for performing PCR and other amplification reactions, e.g., the ABI Prism®7700 sequence detection system from Applied Biosystems, the iCycler iQ® real-time PCR detection system from Bio-Rad, or the DNA Engine Opticon® continuous fluorescence detection system from MJ Research, Inc. In particularly preferred embodiments, the PCR reactions were performed in a Perkin-Elmer 2400 thermocycler (Applied Biosystems, Foster City, Calif.).
Thermostable enzymes (including Thermus aquaticus Taq DNA polymerase, as well as enzymes substantially lacking 5′ to 3′ nuclease activity), appropriate buffers, etc. are also widely commercially available, e.g., from Clontech (Palo Alto, Calif., USA), Invitrogen (Carlsbad, Calif., USA), Sigma-Aldrich (St. Louis, Mo., USA), and New England Biolabs (Beverley Ma, USA). For example, thermostable polymerases lacking 5′ to 3′ nuclease activity are commercially available, e.g., TitaniumTM Taq (Clontech, Palo Alto, Calif., USA, www.clontech.com), KlenTaq DNA polymerase (Sigma-Aldrich, St. Louis, Mo., USA, www.sigma-aldrich.com), VentTM and DeepVentTM DNA polymerase (New England Biolabs, Beverley Ma, USA, www.neb.com), Tgo DNA polymerase and FastStart DNA polymerase (Roche, Indianapolis, Ind., USA www.roche-applied-science.com), ABgene's Thermoprime Plus DNA Polymerase (Rochester, N.Y., USA), SuperTaq or SuperTaq PlusTM (Ambion, Austin, Tx, USA), FideliTaqTM DNA Polymerase (USB Corp., Cleveland, Ohio, USA, www.usb.com), Tfl DNA Polymerase (Promega, Madison, Wis., www.promega.com), and PfuTurbo® Cx Hotstart DNA Polymerase (Stratagene, La Jolla, Calif., USA, www.stratagene.com).
While it is preferred that the amplification reaction is the PCR reaction, there are other suitable amplification techniques such as CPR (Cycling Probe Reaction), bDNA (Branched DNA Amplification), SSR (Self-Sustained Sequence Replication), SOA (Strand Displacement Amplification), QBR (Q-Beta Replicase), Re-AMP (Formerly RAMP), NASBA (Nucleic Acid Sequence Based Amplification), RCR (Repair Chain Reaction), LCR (Ligase Chain Reaction), TAS (Transorbtion Based Amplification System), and HCS (amplifies ribosomal RNA), all of which may be used as the amplification method.
In one embodiment of the invention, this assay comprises the steps of exposing a nucleic acid isolated from a biological sample to oligonucleotide primers chosen from the group consisting of SEQ ID NO:1 through SEQ ID NO:8 and SEQ ID NO: 11 through SEQ ID NO:14, including permutations of each of these primers where one or other of the “N,” “Y,” “R,” “B,” “D,” “V” residues in SEQ ID NO:1 through 8 is fixed as a specific nucleotide. In specific embodiments, the PCR or transcription reaction is conducted directly on a bacterial lysate without isolating the nucleic acid therefrom. In other embodiments, nucleic acid is isolated from the sample, RNA is prepared therefrom and cDNA created from RNA. The reaction is allowed to incubate using PCR or other amplification or transcription conditions and the sample may then be examined for the presence of an amplification or other reaction product. In addition, these methods also could be performed using primer pairs specific for the cld gene (e.g., including all permutations of any one or more of the primers of SEQ ID NO:15 through SEQ ID NO:24.)
In PCR, the method begins with exposing a biological sample to a primer pair specific for the gene of interest, e.g., the perchlorate reductase gene or the cld gene. More particularly, nucleic acid is isolated from the biological sample and contacted with the primer pairs. In certain embodiments, RNA is prepared from the nucleic acids. RNA is preferably isolated from biological samples by adding a guanidinium solution. Other methods known in the art of isolating RNA would also be suitable.
To perform the method of the present invention, one must first select a probe and primer pair of the present invention and expose the perchlorate reductase cDNA to the primer pair. After amplification, the PCR product is detected. In certain embodiments, performing PCR with SEQ ID NO:1 and 2 alone is sufficient. In other preferred embodiments, however, it is contemplated that a nested PCR is performed in which the amplification product from the PCR reaction with SEQ ID NO:1 and 2 (or alternatively, SEQ ID NO:11 and 12) is used as a template for a second PCR reaction in which the probes are primers SEQ ID NO:3 and 4. Additional nested PCR reactions can be performed using the other primers described herein (including permutations of each of these primers where one or other of the “N,” “Y,” “R,” “B,” “D,” “V” residues in SEQ ID NO:1 through 8 is fixed as a specific nucleotide).
In specific embodiments, it is contemplated that a reverse transcription reaction is carried out to determine the presence of perchlorate reductase mRNA in the sample. The reverse transcription reaction performed alone would be sufficient to identify the presence of the DPRB in the biological sample being tested. However, in other specific embodiments, it is contemplated that the reaction product from the reverse transcription reaction is employed as a template for a further PCR reaction in which a pair of primers described herein are used. Thus, in certain embodiments, it is contemplated that a method is carried out in which a reverse transcription reaction is carried out using a primer selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:8 and SEQ ID NO:11 through SEQ ID NO:14 (including permutations of each of these primers where one or other of the “N,” “Y,” “R,” “B,” “D,” “V” residues in SEQ ID NO:1 through 8 is fixed as a specific nucleotide). In yet another embodiment, a contemplated method involves performing such a reverse transcription reaction, isolating the product of the reaction and using it as a template for a PCR reaction using a pair of primers selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:8 and SEQ ID NO:11 through SEQ ID NO:14 (including permutations of each of these primers where one or other of the “N,” “Y,” “R,” “B,” “D,” “V” residues in SEQ ID NO:1 through 8 is fixed as a specific nucleotide).
In performing the reverse transcription, once RNA is isolated from the biological sample, and exposed to reverse transcriptase enzyme and deoxyribonucleotides so that a cDNA molecule may be created that corresponds to the initial RNA molecule. Exemplary reverse transcriptases that may be used include, but are not limited to AMV Reverse Transcriptase (GEHealthcare and Amersham Biosciences, Piscataway, N.J., USA, AMV Reverse Transcriptase (Stratagene, La Jolla, Calif., USA www.stratagene.com), AMV RT (CHIMERx, Madison, Wis. USA, www.chimerx.com), cloned AMV Reverse Transcriptase (Invitrogen, Carlsbad, Calif., USA www.invitrogen.com), AMV Reverse Transcriptase (Ambion, Austin Tx, USA, www.ambion.com), AMV Reverse Transcriptase (www.MJResearch.com), AMV Reverse Transcriptase (Promega, Madison Wis., USA www.promega.com), and Reverse Transcriptase AMV (Roche, Indianapolis, Ind., USA, www.roche-applied-science.com)
For a PCR reaction, one would choose to use a pair of primers and examine the final product for presence of a PCR amplification product. This examination could involve examining the products of the reaction on an electrophoretic gel and determining whether an amplified product of the appropriate size had been created. One of skill in the art of molecular biology will be aware of many protocols designed to optimize PCR reactions. Particularly useful protocols are described in PCR Protocols, Ed. M. Innis, et al. Academic Press, San Diego.
The PCR reaction can be coupled to, for example, an ELISA detection procedure. In such a procedure, one would anchor the PCR amplification product to a solid support and examine the support for the presence of the PCR product. This procedure could be done in several ways. For example, one first attaches the amplified product to a solid support, such as a microtiter dish. For example, a streptavidin-coated plate may be provided. One of the selected primers may be attached to a biotin molecule so that an amplification product will be labelled with biotin and bind to the streptavidin plate. This is yet a further use of the probes of the present invention in that the primers/probes of the invention can be used to “fish-out” and identify/isolate the amplified product. For such embodiments, hybridization conditions discussed above could be used. In these embodiments, the plate and product are then exposed to perchlorate-reductase-specific oligonucleotide probe of the invention containing a segment of the perchlorate-reductase sequence. This probe is attached to a marker enzyme, such as horseradish peroxidase (HRP), which may be detected via its enzymatic properties.
In another method, one would attach a protein molecule capable of binding to the solid support, e.g., BSA, to an oligonucleotide probe/primer of the present invention. The plate is then coated with these protein-attached oligonucleotides, and these oligonucleotides are available to hybridize with an amplified product. This amplified product is preferably attached to a label molecule, such as biotin, that is capable of being detected. In one embodiment, the biotin-labelled PCR product may be complexed to a streptavidin-horse radish peroxidase conjugate. One may then detect this complex with the appropriate substrate.
The amplified products can be identified using various techniques, for example, by inserting a labeled nucleotide into the strands amplified using labeled primers. Examples of standard labeling materials include, but are not limited to, radioactive materials (32P, 35S, 131I, 125I, 14C, 3H), fluorescent labels (e.g., fluorescein, Texas Red, rhodamine, BODIPY, resorufin or arylsulfonate cyanines, digoxygenin, horseradish peroxidase, alkaline phosphatases, acridium ester), chemiluminescent labels (e.g., acridinium esters), biotin, and jack beam urease. Furthermore, the PCR products obtained using non-labeled primers can be identified by combination of gel separation using electrophoresis and a dye-based visualizing technique. Thus it is particularly contemplated that the primers of the invention are labeled with such detectable labels.
A label or quencher can be incorporated into the primers and probes of the invention during oligonucleotide synthesis by using a specialized phosphoramidite including the label or quencher, or a modified base phosphoramidite including an alkyl spacer can be incorporated during oligonucleotide synthesis and the label or quencher can be linked to the spacer after synthesis is complete. As a specific example, fluorescein can be incorporated at the 5′ end of a probe of the invention by using a fluorescein phosphoramidite in the last step of the synthesis. As another specific example, a modified T including a C6 spacer with a primary amino group can be incorporated into the oligonucleotide, and a succinimidyl ester of 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL) can be attached to the primary amino group. (Such modified phosphoramidites are commercially available, e.g., AminoModifier C6 dT from Glen Research.) Similarly, 4-dimethylaminophenylazophenyl-4′-maleimide (DABMI) can be used when the site of attachment is a sulphydryl group.
As other examples, fluorescein can be introduced into oligonucleotides, either by using a fluorescein phosphoramidite that replaces a nucleoside with fluorescein, or by using a fluorescein dT phosphoramidite that introduces a fluorescein moiety at a thymidine ring via a spacer. To link a fluorescein moiety to a terminal location, iodoacetoamidofluorescein can be coupled to a sulphydryl group. Tetrachlorofluorescein (TET) can be introduced during automated synthesis using a 5′-tetrachloro-fluorescein phosphoramidite. Other reactive fluorophore derivatives and their respective sites of attachment include the succinimidyl ester of 5-carboxyrhodamine-6G (RHD) coupled to an amino group; an iodoacetamide of tetramethylrhodamine coupled to a sulphydryl group; an isothiocyanate of tetramethylrhodamine coupled to an amino group; or a sulfonylchloride of Texas red coupled to a sulphydryl group.
C. ASSAYS AND KITS
In one embodiment, the present invention is an assay for the presence of DPRB in a sample. In other embodiments, this assay may be combined in an assay for at least one other microorganism that can effect remediation. For example, in contaminated soils or water samples, it may be desirable to remove the (per)chlorate contamination using DPRB and to remove contamination of other contaminants using other reclamation and/or bioremediation techniques. Preferably, such assays examine a biological sample for presence or absence of the appropriate contaminants as well as microorganisms that can remove such contaminants.
The perchlorate reductase gene may be derived from any DPRB. The presence of this gene in a sample being tested is indicative of the sample possessing bacteria that will effect remediation of the sample and clean-up any (per)chlorate contamination thereof. The organisms from which the perchlorate reductase gene may be detected and whose presence is desired in the samples to effect bioremediation of a (per)chlorate contaminated sample include, but are not limited to, Dechloromonas spp., Azoarcus spp., Dechlorospirillum spp., Dechloromarinus spp., Ideonella spp., Magnetospirillum spp., Pseudomonas spp., Rhodocyclus spp., Rhodospirillum spp., Azospirillum spp., Wolinella spp., and Xanthomonas spp. Exemplary bacteria from these species include for example, Dechloromonas agitata, Dechloromonas aromatica, Azospira suillum, Dechlorospirillum anomalous, Dechloromarinus chlorophilus, and Ideonella dechloratans. These are merely exemplary DPRBs and many others will be known to those of skill in the art.
In another embodiment, the present invention provides a kit for assaying DPRB present in a sample. In a preferred embodiment, the kit comprises a pair of primers selected from SEQ ID NO:1 through SEQ ID NO:8 and SEQ ID NO:11 through SEQ ID NO:14 (including permutations of each of these primers where one or other of the “N,” “Y,” “R,” “B,” “D,” “V” residues in SEQ ID NO:1 through 8 is fixed as a specific nucleotide). In another embodiment, the kit comprises at least one additional pair of primers designed to amplify a 16S RNA from one or more of the organisms indicated above. In a more preferred embodiment of the kit, the kit additionally comprises primers SEQ ID NO:1 or SEQ ID NO:11 and SEQ ID NO:2 or SEQ ID NO:12; in another embodiment, the kit comprises primers SEQ ID NO:3 and SEQ ID NO:4, is still a further embodiment, the kit comprises primers of SEQ ID NO:5 or SEQ ID NO:13 and SEQ ID NO:6 or SEQ ID NO:14; in yet a further embodiment the kit comprises in another embodiment, the kit comprises primers SEQ ID NO:7 and SEQ ID NO:8. Kits that comprise SEQ ID NO:1, SEQ ID NO:2; SEQ ID NO:3 and SEQ ID NO: 4 are specifically contemplated. In some kits the perchlorate reductase primers are provided with primers that detect chlorite dismutase. Specific such chlorite dismutase detection primers include but are not limited to SEQ ID NO:15 through SEQ ID NO:24. Preferably, the primers of each SEQ ID NO are provided in separate containers.
In addition, the kits may comprise one or more enzymes for the PCR amplification and/or for the reverse transcription reactions. Thus, the kits optionally also includes one or more of: a polymerase (e.g., a polymerase having or substantially lacking 5′ to 3′ nuclease activity), a buffer, a standard template for calibrating a detection reaction, instructions for extending the primers to amplify at least a portion of the target nucleic acid sequence or reverse complement thereof, instructions for using the components to amplify, detect and/or quantitate the target nucleotide sequence or reverse complement thereof, or packaging materials. The kits may also preferably include the deoxyribonucleoside triphosphates (typically dATP, dCTP, dGTP, and dTTP, although these can be replaced and/or supplemented with other dNTPs, e.g., a dNTP comprising a base analog that Watson-Crick base pairs like one of the conventional bases, e.g., uracil, inosine, or 7-deazaguanine), an aqueous buffer, and appropriate salts and metal cations (e.g., Mg2+)
The kit may comprise a solid support on which the present the primers. Alternatively, the primers may be bound to a solid support. The solid support may be any support that is typically used to in nucleic acid preparation and analysis. Such supports include, but are not limited to plastic, glass, beads, microtiter plates. Indeed, glass, plastics, metals and the like are often used, and the nucleic acid amplification method of the present invention can be used irrespective of the type of the substrate. In some aspects, the probes of the invention may be effectively used for assaying nucleic acid molecules using a DNA chip or DNA micro-array where a large number of DNA probes are immobilized on a flat substrate. Furthermore, other than the flat DNA chips, the nucleic acid assay method using beads on which DNA probes are immobilized has become popular in recent years, and the nucleic acid amplification method of the present invention is also applicable to preparation of a sample in the nucleic acid detection method using the DNA probes immobilized on the surfaces of beads.
The primers in the kits may be provided in the form of nucleic acid-based arrays. Microarray chips are well known to those of skill in the art (see, e.g., U.S. Pat. Nos. 6,308,170; 6,183,698; 6,306,643; 6,297,018; 6,287,850; 6,291,183, each incorporated herein by reference). These are exemplary patents that disclose nucleic acid microarrays and those of skill in the art are aware of numerous other methods and compositions for producing microarrays. The term “microarray” refers to an ordered arrangement of hybridizable array elements. The array elements are arranged so that there are preferably at least two or more different array elements, more preferably at least 20 array elements, and more preferably at least 100 array elements, on a 1 cm2 substrate surface. The hybridization signal from each of the array elements is individually distinguishable a specific location, or address of the probe. In a preferred embodiment, the array elements comprise polynucleotide primers of the present invention.
In addition to the above, the kits may comprises components as standards. For example, the kits may comprise known perchlorate reductase or a chlorite dismutase sequences such that the signal received from the environmental/biological sample can be compared with that received from the standard to ensure the integrity of the assay components and conditions.
D. EXAMPLES
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1
Materials & Methods
Growth conditions. Dechloromonas agitata and Dechloromonas aromatica were grown both anaerobically and aerobically in basal media as previously described (Bender et al., Appl. Environ. Microbiol. 68:4820-4826, 2002; Bruce et al., Environ. Microbiol., 1:319-329, 1999). For anaerobic cultures, 10 mM acetate and 10 mM perchlorate, chlorate, or nitrate were used as the electron donor and the electron acceptor, respectively. For aerobic growth, (per)chlorate was omitted and oxygen was added to the same basal media. To check for induction under aerobic conditions, 1 mM sodium nitrate, chlorate, or perchlorate was added to aerobically grown cultures.
Nucleic acid extraction and mutant construction. Both genomic DNA and RNA were extracted as previously described using a PUREGENE DNA isolation kit (Gentra Systems Inc., Minneapolis, Minn.) and the RNAwiz reagent (Ambion, Austin, Tex.), respectively (Bender et al., Appl. Environ. Microbiol. 68:4820-4826, 2002). A pcrA mutant of D. aromatica was constructed by replacement of the pcrA gene with a tetracycline resistance cassette. Briefly, a region upstream of the pcrA start codon and a region downstream of the pcrA stop codon were PCR amplified and inserted on either side of a 1.6-kb pBR322 tetracycline resistance cassette that had been cloned into a suicide vector. This construct was used to transform D. aromatica cells; double-recombination mutants in which the pcrA gene on the chromosome had been replaced with the resistance cassette were verified by PCR amplification.
Sequence analysis. DNA sequences obtained from D. agitata lambda library screening (Bender et al., Appl. Environ. Microbiol. 68:4820-4826, 2002), as well as the complete genome sequence of D. aromatica, obtained courtesy of the Joint Genome Institute (http://www.jgi.doe.gov), were subjected to BLAST analysis (Altschul et al., Nucl. Acid Res. 25:3389-3402, 1997). DNA sequence manipulations were performed using the MacVector sequence analysis software for the Macintosh (version 7.0; Oxford Molecular) and the Se—Al sequence alignment editor, v. 1.0 (A. Rambaut, University of Oxford).
Hybridization analyses. Northern blotting was performed using the Northern-Max-Gly glyoxal-based system (Ambion) as previously described (Bender et al., Appl. Environ. Microbiol. 68:4820-4826, 2002). For all growth conditions, 5 μg of total RNA was loaded onto a 1% (wt/vol) glyoxal agarose gel. Following RNA transfer, the blot was hybridized at 50° C. in Easyhyb hybridization solution (Roche Applied Science, Indianapolis, Ind.) with a digoxigenin-labeled probe corresponding to 436 bp in the 5′ half of the D. agitata pcrA gene. This probe was generated via PCR at an annealing temperature of 55° C. with the following primers: PR-750F (5′-CGCGAAGGTAGTCAGCATCT-3′; SEQ ID NO:26) and PR-1185R (5′-TCCATCCTGCAACTTGACCT-3′). For DNA slot blotting, genomic DNAs from known DPRB and non-perchlorate-reducing close relatives were blotted as previously described (Bender et al., Appl. Environ. Microbiol. 68:4820-4826, 2002). The blot was hybridized at 45° C. with the same perchlorate reductase probe used in the Northern blot analysis.
Phylogenetic analysis. Protein sequences from the α-subunits of known DMSO reductase enzymes were obtained from the GenBank database (Bensen et al., Nucleic Acid Res. 32:D35-D40, 2004) and aligned with the α-subunit of perchlorate reductase using the CLUSTALW 1.82 program (Thompson et al., Nucl. Acid Res. 22:4773-4680, 1994). A phylogenetic tree was constructed with the PAUP* v 4.0 program (D. L. Swofford, Sinauer Associates) using distance as the criterion and neighbor joining as the drawing method.
GenBank accession numbers. The GenBank accession numbers for the D. agitata perchlorate reductase genes are as follows: pcrA, AY180108; pcrB, AY953269; pcrC, AY953270; and pcrD, AY953271. The accession numbers for the protein sequences shown in FIG. 2 are as follows: SerB, Q9S1G9; ClrB, P60069; EbdB, CAD58340; DdhB, AAN46633; and NarH, CAD22070. The accession number for the Nitrosomonas europaea cytochrome c554 shown in FIG. 3 is NP—842334. The GenBank accession numbers for the 16S rRNA gene sequences of the organisms shown in FIG. 7 are as follows: D. agitata, AF047462; Rhodocyclus tenuis, D16209; D. aromatica, AY032610; Dechloromonas sp. Strain JJ, AY032611; Dechlorospirillum anomolous strain WD, AF170352; Magnetospirillum magnetotacticum, Y10110; Pseudomonas sp. strain PK, AF170358; Pseudomonas stutzeri, U26415; Dechloromarinus chlorophilus strain NSS, AF170359; Azospira suillum, AF170348; Dechloromonas sp. strain LT-1, AY124797; and I. dechloratans, X72724.







TABLE 1







BLAST analysis of the pcrABCD translation products











% Amino




acid


Gene
GenBank BLAST hit
identity





pcrA
CAD22069, Haloarcula marismortui nitrate reductase α-subunit (NarG)
41



CAF21906, Haloferax mediterranei nitrate reductase α-subunit (NarG)
40



AAN46632, Rhodovulum sulfidophilum dimethyl sulfide dehydrogenase α-subunit (DdhA)
34



Q9S1H0, Thauera selenatis selenate reductase α-subunit (SerA)
33



P60068, Ideonella dechloratans chlorate reductase α subunit (ClrA)
33


pcrB
Q9S1G9, Thauera selenatis selenate reductase β-subunit (SerB)
54



P60069, Ideonella dechloratans chlorate reductase β-subunit (ClrB)
54



CAD58340, Azoarcus sp. strain EbN1 ethylbenzene dehydrogenase β-subunit (EbdB)
53



AAN46633, Rhodovulum sulfidophilum dimethyl sulfide dehydrogenase β-subunit (DdhB)
52


pcrC
NP_842334, Nitrosomonas europaea cytochrome c554 precursor
49


pcrD
Q9S1G8, Thauera selenatis selenate reductase (SerD)
35



AAN46634, Rhodovulum sulfidophilum dimethyl sulfide dehydrogenase δ-subunit (DdhD)
29



CAD58338, Azoarcus sp. strain EbN1 ethylbenzene dehydrogenase δ-subunit (EbdD)
25



CAD22073, Haloarcula marismortui nitrate reductase molybdenum chaperone (NarJ)
25









Example 2
Results And Discussion
Identification of pcrABCD genes. In the course of characterizing the chlorite dismutase (cld) gene (Bender et al., Appl. Environ. Microbiol. 68:4820-4826, 2002), we identified a proximal operon putatively encoding perchlorate reductase in the genomes of two DPRB, D. agitata and D. aromatica. The orientation of the perchlorate reductase genes was the same in both DPRB with exception of the position of the cld gene (FIG. 1). BLAST analysis of the open reading frames, designated pcrABCD, revealed amino acid similarities to subunits of microbial nitrate reductase, selenate reductase (serABDC), dimethyl sulfide dehydrogenase (ddhABDC), ethylbenzene dehydrogenase (ebdABCD), and chlorate reductase (clrABDC), all of which are members of the type II DMSO reductase family (Table 1). While the serABDC (Kengen et al., J. Bacteriol 181:6706-6711, 1999), ddhABDC (Kisker et al., Annu Rev. Biochem., 66:233-267, 1997), and clrABDC (Danielsson-Thorell, Appl. Environ. Microbiol. 69:5585-5592, 2003) operons all have the same gene order, the pcrABCD operon mimics the ebdABCD (McEwan et al., Geomicrobiol., J., 19:3-21, 2002) operon arrangement.
pcrA. Translational analysis of the 2,784-bp pcrA gene identified a molybdopterin-binding domain (data not shown), as well as a twin-arginine signal motif, (S/T)RRXFLK (FIG. 1). Previous studies have suggested that the twin-arginine motif tags proteins involved in electron transfer reactions, whose prosthetic groups are formed in the cytoplasm prior to secretion, for transport to the periplasm via Sec-independent transport (Tat pathway) (Berks et al., Mol. Microbiol., 35:260-274, 2000). This motif is also commonly found in electron transfer proteins possessing a pterin molybdenum cofactor and iron-sulfur (Fe—S) centers (Berks, Mol. Microbiol., 22:393-404, 1996). Since the perchlorate reductase of GR-1 was located in the periplasm and contained molybdenum and Fe—S centers (Kengen et al., J. Bacteriol 181:6706-6711, 1999), the presence of this signal peptide further supports identification of the pcrA gene as the gene encoding the α-subunit of the perchlorate reductase. In addition, the calculated molecular mass of the PcrA subunit is 105 kDa, a value that corresponds well to the 95 kDa predicted for the α-subunit of the purified perchlorate reductase from GR-1 (Kengen et al., J. Bacteriol 181:6706-6711, 1999)
pcrB. The inferred amino acid sequence of the 1,002-bp pcrB gene product indicated the presence of four cysteine-rich clusters for Fe—S center binding, a feature shared with β-subunits of type II DMSO reductase enzymes (FIG. 2). This cysteine organization has been shown to bind one 3Fe-4S center and three 4Fe -4S centers in both dimethyl sulfide dehydrogenase (McDevitt et al., Biochemistry, 41:15234-15244, 2002) and nitrate reductase (Jormakka et al., Structure, 12:95-104, 2004) β-subunits. Based on data for the β-subunit of the Escherichia coli nitrate reductase (Guigliarelli et al., Biochemistry, 35:4828-4836, 1996), these Fe—S centers may be responsible for electron transfer to the molybdopterin-containing α-subunit of perchlorate reductase.
The predicted N-terminal amino acid sequences of the D. agitata and D. aromatica PcrB proteins were aligned with the N-terminal sequence of purified PcrB from GR-1 (Kengen et al., J. Bacteriol 181:6706-6711, 1999). This alignment reinforced the identity of the pcrB gene (FIG. 2). The predicted D. agitata PcrB N terminus contained 10 of the 18 residues and the predicted D. aromatica PcrB sequence contained 16 of the 18 residues reported for the purified perchlorate reductase β-subunit from strain GR-1. Since no signal sequence was detected, the β-subunit of perchlorate reductase is likely translocated with the β-subunit in a manner similar to that proposed for selenate reductase (Krafft et al., DNA Seq., 10:365-377, 2000), dimethyl sulfide dehydrogenase (McDevitt et al., Mol. Microbiol., 44:1575-1587, 2002), and chlorate reductase (Danielsson-Thorell, Appl. Environ. Microbiol. 69:5585-5592, 2003). The calculated molecular mass of the PcrB subunit was 37 kDa, a value similar to the 40 kDa reported for the β-subunit of the purified perchlorate reductase from GR-1 (Kengen et al., J. Bacteriol 181:6706-6711, 1999).
pcrC. Although a γ-subunit was not detected in the enzyme analysis of the perchlorate reductase from strain GR-1, a third cytochrome-type subunit responsible for connecting the reductase to the membrane was believed to have been lost during purification of the enzyme (Kengen et al., J. Bacteriol 181:6706-6711, 1999). This observation was borne out by our identification of a 711-bp open reading frame immediately downstream of the pcrB gene in both D. aromatica and D. agitata, whose product exhibited sequence similarity to cytochrome c554 from N. europaea (Table 1). Amino acid alignment indicated that PcrC also has the unique tetraheme organization of cytochrome c554 from N. europaea (Iverson et al., Nat. Struct. Biol., 5:1005-1012, 1998) (FIG. 3). The lack of amino acid sequence similarity between PcrC (ca. 25 kDa) and other type II DMSO reductase γ-subunits was not surprising due to the overall sequence diversity noted in the SerC, EbdC, DhdC, and ClrC subunits (Danielsson-Thorell, Appl. Environ. Microbiol. 69:5585-5592, 2003).
The ProteinPredict server (http://cubic.bioc.columbia.edu/pp/) indicated that the pcrC translation product is not a membrane-bound protein and therefore cannot link the PcrAB complex to the membrane. However, further analysis of the D. aromatica genome revealed the presence of a NirT-type cytochrome gene downstream of the chlorite dismutase gene. The cytochrome may link the periplasmic PcrABC reductase to the membrane quinol pool (FIG. 4). Membrane-bound NirT-type cytochromes have been shown to shuttle electrons to the periplasmic nitric reductase of P. stutzeri, the Fe3+ and fumarate reductases of Shewanella putrefaciens, and the periplasmic nitrate reductase (McEwan et al., Geomicrobiol., J., 19:3-21, 2002; Richardson, Microbiology 146:551-571, 2000; Roldan et al., J. Biol. Chem., 273:28785-28790, 1998). The model predicted for perchlorate reduction (FIG. 4) differs from the model projected for selenate reduction (McEwan et al., Geomicrobiol., J., 19:3-21, 2002) by replacement of the bc1 complex with a NirT-type cytochrome.
pcrD. Based on sequence identity with SerD, DdhD, EbdD, and NarJ, the final 675-bp pcrD gene likely encodes a system specific molybdenum chaperone protein (ca. 25 kDa) (Table 1). This finding is supported by the absolute requirement for molybdenum for active perchlorate reduction (Chaudhuri et al., Appl. Environ. Microbiol., 68:4425-2230, 2002). The SerD, DdhD, and EbdD proteins are believed to be involved in assembly of the mature molybdenum-containing selenate reductase, dimethyl sulfide dehydrogenase, and ethylbenzene dehydrogenase, respectively, prior to periplasmic translocation via the Tat pathway. However, these proteins are not believed to be parts of the active enzymes (Krafft et al., DNA Seq., 10:365-377, 2000, McDevitt et al., Mol. Microbiol., 44:1575-1587, 2002, Rabus et al., Arch. Microbiol., 178:506-516, 2002).
Expression and mutagenesis of pcrA. Both expression analysis and mutagenesis of the pcrA gene verified the identity of the pcrABCD operon. Northern analysis of D. agitata RNA indicated that there was pcrA gene expression in anaerobic perchlorate- and chlorate-grown cultures (data not shown). However, the presence of perchlorate, chlorate, or nitrate was not enough to induce pcrA expression in aerobic cultures and, as such, indicates the ability of oxygen to completely inhibit pcrA expression, as suggested by the previously documented inhibitory effects of oxygen on perchlorate reduction (Chaudhuri et al., Appl. Environ. Microbiol., 68:4425-2230, 2002, 22).
Functional proof that the pcrA gene is involved in perchlorate reduction was obtained by mutational knockout in D. aromatica, in which insertional inactivation of the pcrA gene with a tetracycline resistance cassette abolished both perchlorate and chlorate reduction (FIG. 5). However, as expected, the D. aromatica pcrA mutant was still able to grow aerobically (data not shown), as well as anaerobically via nitrate reduction, indicating that there are separate metabolic pathways for each electron acceptor (FIG. 5).
Phylogenetic analysis of PcrA. Based on the biochemical analysis of the purified enzyme from strain GR-1 (Kengen et al., J. Bacteriol 181:6706-6711, 1999), perchlorate reductase was identified as a member of the type II DMSO reductase family (McEwan et al., Geomicrobiol., J., 19:3-21, 2002). Our sequence analysis of the perchlorate reductase genes also supported this identification. Enzymes in the prokaryotic type II DMSO reductase family reside in the periplasm and have a common pterin molybdenum cofactor known as bis(molybdopterin guanine dinucleotide) Mo (Kisker et al., Annu Rev. Biochem., 66:233-267, 1997, McDevitt et al., Mol. Microbiol., 44:1575-1587, 2002, McEwan et al., Geomicrobiol., J., 19:3-21, 2002). DMSO reductase enzymes are involved in a myriad of reduction capabilities, including the dissimilatory reduction of toxic elements such as selenate and arsenate (McEwan et al., Geomicrobiol., J., 19:3-21, 2002). Using α-subunit protein sequences from known microbial DMSO enzymes (McDevitt et al., Mol. Microbiol., 44:1575-1587, 2002, McEwan et al., Geomicrobiol., J., 19:3-21, 2002) and from the PcrA sequences resulting from this study, a phylogenetic tree was constructed (FIG. 6), and this tree had a topology similar to that of a DMSO reductase family tree constructed by McEwan and coworkers (McEwan et al., Geomicrobiol., J., 19:3-21, 2002).
The type I, type II, and type III DMSO enzymes form separate clades in the tree. The type I enzymes include formate dehydrogenase (FDH), periplasmic nitrate reductase (NapA), bacterial assimilatory nitrate reductase (NasA), and arsenite oxidase (AsoA) (McDevitt et al., Mol. Microbiol., 44:1575-1587, 2002, McEwan et al., Geomicrobiol., J., 19:3-21, 2002). Type II enzymes, such as ethylbenzene dehydrogenase (EbdA), dimethyl sulfide dehydrogenase (DdhA), selenate reductase (SerA), chlorate reductase (ClrA), nitrate reductase (NarG), and perchlorate reductase (PcrA), share a heterotrimeric structure and have conserved cysteine residues for Fe—S binding in the β-subunit (McDevitt et al., Mol. Microbiol., 44:1575-1587, 2002, McEwan et al., Geomicrobiol., J., 19:3-21, 2002). The type III enzymes are represented by the monomeric proteins biotin sulfoxide reductase (BisC), dimethyl sulfoxide reductase (DorA), and trimethylamine-N-oxide reductase (TorA) (20, McEwan et al., Geomicrobiol., J., 19:3-21, 2002). The type II enzyme dimethyl sulfoxide reductase (DmsA) and the type II enzymes polysulfide and thiosulfate reductases (PsrA/PhsA) form unaffiliated lineages (McDevitt et al., Mol. Microbiol., 44:1575-1587, 2002, McEwan et al., Geomicrobiol., J., 19:3-21, 2002).
Our analysis indicated that PcrA forms its own monophyletic group in the type II DMSO enzymes and has a common ancestor with E. coli and Bacillus subtilis NarG, L dechloratans ClrA, Thauera selenatis SerA, Rhodovulum sulfidophilum DdhA, and Azoarcus sp. strain EBI EbdA. The alignment used for tree construction indicated that PcrA contains the type II DMSO signature motif [HX3CX2CX(n)C] for binding one 4Fe-4S center in domain I (data not shown) (Jormakka et al., Structure, 12:95-104, 2004). Based on the NarG analysis of Jormakka and coworkers, type II DMSO enzymes have also been shown to contain a conserved Asp residue for Mo ion binding (Jormakka et al., Structure, 12:95-104, 2004). This residue is present at position 212 in PcrA.
The tree topology also indicated that PcrA is more closely related to NarG from B. subtilis and E. coli than to ClrA from L dechloratans, further emphasizing the differences between the perchlorate and chlorate reductases. The distance between the perchlorate and chlorate reductases indicates that they are distinct enzymes, which was supported by our molecular probing of genomic DNAs from perchlorate and chlorate reducers, as well as from close relatives unable to reduce either electron acceptor (FIG. 7). The slot blot analysis resulted in hybridization signals for the pcrA gene from perchlorate reducers alone. No signal was observed for the close relatives or for Pseudomonas sp. strain PK, D. chlorophilus, or I. dechloratans, organisms that are capable of chlorate reduction but not perchlorate reduction (FIG. 7). This finding supports the hypothesis that two distinct metabolic pathways involved in the reduction of these analogous compounds evolved.
From the current study, it is clear that a more complete understanding of perchlorate reduction and other environmentally significant pathways is pivotal for obtaining knowledge applicable to the design of future bioremediation strategies. Perchlorate reductase and other members of the type II DMSO reductase family play a role in a broad range of substrate reductions and oxidations, and differences in various active sites are the major differences between family members. The different active sites indicate that there was a common reductase ancestor which acquired mutations advantageous for utilization of specific substrates. Thus, it is possible that directed mutagenesis of the active sites of DMSO enzymes could lead to creation of novel enzymes useful for biotechnological as well as bioremediation applications.
The discussion in the present example reveals the significance of using DPRB in environmental bioremediation techniques. The previous examples have provided detailed and exemplary methods for identifying the DPRB in accordance with the present invention. It should be understood that the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
The references cited herein throughout, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are all specifically incorporated herein by reference.










Sequences for sequence listing












SEQ
Genbank






ID
Accession
Sequence




NO:
No.
Type
Description
Sequence





1
N/A
DNA
DPR.F primer
GCNCCNCCNGTNGCNTTYATG



 


2
N/A
DNA
DPR.R primer
RTCRTTNACYTCNGCRTGDAT


 


3
N/A
DNA
DPR1.F primer
ACAGCGABBTGCTDDTGCG


 


4
N/A
DNA
DPR1.R primer
VGCCGAAGGVADAACCABV


 


5
N/A
DNA
UPR.F primer
GAYCCNGCNCTNGARGGNAAR


 


6
N/A
DNA
UPR.R primer
CCADATNGTNGTYTGRCA


 


7
N/A
DNA
UPR1.F primer
AGACCACBATCTGGACCTATGTC


 


8
N/A
DNA
UPR1.R primer
GGVADAACCABVTCVGAATAYA


 


9
N/A
DNA
primer
GCGCGGAATTC


 


10
N/A
DNA
primer
GCGCGCAAGCTT


 


11
N/A
DNA
primer
GC(I/N)CC(I/N)CC(I/N)GT(I/N)GC






(I/N)TTYATG


 


12
N/A
DNA
primer
RTCRTT(I/N)ACYTC(I/N)GCRTGDAT


 


13
N/A
DNA
primer
GAYCC(I/N)GC(I/N)CT(I/N)GARGG






(I/N)AAR


 


14
N/A
DNA
primer
CCADAT(I/N)GT(I/N)GTYTGRCA


 


15
N/A
DNA
primer
GARCGCAARRGNGCNGCNGMNGARGT


 


16
N/A
DNA
primer
TCRAARTANGTDATEAARTC


 


17
N/A
DNA
primer
TYGAVAARCAYAAGGAHAAVGT


 


18
N/A
DNA
primer
GAGTGGTAVARYTTVCGYTT


 


19
N/A
DNA
primer
GANCGNAANNGNGCNGCNGNNGANGT


 


20
N/A
DNA
primer
TCNAANTANGTNATNAANTC


 


21
N/A
DNA
primer
GARCGCAARRG(I/N)GCIGC(I/N)GM






(I/N)GARGT


 


22
N/A
DNA
primer
TCRAARTA(I/N)GTVATRAARTC


 


23
N/A
DNA
primer
GA(I/N)CG(I/N)AA(I/N)(I/N)G






(I/N)GC(I/N)GC(I/N)G(I/N)(I/N)






GA(I/N)GT


 


24
N/A
DNA
primer
TC(I/N)AA(I/N)TA(I/N)GT(I/N)AT






(I/N)AA(I/N)TC


 


25
N/A
DNA
primer
GTCGTTNACTTCNGCGTGAAT


 


26
N/A
DNA
primer
CGCGAAGGTAGTCAGCATCT


 


27
AY180108
DNA
D. agitata
atggcacgactgagtcgcagagattttttg





(pcrA)
aaggcatccgccgccactctgctgggtaat






tcacttaccttcaaaacattagcggccacc






atggatttatccggcgccttcgaatattcg






ggctgggaaaattttcatcgcaaccagtgg






tcgtgggacaagaaaactcgcggagcgcac






ctgatcaactgtactggggcttgcccgcac






ttcgtttatacgaaagatggtgtggtcatt






cgcgaggagcagtccaaggacattccgccg






atgccgaatatcccggaattgaatccccgg






gggtgcaacaagggggagtgcgcacaccac






tacatgtatggtccgcatcgcctgaaatat






cctttgatccgggttggcgaacgcggggag






ggcaagtggcggcgtgccacgtgggaggag






gcactagaccttatctcagacaagataatc






gacacgatcaagaatcactctcccgattgc






atcagtgtctactcacctcttccgggcaca






gcaccagtatcgttctctgctggacacagg






tttgcgcactatatcggcgcccatacacat






accttctttgactggtacagtgaccatccc






actggtcagacacagacgtgcggtgtccag






ggcgactcagcggagtgctccgactggttc






aactcaaaatacataatcctctggggggca






aaccccactcagactcgtattccagatgcg






catttcctgtcagaggcgcagttgaacggc






gcgaaggtagtcagcatctccccggatttc






aactcttctaccatcaaggcggaccgttgg






attcaccctctgcccggtactgatggcgcc






ctcgcactggccatggcgcatgtgatcatc






aaggaaaagctctacgacgcccacaacctc






aaggaacaaactgatctaccgtatttgatc






agaagggacaccaagcggttcttacgtgaa






gctgatgtggttgccgggggatccaaagat






aagttctatatttgggacagcaagactggc






aagccggtaatcacgaaaggttcctggggc






gaccaaccagaacaaaaagctccgcctgtc






gcatttatgggaaggaacacccatacattc






ccaaagggctatatcgccctggagaatctg






gaccccgccctggaaggcaaatttcaggtc






aagttgcaggatggaaatacagttgaagtc






agaccagtattcgaaattctgaaatcgcgc






atcgaggccgacaacaatatcgccaaggca






gcgaaaatcaccggggtgccggcaaagacc






atcattgaagtggccagagagtatgcaaca






acgcagccggcgatgatcatttgcggtggc






ggcactatgcactggtattacagcgacgtg






ctgttacgtgcgatgcatctgctaaccgca






ctcgttggcagcgaaggcaagaatggcggc






gggatgaatcattacatcggacagtggaag






cctgtcttcctgccgggtgtggctgctctc






gccttccccgaaggccctgcgaacgaacgg






tcctgccagacgacgatctggacttacatc






catgccgaagttaacgacgagatggcaaat






gtcgggatcgataccgacaagtatctgatg






cacgcgatcgatacgcgccaaatgccgaat






taccctcgagacggcagagatcccaaagta






tttatcgtctatcgcggcaactggctaaac






caagccaagggacagaaatatgtcttgcgc






aatctctggcccaagctggatttgattgtt






gacatcaatatccgcatggactcaacggcg






ctgtattcggatgttgtgctgccttccgct






cactggtacgaaaagctcgacctcaacgtc






accgccgagcacacctatatcaacatgacg






gaaccggcaatcaagccgatgtgggaatcc






aagaccgattggcagattttcctcgcactc






gccaaacgcgttgaaatgtcagccaagcgc






aagagttttgagaggttttacgacgaacaa






ttcaagtgggcacgcgacctgacgaacctg






tggaaccagatgaccatggacggcaagttg






gccgaggatgaggctgctgcccaatacatt






ctggacactgccccccattcgaaaggcatt






acgctccagatgctgagggaaaaaggtgag






cgcttcaaggcgaattggacttcgccgatg






aaggaaggcgtgccctataccccgttccag






aattacattgtcgacaagaagccttggccg






acactcacggggcgccagcagttctatctg






gaccatgaagtgttcttcgaaatgggtgtc






gaattgccgacctacaaggctccggtcgat






gcggacaaattccccttccgcttcaactcg






ccacacagtcgtcattcgatccactcgacc






ttcaaggatagtgtgctgatgctacggctt






cagcgcggcggtccctgcgtagaaatctcg






ccgatcgatgccacggcaattggagtcaag






gacaacgactgggtagaaatctggaatagc






cacggcaaggtgatctgccgggccaagatc






cgtgccggtgagcaacgtggccgcgtctcc






atgtggcacaccccggaactgtacatggat






ctcctcgaaggcagcacgcaaagcgtatgc






cccgtccgcatcacgccaacgcacttggtg






ggcaactacggacatttggtgtttcgcccg






aactactacggaccggccggcagccagcga






gacgttcgtgtcgatgtgaaacgctatatc






ggcgcaactccgatcagtctctag


 


28
AY180108
AA
D. agitata
MARLSRRDFLKASAATLLGNSLTFKTLAAT





(pcrA)
MDLSGAFEYSGWENFHRNQWSWDKKTRGAH






LINCTGACPHFVYTKDGVVIREEQSKDIPP






MPNIPELNPRGCNKGECAHHYMYGPHRLKY






PLIRVGERGEGKWRRATWEEALDLISDKII






DTIKNHSPDCISVYSPLPGTAPVSFSAGHR






FAHYIGAHTHTFFDWYSDHPTGQTQTCSVQ






GDSAECSDWFNSKYIILWGANPTQTRIPDA






HFLSEAQLNGAKVVSISPDFNSSTIKADRW






IHPLPGTDGALALAMAHVIIKEKLYDAHNL






KEQTDLPYLIRRDTKRFLREADVVAGGSKD






KFYIWDSKTGKPVITKGSWGDQPEQKAPPV






AFMGRNTHTFPKGYIALENLDPALEGKFQV






KLQDGNTVEVRPVFEILKSRIEADNNIAKA






AKITGVPAKTIIEVAREYATTQPAMIICGG






GTMHWYYSDVLLRAMHLLTALVGSEGKNGG






GMNHYIGQWKPVFLPSVAALAFPEGPANER






SCQTTIWTYIHAEVNDEMANVGIDTDKYLM






HAIDTRQMPNYPRDGRDPKVFIVYRGNWLN






QAKGQKYVLRNLWPKLDLIVDINIRMDSTA






LYSDVVLPSAHWYEKLDLNVTAEHTYINMT






EPAIKPMWESKTDWQIFLALAKRVEMSAKR






KSFERFYDEQFKWARDLTNLWNQMTMDGKL






AEDEAAAQYILDTAPHSKGITLQMLREKGE






RFKANWTSPMKEGVPYTPFQNYIVDKKPWP






TLTGRQQFYLDHEVFFEMGVELPTYKAPVD






ADKFPFRFNSPHSRHSIHSTFKDSVLMLRL






QRGGPCVEISPIDATAIGVKDNDWVEIWNS






HGKVICRAKIRAGEQRGRVSMWHTPELYMD






LLEGSTQSVCPVRITPTHLVGNYGHLVFRP






NYYGPAGSQRDVRVDVKRYIGATPISL


 


29
AY953269
DNA
D. agitata
atgtcaaatatgacgaagtcgcctaaacgc





(pcrB)
caattggcatatgttgccgacctgaataag






tgcattggctgccagacatgcactgtcgca






tgcaagactctttggactagtggacctgga






caggactacatgtattggcggaatgtcgaa






accgccccaggtttggggtatccaagaaac






tggcagagcaagggcggtggatacaaggat






ggagtcctgcagaaagggaagattccgccg






atgatcgattatggcgttcccttcgaattc






gactacgccggtcggcttttcgaagggaaa






aaggaaagagcgcgaccgagtccgacgcct






cggtatgctcccaattgggatgaggatcag






ggagcgggcgaatatcccaacaactccttt






ttctatgtgccgcggatgtgcaaccactgc






gccaagccggcatgtcttgaagcctgcccg






aatgaagcgatatacaagcgcgagcaagac






ggcctggtggtgatccatcaggagaagtgc






aaaggggctcaggcatgtatccagtcctgc






ccatatgccaaaccatactttaatgctcag






gtcaataaagccaacaagtgcatcggttgc






ttcccgcggattgaaaaaggggttgcaccg






gcatgcgttgctgagtgtgcgggaagagcc






atgcatgtcggcttcatcgatgaccaagaa






agctccgtgttcaagctggtcaagcggttc






ggcgtggcactgcctttgcaccccgagtac






ggcaccgaacccaacgtgttctatgtccct






cccgttctcggaccgcgcgtagaaatgccg






aatggcgaacataccgccgacccgaaaatt






tcgatgactcagcttgaacagttgtttggc






aagcaggtccgcgaggttttgaagacgctg






caggccgaacgcgagaaaaagatcaagaac






cagccgtctgaactgatggacatcctgatc






ggccgacgcagtgcggacatgatgatttcg






cctatgacctga


 


30
AY953269
AA
D. agitata
MSNMTKSPKRQLAYVADLNKCIGCQTCTVA





(pcrB)
CKTLWTSGPGQDYMYWRNVETAPGLGYPRN






WQSKGGGYKDGVLQKGKIPPMIDYGVPFEF






DYAGRLFEGKKERARPSPTPRYAPNWDEDQ






GAGEYPNNSFFYVPRMCNHCAKPACLEACP






NEAIYKREQDGLVVIHQEKCKGAQACIQSC






PYAKPYFNAQVNKANKCIGCFPRIEKGVAP






ACVAECAGRAMHVGFIDDQESSVFKLVKRF






GVALPLHPEYGTEPNVFYVPPVLGPRVEMP






NGEHTADPKISMTQLEQLFGKQVREVLKTL






QAEREKKIKNQPSELMDILIGRRSADMMIS






PMT


 


31
AY953270
DNA
D. agitata
atgaaaagaatcgttgctctgttaagcacg





(pcrC)
ttgctcgttaccgcgatgccgagcatgggc






gtagcccaacagaccgaatatttaggcttt






cgcgcatgcacaaagtgtcacgatgcccag






ggtgatacctggcggacatcggcacatgcc






aaagcctttgagt


 


32
AY953270
AA
D. agitata
MKRIVALLSTLLVTAMPSMGVAQQTEYLGF





(pcrC)
RACTKCHDAQGDTWRTSAHAKAFE


 


33
AY953271
DNA
D. agitata
atgaatagtattaccgacgatcgactcgtc





(pcrD)
ttggcggccgataaagcgttatgccgaagc






cacatttattcattgttggccagtggattt






ggatatcccgacgaatctggttatcaggga






ttttcggacggcaagtttattgatgaaatc






cgtcaggaactcggggtatgttggggtgaa






ctcttggagcattttaatgaggtacttgct






cctgggttgaaacttgcctgctcgcgcgaa






gtattcgaatcacagttcttgagtgcattc






gagaccaatatgccatcacctagcgcctcc






ttgaatgaaggtgttcacatatttaaatcg






gaccgaccaaatttattgctcgaactgaag






ggtttttacagcaacttcggcttgcaggtc






gatagcaaaggcaatgagcttgaagacact






ctgacagcagaacttgatttcatgcagttt






cttgcgttgaaacaggcacaggcccatctc






gaaggcatgtcggctgatgcctacaagttg






gcgcagaaggattttctggagcggcaccta






tgtgcttggttgccattggtgcggagggaa






atcgccgaaaaggtgactaccccattcttc






gtcactcttgctgagtttgcagaaagcttt






gcgcttgctaatttaagagaattgaaagaa






gagctaggagaataa


 


34
AY953271
AA
D. agitata
MNSITDDRLVLAADKALCRSHIYSLLASGF





(pcrD)
GYPDESGYQGFSDGKFIDEIRQELGVCWGE






LLEHFNEVLAPGLKLACSREVFESQFLSAF






ETNMPSPSASLNEGVHIFKSDRPNLLLELK






GFYSNFGLQVDSKGNELEDTLTAELDFMQF






LALKQAQAHLEGMSADAYKLAQKDFLERHL






CAWLPLVRREIAEKVTTPFFVTLAEFAESF






ALANLRELKEELGE


 


35
Q9S1G9
AA
T. selenatis
MSQRQLAYVFDLNKCIGCHTCTMACKQLWT





(SerB)
NRDGREYMYWNNVESRPGKGYPKNWEQKGG






GFDKDGKLKTNGIIPIRADYGGTWNYNLLE






TLVEGKSNQVVPDEKPTWGPNWDEDEGKGE






FPNNHYFYLPRICNHCSNPACLAACPTKAI






YKREEDGLVVVDQSRCKGYRYCVKACPYGK






MYFNLQKGTSEKCIGCYPRVEKGEAPACVK






QCSGRIRFWGYRDDKDGPIYKLVDQWKVAL






PLHAEYGTEPNVFYVPPMNTTPPPFEEDGR






LGDKPRIPIEDLEALFGPGVKQALATLGGE






MAKRRKAQASELTDILIGYTNKDRYGI


 


36
P60069
AA
I. dechloratans
MSQRQVAYVFDLNKCIGCHTCTMACKQLWT





(ClrB)
NRDGREYMYWNNVETRPGKGYPKNWEGKGG






GFDQEGKLKTNGIIPIMADYGGRIGDFNLN






EVLLEGKADQVVPHEKATWGPNWDEDEGKG






EFPNNHSFYLPRICNHCSNPACLAACPTKA






IYKRPEDGIVVVDQTRCRGYRYCVKACPYG






KMYFNLQKGKSEKCIGCYPRVEKGEAPACV






KQCSGRIRFWGYRDDKNGPIYKLVEQWKVA






LPLHAEYGTEPNVFYVPPMNTTPPPFEEDG






RLGDKPRIPIEDLEALFGPGVKQALATLGG






EMAKRRKAQASELTDILIGFTNKDRYGV


 


37
CAD58340
AA
Azoarcus sp.
MTYVQDGNKSELRKAKRQLVTVIDLNKCLG





EbN1 (EbdB)
CQTCTVACKNIWTKRPGTEHMRWNNVTTYP






GKGYPRDYERKGGGFLRGEPQPGVLPTLID






SGDDFQFNHKEVFYEGKGQTVHFHPTSKST






GKDPAWGYNWDEDQSSSKWPNPFFFYLARM






CNHCTNPACLAACPTGAIYKREDNGIVLVD






QERCKGHRHCVEACPYKAIYFNPVSQTSEK






CILCYPRIEKGIANACNRQCPGRVRAFGYL






DDTTSHVHKLVKKWKVALPLHAEYGTGPNI






YYVPPMGARGFGEDGEITDKTRIPLDVLES






LFGPEVKRVLAVLHTERENMRAGRGSELMD






LLISKKWSDRFGGFTNDPLTQS


 


38
AAN46633
AA
R.
MVKRQISMVLDLNKCIGCQTCTSACKLQWT





sulfidophilum
NRNGREYMYWNNVETHPGPGYPRNYEHSGG





(DdhB)
GFDEEGALKIGITPSAEDYGIPWEYNYEEA






LMTGTDPWLRPNVKPTWGANWNEDEGRGEY






PNSYYFYLPRICNHCANPGCLAACARNAIY






KRQEDGIVLVDQERCRGYRYCITACPYKKV






YFNEQISKAEKCIFCYPRIEKGLPTACAKQ






CVGRIRFIGYLDDEASPVHLLVERYKVAIP






LHPEWGTKPSVFYVPPLAPPRIGDDGEPTE






ETRVPLAYLKELFGEAVVPALETLKTERAK






KQSGAESELMDTLIGYRHPEMFKLS


 


39
CAD22070
AA
H. marismortui
MSSDQQDDQGEEDTLVNVADGVDHQVAMVM





(NarH)
DLNKCIGCQTCTIACKNLWTEDGGSEYMYW






NNVETKPGEGYPRGWENSGGGWKSGEHKER






QPGEIPDEEDYGRAWEFNHEEIMYEGSDEP






LRPRDGAEWGPNWDEDQSASEYPNSYYFYL






PRICNHCTHPSCVEACPRSALYKRQEDGIV






LVDQDRCRGYRYCVEGCPYKKVYYNTVSKK






SEKCIFCYPRIEGEGPDSETFAPACAEECP






PQLRLVGFLDDEEGPIHKLVNEYEVALPLH






PEFRTQPNVYYIPPFAPGQHTEDGETVDID






RIPRQYLRDLFGDGVDQALDTIERERQRAR






QGEDSELMELLQDKNPAKQYRLEVFDDE


 


40
NP_842334
AA
N. europaea
MKIMIACGLVAAALFTLTSGQSLAADAPFE





(cytochrome
GRKKCSSCHKAQAQSWKDTAHAKAMESLKP





c554)
NVKKEAKQKAKLDPAKDYTQDKDCVGCHVD






GFSQKGGYTIESPKPMLTGVGCESCHGPGR






NFRGDHRKSGQAFEKSGKKTPRKDLAKKGQ






DFHFEERCSACHLNYEGSPWKGAKAPYTPF






TPEVDAKYTFKFDEMVKEVKAMHEHYKLEG






VFEGEPKFKFHDEFQASAKPAKKGK


 


41
AF047462
DNA
D. Aginata
tgaacgctggcggcatgccttacacatgca






agtcgaacggcagcacggacttcggtctgg






tggcgagtggcgaacgggtgagtaatgtat






cggaacgtacctttcagtgggggataacgt






agcgaaagttacgctaataccgcatattct






gtgagcaggaaagcaggggatcgcaagacc






ttgcgctgattgagcggccgatatcagatt






agctagttggtggggtaaaggcctaccaag






gctacgatctgtagcgggtctgagaggatg






atccgccacactggaactgagacacggtcc






agactcctacgggaggcagcagtggggaat






tttggacaatgggggcaaccctgatccagc






catgccgcgtgagtgaagaaggccttcggg






ttgtaaagctctttcggccgggaagaaatc






gcatcagctaatacctggtgtggatgacgg






tacccgaataagaagcaccggctaactacg






tgccagcagccgcggtaatacgtagggtgc






gagcgttaatcggaattactgggcgtaaag






cgtgcgcaggcggttgtgtaagacaggcgt






gaaatccccgggctcaacctgggaactgcg






cttgtgactgcacagctagagtacggcaga






ggggggtggaattccacgtgtagcagtgaa






atgcgtagagatgtggaggaacaccgatgg






cgaaggcagccccctgggccgatactgacg






ctcatgcacgaaagcgtgggtagcaaacag






gattagataccctggtagtccacgccctaa






acgatgtcaactaggtgttgggtgggtaaa






accacctagtaccgtagctaacgcgtgaag






ttgaccgcctggggagtacggccgcaaggt






taaaactcaaaggaattgacggggacccgc






acaagcggtggatgatgtggattaattcga






tgcaacgcgaaaaaccttacctacccttga






catgtccagaagcccggagagatttgggtg






tgcccgaaagggaactggaacacaggtgct






gcatggctgtcgtcagctcgtgtcgtgaga






tgttgggttaagtcccgcaacgagcgcaac






ccttgtcgttaattgccatcatttagttgg






gcactttaacgagactgccggtgacaaacc






ggaggaaggtggggatgacgtcaagtcctc






atggcccttatgggtagggcttcacacgtc






atacaatggtcggtacagagggttgccaag






ccgcgaggtggagccaatcccagaaagccg






atcgtagtccggattgcaggctgcaactcg






cctgcatgaagtcggaatcgctagtaatcg






cggatcagcatgtcgcggtgaatacgttcc






cgggtcttgtacacaccgcccgtcacacca






tgggagcgggttccgccagaagatggatgc






ctaaccgcaaggagggcgcttaccacggcg






gggttcgtgactggggtgaagtcgtaacaa






ggtagccgtaggggaacctgcggctggatc






acctcc


 


42
D16209
DNA
R. tenuis
attgaacgctggcggcatgccttacacatg






caagtcgaacggcagcacgggagcaatcct






ggtggcgagtggcgaacgggtgagtaatgc






atcggaacgtgccctgaagtgggggataac






gtagcgaaagttacgctaataccgcatatt






ctgtgagcaggaaagcaggggatcgcaaga






ccttgcgctttgggagcggccgatgtcgga






ttagctagttggtggggtaaaggcctacca






aggccacgatccgtagcgggtctgagagga






tgatccgccacactgggactgagacacggc






ccagactcctacgggaggcagcagtgggga






attttggacaatgggcgaaagcctgatcca






gccatgccgcgtgagtgaagaaggccttcg






ggttgtaaagctctttcggcggggaagaaa






ttgctcaggataataccctgagtagatgac






ggtacccgaagaagaagcaccggctaacta






cgtgccagcagccgcggtaatacgtagggt






gcgagcgttaatcggaattactgggcgtaa






agcgtgcgcaggcggttgtgtaagacagac






gtgaaatccccgggctcaacctgggaactg






cgtttgtgactgcacgactagagtgtggca






gaggggggtggaattccacgtgtagcagtg






aaatgcgtagagatgtggaggaacaccgat






ggcgaaggcagccccctgggccaatactga






cgctcatgcacgaaagcgtggggagcaaac






aggattagataccctggtagtccacgccct






aaacgatgtcaactaggtgttggtggggtt






aaacccattagtgccgtagctaacgcgtga






agttgaccgcctggggagtacggccgcaag






gttaaaactcaaaggaattgacggggaccc






gcacaagcggtggatgatgtggattaattc






gatgcaacgcgaaaaaccttacctaccctt






gacatgtcaggaatcctgaagagattcggg






agtgcccgaaagggagcctgaacacaggtg






ctgcatggctgtcgtcagctcgtgncgtga






gatgttgggttaagtcccgcaacgagcgca






acccttgtcattaattgccatcatttagtt






gggcactctaatgaaactgccggtgacaaa






ccggaggaaggtggggatgacgtcaagtcc






tcatggcccttatgggtagggcttcacacg






tcatacaatggtcggtacagagggttgcca






agccgcgaggtggagccaatcacagaaagc






cgatcgtagtccggattgcagtctgcaact






cgactgcatgaagtcggaatcgctngtaat






cgcggatcagcatgtcgcggtgaatacgtt






cccgggtcttgtacacaccgcccgtcacac






catgggagcgggttctgccagaagtagtta






gcctaaccgcaaggagggcgattaccacgg






cagcgttcgtgactggggtg


 


43
AY032610
DNA
D. aromatica
gagtttgatcctggctcagattgaacgctg






gcggcatgccttacacatgcaagtcgaacg






gcagcacgggagcaatcctggtggcgagtg






gcgaacgggtgagtaatatatcggaacgta






cctttcagtgggggataacgtagcgaaagt






tacgctaataccgcatattctgtgagcagg






aaagcaggggatcgcaagaccttgcgctga






ttgagcggccgatatcagattagctagttg






gtgaggtaaaggctcaccaaggcgacgatc






tgtagcgggtctgagaggatgatccgccac






actggaactgagacacggtccagactccta






cgggaggcagcagtggggaattttggacaa






tgggggcaaccctgatccagccatgccgcg






tgagtgaagaaggccttcgggttgtaaagc






tctttcagccgggaagaaaacgcatgggtt






aataccctgtgtggatgacggtaccggaat






aagaagcaccggctaactacgtgccagcag






ccgcggtaatacgtagggtgcgagcgttaa






tcggaattactgggcgtaaagcgtgcgcag






gcggtttgttaagataggcgtgaaatcccc






gggctcaacctgggaactgcgtttatgact






ggcaggctagagtatggcagaggggggtgg






aattccacgtgtagcagtgaaatgcgtaga






gatgtggaggaacaccgatggcgaaggcag






ccccctgggccaatactgacgctcatgcac






gaaagcgtgggtagcaaacaggattagata






ccctggtagtccacgccctaaacgatgtca






actaggtgttgggagggtaaaaccttttag






taccggagctaacgcgtgaagttgaccgcc






tggggagtacggccgcaaggttaaaactca






aaggaattgacggggacccgcacaagcggt






ggatgatgtggattaattcgatgcaacgcg






aaaaaccttacctacccttgacatgtccgg






aagcccttagagatttgggtgtgctcgaaa






gagagccggaacacaggtgctgcatggctg






tcgtcagctcgtgtcgtgagatgttgggtt






aagtcccgcaacgagcgcaacccttgtcgt






taattgccatcatttagttgggcactttaa






cgagactgccggtgacaaaccggaggaagg






tggggatgacgtcaagtcctcatggccctt






atgggtagggcttcacacgtcatacaatgg






tcggtacaaagggttgccaacccgcgaggg






ggagctaatcccagaaagccgatcgtagtc






cggatcgtaggctgcaactcgcctgcgtga






agtcggaatcgctagtaatcgtggatcagc






atgtcacggtgaatacgttcccgggtcttg






tacacaccgcccgtcacacaatgggagcgg






gttccgccagaagtaggtagcctaaccgca






aggggggcgcttaccacggcggggttcgtg






actggggtgaagtcgtaacaaggtagccgt






aggggaacctgcggctggatcacctcct


 


44
AY032611
DNA
Dechloromonas
tttgatcctggctcagattgaacgctggcg





sp. Strain JJ
gcatgccttacacatgcaagtcgaacggca






gcacgggagcaatcctggtggcgagtggcg






aacgggtgagtaatgtatcggaacgtacct






ttcagtgggggataacgtagcgaaagttac






gctaataccgcatattctgtgagcaggaaa






gcaggggatcgcaagaccttgcgctgattg






agcggccgatatcagattagctagttggtg






aggtaaaggctcaccaaggcgacgatctgt






agcgggtctgagaggatgatccgccacact






ggaactgagacacggtccagactcctacgg






gaggcagcagtggggaattttggacaatgg






gggcaaccctgatccagccatgccgcgtga






gtgaagaaggccttcgggttgtaaagctct






ttcggccgggaagaaatcgcatgggttaat






accctgtgtggatgacggtaccggaataag






aagcaccggctaactacgtgccagcagccg






cggtaatacgtagggtgcgagcgttaatcg






gaattactgggcgtaaagcgtgcgcaggcg






gtttagtaagacaggcgtgaaatccccggg






ctcaacctgggaactgcgcttgtgactgct






aagctagagtacggcagaggggggtggaat






tccacgtgtagcagtgaaatgcgtagagat






gtggaggaacaccgatggcgaaggcagccc






cctgggccgatactgacgctcatgcacgaa






agcgtgggtagcaaacaggattagataccc






tggtagtccacgccctaaacgatgtcaact






aggtgttgggtgggtaaaaccatttagtac






cggagctaacgcgtgaagttgaccgcctgg






ggagtacggccgcaaggttaaaactcaaag






gaattgacggggacccgcacaagcggtgga






tgatgtggattaattcgatgcaacgcgaaa






aaccttacctacccttgacatgtccagaag






cccgaagagatttgggtgtgcccgaaaggg






agctggaacacaggtgctgcatggctgtcg






tcagctcgtgtcgtgagatgttgggttaag






tcccgcaacgagcgcaacccttgtcgttaa






ttgccatcatttagttgggcactttaacga






gactgccggtgacaaaccggaggaaggtgg






ggatgacgtcaagtcctcatggcccttatg






ggtagggcttcacacgtcatacaatggtcg






gtacagagggttgccaagccgcgaggtgga






gccaatcccagatagccgatcgtagtccgg






atcgtaggctgcaactcgcctgcgtgaagt






cggaatcgctagtaatcgcggatcagcatg






tcgcggtgaatacgttcccgggtcttgtac






acaccgcccgtcacaccatgggagcgggtt






ccgccagaagtaggtagcctaaccgcaagg






ggggcgcttaccacggcggggttcgtgact






ggggtgaagtcgtaacaaggtagccgtagg






ggaacctgcggctggatcacctccc


 


45
AF170352
DNA
D. anomolous
tttgagcctggctcagaacgaacgctggcg





strain WD
gcaggcctaacacatgcaagtcgaacgaag






tcttcggacttagtggcgcacgggtgagta






acacgtgggaatataccttacgctggggga






taacatcgggaaactgatgctaataccgca






tacgcccttcgggggaaagatttatcggcg






aaagattagcccgcgtccgattagctagtt






ggtgaggtaatggctcaccaaggctccgat






cggtagctggtctgagaggatgaccagcca






cactgggactgagacacggcccagactcct






acgggaggcagcagtggggaatattggaca






atgggcgcaagcctgatccagccatgccgc






gtgagtgatgaaggccttagggttgtaaag






ctctttcacccacgacgatgatgacggtag






tgggagaagaagccccggctaacttcgtgc






cagcagccgcggtaatacgaagggggctag






cgttgttcggaattactgggcgtaaagcgc






acgcaggcggttcggtcagtcagaagtgaa






agccccgggcttaacctgggaactgctttt






gatactgccgagcttgaatcacggagaggg






tagtggaattccgagtgtagaggtgaaatt






cgtagatattcggaagaacaccagtggcga






aggcgactacctggccgtcgattgacgctc






atgtgcgaaagcgtggggagcaaacaggat






tagataccctggtagtccacgccgtaaacg






atgagtgctagttgttggggtgcatgcacc






tcagtgacgcagctaacgcgttaagcactc






cgcctggggagtacggccgcaaggttaaaa






ctcaaaggaattgacgggggcccgcacaag






cggtggagcatgtggtttaattcgaagcaa






cgcgcagaaccttaccagcccttgacatgg






gaactatgggtccgagagattggatccttc






acttcgggtgggttccacacaggtgctgca






tggctgtcgtcagctcgtgtcgtgagatgt






tgggttaagtcccgcaacgagcgcaaccct






catcgtcagttgccatcattcagttgggca






ctctgacgaaactgccggtgacaagccgga






ggaaggtggggatgacgtcaagtcctcatg






gcccttacgggctgggctacacacgtgcta






caatggtggtgacaatgggtcgcgagctcg






cgagagttagctaatccccaaaagccatct






cagttcggattgtactctgcaactcgagtg






catgaagtcggaatcgctagtaatcgtgga






tcagcatgccacggtgaatacgttcccggg






ccttgtacacaccgcccgtcacaccatggg






agttggctttacccgaagccggtgcgctaa






ccgcaaggaggcagccgaccacggtaaggt






cagcgactggggtgaagtcgtaacaaggta






gccgtaggggaacctgcggccggatcacct






cc


 


46
Y10110
DNA
M.
aacgaacgctggcggcaggcttaacacatg





magnetotacticum
caagtcgaacgaagtcttcggacttagtgg






cgcacgggtgagtaacacgtgggaatatac






ctcttggtggggaataacgtcgggaaactg






acgctaataccgcatacgcccttcggggga






aagatttatcgccgagagattagcccgcgt






ccgattagctagttggtgaggtaatggctc






accaaggcgacgatcggtagctggtctgag






aggatgatcagccacactgggactgagaca






cggcccagactcctacgggaggcagcagtg






gggaatattggacaatgggcgaaagcctga






tccagccatgccgcgtgagtgatgaaggcc






ttagggttgtaaagctctttcacccacgac






gatgatgacggtagtgggagaagaagcccc






ggctaacttcgtgccagcagccgcggtaat






acgaagggggctagcgttgttcggaattac






tgggcgtaaagcgcacgcaggcggtggtca






tagtcagaagtgaaagccctgggctcaacc






cgggaattgcttttgatactggaccgctag






aatcacggagagggtagtggaattccgagt






gtagaggtgaaattcgtagatattcggaag






aacaccagtggcgaaggcgactacctggcc






gtcgattgacgctcatgtgcgaaagcgtgg






ggagcaaacaggattagataccctggtagt






ccacgccgtaaacgatgagtgctagttgtt






ggggtgcatgcacctcagtgacgcagctaa






cgcgttaagcactccgcctggggagtacgg






ccgcaaggttaaaactcaaaggaattgacg






ggggcccgcacaagcggtggagcatgtggt






ttaattcgaagcaacgcgcagaaccttacc






agcccttgacatgggacgtatgtttgccag






agatggtgacttgtcttcggacgcgtccac






acaggtgctgcatggctgtcgtcagctcgt






gtcgtgagatgttgggttaagtcccgcaac






gagcgcaaccctcatcttcagttgccatca






tttagttgggcactctgaagaaactgccgg






tgacaagccggaggaaggtggggatgacgt






caagtcctcatggcccttacgggctgggct






acacacgtgctacaatggtggtgacagtgg






gtcgctaactcgcgagagtatgctaatccc






taaaagccatctcagttcggattgcactct






gcaactcgagtgcatgaagtcggaatcgct






agtaatcgtggatcagcatgccacggtgaa






tacgttcccgggccttgtacacaccgcccg






tcacaccatgggagttggctttacccgaag






ccggtgcgctaaccgcaaggaggcagccga






ccacggtaaggtcagcgactggggtgaagt






cgtaacaaggtagccgtaggggaacctgcg






gct


 


47
AF170358
DNA
Pseudomonas sp.
atgaagagggcttgctctctgattcagcgg





strain PK
cggacgggtgagtaatgcctaggaatctgc






ctgatagtgggggacaacgtttcgaaagga






acgctaataccgcatacgtcctacgggaga






aagcaggggaccttcgggccttgcgctatc






agatgagcctaggtcggattagctagttgg






tgaggtaacggctcaccaaggcgacgatcc






gtaactggtctgagaggatgatcagtcaca






ctggaactgagacacggtccagactcctac






gggaggcagcagtggggaatattggacaat






gggcgaaagcctgatccagccatgccgcgt






gtgtgaagaaggtcttcggattgtaaagca






ctttaagttgggaggaagggcattaaccta






atacgttagtgttttgacgttaccgacaga






ataagcaccggctaacttcgtgccagcagc






cgcggtaatacgaagggtgcaagcgttaat






cggaattactgggcgtaaagcgcgcgtagg






tggtttgttaagttgaatgtgaaagccccg






ggctcaacctgggaactgcatccaaaactg






gcaagctagagtatggcagagggtggtgga






atttcctgtgtagcggtgaaatgcgtagat






ataggaaggaacaccagtggcgaaggcgac






cacctgggctaatactgacactgaggtgcg






aaagcgtggggagcaaacaggattagatac






cctggtagtccacgccgtaaacgatgtcga






ctagccgttgggatccttgagatcttagtg






gcgcagctaacgcattaagtcgaccgcctg






gggagtacggccgctaggttaaaactctaa






tgaattgacgggggcccgcacaagcggtgg






agcatgtggtttaattcgaagcaacgcgaa






gaaccttaccaggccttgacatgcagagaa






ctttccagagatggattggtgccttcggga






actctgacacaggtgctgcatggctgtcgt






cagctcgtgtcgtgagatgttgggttaagt






cccgtaacgagcgcaacccttgtccttagt






taccagcacgttaaggtgggcactctaagg






agactgccggtgacaaaccggaggaaggtg






gggatgacgtcaagtcatcatggcccttac






ggcctgggctacacacgtgctacaatggtc






ggtacaaagggttgccaagccgcgaggtgg






agctaatcccataaaaccgatcgtagtccg






gatcgcagtctgcaactcgactgcgtgaag






tcggaatcgctagtaatcgtgaatcagaat






gtcacggtgaatacgttcccgggccttgta






cacaccgcccgtcacaccatgggagtgggt






tgctccagaagtagctagtctaaccttcgg






ggggacggttaccacggaggtattcatgac






tggggtgaagtcgtaacaaggtagccgtag






gggaacctgcggctggatcacctcctt


 


48

DNA
P. stutzeri
gaacgctggcggcaggcctaacacatgcaa






gtcgagcggatgaagagagcttgctctctg






attcagcggcggacgggtgagtaatgccta






ggaatctgcctgatagtgggggacaacgtt






tcgaaaggaacgctaataccgcatacgtcc






tacgggagaaagcaggggaccttcgggcct






tgcgctatcagatgagcctaggtcggatta






gctagttggtgaggtaacggctcaccaagg






cgacgatccgtaactggtctgagaggatga






tcagtcacactggaactgagacacggtcca






gactcctacgggaggcagcagtggggaata






ttggacaatgggcgaaagcctgatccagcc






atgccgcgtgtgtgaagaaggtcttcggat






tgtaaagcactttaagttgggaggaagggc






attaacctaatacgttagtgttttgacgtt






accgacagaataagcaccggctaacttcgt






gccagcagccgcggtaatacgaagggtgca






agcgttaatcggaattactgggcgtaaagc






gcgcgtaggtggtttgttaagttgaatgtg






aaagccccgggctcaacctgggaactgcat






ccaaaactggcaagctagagtatggcagag






ggtggtggaatttcctgtgtagcggtgaaa






tgcgtagatataggaaggaacaccagtggc






gaaggcgaccacctgggctaatactgacac






tgaggtgcgaaagcgtggggagcaaacagg






attagataccctggtagtccacgccgtaaa






cgatgtcgactagccgttgggatccttgag






atcttagtggcgcagctaacgcattaagtc






gaccgcctggggagtacggccgcaaggtta






aaactcaaatgaattgacgggggcccgcac






aagcggtggagcatgtggtttaattcgaag






caacgcgaagaaccttaccaggccttgaca






tgcagagaactttccagagatggattggtg






ccttcgggaactctgacacaggtgctgcat






ggctgtcgtcagctcgtgtcgtgagatgtt






gggttaagtcccgtaacgagcgcaaccctt






gtccttagttaccagcacgttaaggtgggc






actctaaggagactgccggtgacaaaccgg






aggaaggtggggatgacgtcaagtcatcat






ggcccttacggcctgggctacacacgtgct






acaatggtcggtacaaagggttgccaagcc






gcgaggtggagctaatcccataaaaccgat






cgtagtccggatcgcagtctgcaactcgac






tgcgtgaagtcggaatcgctagtaatcgtg






aatcagaatgtcacggtgaatacgttcccg






ggccttgtacacaccgcccgtcacaccatg






ggagtgggttgctccagaagtagctagtct






aaccttcggggggacggttaccacggagtg






attcatgactggggtg


 


49
AF170359
DNA
Dechloromarinus
gagtttgatcctggctcagattgaacgctg





chlorophilus
gcggtatgcttaacacatgcaagtcgaacg





strain NSS
gcagcacgagagagcttgctctcttggtgg






cgagtggcggacgggtgagtaacgcgtaag






aatctgcctggtagtgggggataactcggg






gaaactcgagctaataccgcatacgcccta






cgggggaaagtgggggaccttcgggcctca






cgctattagatgagcttgcgttggattagc






tagttggtagggtaatggcctaccaaggcg






acgatccatagctggtctgagaggacgatc






agccacactgggactgagacacggcccaga






ctcctacgggaggcagcagtggggaatatt






ggacaatgggcgcaagcctgatccagcaat






accgcgtgtgtgaagaaggcctgcgggttg






taaagcactttcaattgtgaagaaaagctt






ggggctaatatcctcgagtcttgacgttaa






ctttagaagaagcaccggctaactctgtgc






cagcagccgcggtaatacagagggtgcgag






cgttaatcggaattactgggcgtaaagcgt






gcgtaggcggtttagtaagtcagatgtgaa






agccctgggcttaacctgggaactgcattt






gaaactgctgaactagagtgtggtagaggg






aagtggaattccgggtgtagcggtgaaatg






cgtagatatccggaggaacaccagtggcga






aggcgacttcctggaccaacactgacgctg






aggtacgaaagcgtgggtagcaaacaggat






tagataccctggtagtccacgccgtaaacg






atgtcaactagttgttgggagcattttggc






ttttagtaacgtagctaacgcgataagttg






accgcctggggagtacggccgcaaggttaa






aactcaaaggaattgacgggggcccgcaca






agcggtggagcatgtggtttaattcgatgc






aacgcgaagaaccttaccagcccttgacat






cctgcgaactttctagagatagattggtgc






tttcgggaacgcagtgacaggtgctgcatg






gctgtcgtcagctcgtgtcgtgagatgttg






ggttaagtcccgtaacgagcgcaacccttg






tccttagttgccagcatttcgggatgggaa






ctctagggagactgccggtgataaaccgga






ggaaggtggggatgacgtcaagtcatcatg






gcccttatgggctgggctacacacgtgcta






caatggccggtacagagggccgcaaacccg






cgagggggagcaaatctcacaaaaccggtc






gtagtccggatcgcagtctgcaactcgact






gcgtgaagttggaatcgctagtaatcgcga






atcagaatgtcgcggtgaatacgttcccgg






gccttgtacacaccgcccgtcacaccatgg






gagtgggttgcaaaagaagtaggtagtcta






accttcgggaggacgcttaccactttgtga






ttcatgactggggtgaagtcgtaacaaggt






a


 


50
AF170348
DNA
A. suillum
agagtttgatcctggctcagattgaacgct






ggcggcatgccttacacatgcaagtcgaac






ggcagcacgggagcttgctcctggtggcga






gtggcgaacgggtgagtaatacatcggaac






gtacccaggagtgggggataacgtagcgaa






agttacgctaataccgcatattctgtgagc






aggaaagcgggggatcgcaagacctcgcgc






tcttggagcggccgatgtcggattagctag






ttggtgaggtaaaagctcaccaaggcgacg






atccgtagcaggtctgagaggatgatctgc






cacactgggactgagacacggcccagactc






ctacgggaggcagcagtggggaattttgga






caatgggggcaaccctgatccagccatgcc






gcgtgagtgaagaaggccttcgggttgtaa






agctctttcggcggggaagaaatggcaacg






gctaatatccgttgttgatgacggtacccg






cataagaagcaccggctaactacgtgccag






cagccgcggtaatacgtagggtgcgagcgt






taatcggaattactgggcgtaaagcgtgcg






caggcggtttcgtaagacagacgtgaaatc






cccgggctcaacctgggaactgcgtttgtg






actgcgaggctagagtacggcagagggggg






tagaattccacgtgtagcagtgaaatgcgt






agagatgtggaggaataccgatggcgaagg






cagccccctgggttagtactgacgctcatg






cacgaaagcgtggggagcaaacaggattag






ataccctcgtagtccacgccctaaacgatg






tcaactaggtgttggaagggttaaaccttt






tagtaccgcagctaacgcgtgaagttgacc






gcctggggagtacggccgcaaggttaaaac






tcaaaggaattgacggggacccgcacaagc






ggtggatgatgtggattaattcgatgcaac






gcgaaaaaccttacctacccttgacatgcc






aggaactttccagagatggattggtgcccg






aaagggagcctggacacaggtgctgcatgg






ctgtcgtcagctcgtgtcgtgagatgttgg






gttaagtcccgcaacgagcgcaacccttgt






cattaattgccatcattcagttgggcactt






taatgagactgccggtgacaaaccggagga






aggtggggatgacgtcaagtcctcatggcc






cttatgggtagggcttcacacgtcatacaa






tggtcggtacagagggttgccaagccgcga






ggtggagccaatcccagaaagccgatcgta






gtccggatcgcagtctgcaactcgactgcg






tgaagtcggaatcgctagtaatcgcggatc






agcatgtcgcggtgaatacgttcccgggtc






ttgtacacaccgcccgtcacaccatgggag






tgggttctaccagaagtagttagcctaacc






gtaaggagggcgattaccacggtaggattc






atgactggggtgaagtcgtaacaaggtagc






cgtatcggaaggtgcggctggatcacctcc






tt


 


51
AY124797
DNA
Dechloromonas
gtttgatcctcgctcagattgaacgctggc





sp. strain LT-1
ggcatgccttacacatgcaagtcgaacggc






agcacggacttcggtctggtggcgagtggc






gaacgggtgagtaatgcatcggaacgtacc






cggaagtgggggataactatccgaaaggat






ggctaataccgcatattctgtacgcaggaa






agagggggatcttcggacctcttgctttcg






gagcggccgatgtcagattagctagttggt






ggggtaaaggcctaccaaggcgacgatctg






tagcgggtctgagaggatgatccgccacac






tggaactgagacacggtccagactcctacg






ggaggcagcagtggggaattttggacaatg






ggcgcaagcctgatccagccatgccgcgtg






agtgaagaaggccttcgggttgtaaagctc






tttcggccgggaaaaaatcggtgaggctaa






tatcctcatcggatgatggtaccggactaa






gaagcaccggctaactacgtgccagcagcc






gcggtaatacgtagggtgcgagcgttaatc






ggaattactgggcgtaaagcgtgcgcaggc






ggttttttaagacaggcgtgaaatccccgg






gcttaacctgggaactgcgtttgtgactgg






aaggctagagtacggcagaggggggtggaa






ttccacgtgtagcagtgaaatgcgtagaga






tgtggaggaacaccaatggcgaaggcagcc






ccctgggtcgatactgacgctcatgcacga






aagcgtggggagcaaacaggattagatacc






ctggtagtccacgccctaaacgatgtcaac






taggtgttgggtgggtaaaaccatttagta






ccggagctaacgcgtgaagttgaccgcctg






gggagtacggccgcaaggttaaaactcaaa






ggaattgacggggacccgcacaagcggtgg






atgatgtggattaattcgatgcaacgcgaa






aaaccttacctacccttgacatgccaggaa






cttgccagagatggcttggtgcccgaaagg






gagcctggacacaggtgctgcatggctgtc






gtcagctcgtgtcgtgagatgttgggttaa






gtcccgcaacgagcgcaacccttgtcgtta






attgccatcattaagttgggcactttaacg






agactgccggtgacaaaccggaggaaggtg






gggatgacgtcaagtcctcatggcccttat






gggtagggcttcacacgtcatacaatggtc






ggtacagagggttgccaagccgcgaggtgg






agccaatcccagaaagccgatcgtagtccg






gatcgtaggctgcaactcgcctgcgtgaag






tcggaatcgctagtaatcgcggatcagcat






gtcgcggtgaatacgttcccgggtcttgta






cacaccgcccgtcacaccatgggagcgggt






tccgccagaagtaggtagcctaaccgcaag






gggggcgcttaccacggcggggttcgtgac






tggggtgaagtcgtaacaaggtagccgtag






gggaacctgcggctggatcacct


 


52
X72724
DNA
I. dechloratans
attgaacgctggcggcatgccttacacatg






caagtcgaacggtaagcgggcaacctggcg






acgagtggcgaacgggtgagtaatgcatcg






gaacgtgcccagtagtgggggatagcccgg






cgaaagccggattaataccgcatacgacct






gagggtgaaagggggggatcgcaagacctc






tcgctattggagcggccgatgtcagattag






gtagttggtggggtaaaggcctaccaagcc






gacgatctgtagctggtctgagaggacgac






cagccacactgggactgagacacgccccag






actcctacgggaggcagcagtggggaattt






tggacaatgggcgcaagcytgatccagcca






tgccgcgtgcggaagaaggccttcgggttg






taaaccgcttttgtcagggaagaaatcttc






tgggctaatacctcgggaggatgacggtac






ctgaagaataagcaccggctaactacgtgc






cagcagccgcggtaatacgtagggtgcaag






cgttaatcggaattactgggcgtaaagcgt






gcgcaggcggttttgtaagacagaggtgaa






atccccgggctcaacctgggaactgccttt






gtgactgcaaggcttgagtgcgcagagggg






gatggaattccgcgtgtagcagtgaaatgc






gtagatatgcggaggaacaccgatggcgaa






ggcaatcccctgggcctgcactgacgctca






tgcacgaaagcgtggggagcaaacaggatt






agataccctggtagtccacgccctaaacga






tgtcaactggttgttgggaaggttccttct






cagtaacgtagctaacgcgtgaagttgacc






gcctggggagtacggccgcaaggttgaaac






tcaaaggaattgacggggacccgcacaagc






ggtggatgatgtggtttaattcgatgcaac






gcgaaaaaccttacctacccttgacatggc






aggaatcctgaagagatttgggagtgctcg






aaagagaacctgcacacaggtgctgcatgg






ccgtcgtcagctcgtgtcgtgagatgttgg






gttaagtcccgcaacgagcgcaacccttgt






cattagttgctacgaaagggcactctaatg






agactgccggtgacaaaccggaggaaggtg






gggatgacgtcaggtcctcatggcccttat






gggtagggctacacacgtcatacaatggcc






ggtacagagggctgccaayccgcgaggggg






agccaatcccagaaaaccggtcgtagtccg






gatcgcagtctgcaactcgactgcgtgaag






tcggaatcgctagtaatcgcggatcagctt






gccgcggtgaatacgttcccgggtcttgta






cacaccgcccgtcacaccatgggagcgggt






tctgccagaagtagttagcctaaccgcaag






gagggcgattaccacggcagggttcgtgac






tggggtgaagtcgtaacaaggtagccgtat






cggaaggtgcggctggatcacctcctttct









The present invention is directed to metabolic primers for the detection of perchlorate-reducing bacteria and methods and compositions for use of the same. Certain specific aspects can be described according to the following paragraphs:
Paragraph 1: A composition comprising a first primer and a second primer, wherein the first primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:1 or SEQ ID NO:11 and the second primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:2 or SEQ ID NO:12, wherein said first and second primers are capable of hybridizing to a perchlorate reductase gene.
Paragraph 2: The composition of paragraph 1, further comprising a third primer and a fourth primer, wherein the third primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:3 and the fourth primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:4, wherein said third and fourth primers are capable of hybridizing to a perchlorate reductase gene.
Paragraph 3: The composition of paragraph 1, further comprising a third primer and a fourth primer, wherein the third primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:5 or SEQ ID NO:13 and the fourth primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:6 or SEQ ID NO:14, wherein said third and fourth primers are capable of hybridizing to a perchlorate reductase gene.
Paragraph 4: The composition of paragraph 1, further comprising a third primer and a fourth primer wherein the third primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:7 and the fourth primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:8 wherein said third and fourth primers are capable of hybridizing to a perchlorate reductase gene.
Paragraph 5: A composition comprising a first primer and a second primer, wherein the first primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:3 and the second primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:4, wherein said first and second primers are capable of hybridizing to a perchlorate reductase gene.
Paragraph 6: The composition of paragraph 5, further comprising a third primer and a fourth primer, wherein the third primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:5 or SEQ ID NO:13 and the fourth primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:6 or SEQ ID NO:14, wherein said third and fourth primers are capable of hybridizing to a perchlorate reductase gene.
Paragraph 7: The composition of paragraph of paragraph 5, further comprising a third primer and a fourth primer, wherein the third primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:7 and the fourth primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:8 wherein said third and fourth primers are capable of hybridizing to a perchlorate reductase gene.
Paragraph 8: A composition comprising a first primer and a second primer, wherein the first primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:5 or SEQ ID NO:13 and the second primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:6 or SEQ ID NO:14, wherein said first and second primers are capable of hybridizing to a perchlorate reductase gene.
Paragraph 9: The composition of paragraph of paragraph 8, further comprising a third primer and a fourth primer, wherein the third primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:7 and the fourth primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:8 wherein said third and fourth primers are capable of hybridizing to a perchlorate reductase gene.
Paragraph 10: The composition of paragraph 2, further comprising a fifth primer and a sixth primer, wherein the fifth primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:5 or SEQ ID NO:13 and the sixth primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:6 or SEQ ID NO:14, wherein said fifth and sixth primers are capable of hybridizing to a perchlorate reductase gene.
Paragraph 11: The composition of paragraph 10, further comprising a seventh primer and an eighth primer, wherein the seventh primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:7 and the eighth primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:8, wherein said seventh and eighth primers are capable of hybridizing to a perchlorate reductase gene.
Paragraph 12: The composition of paragraph 9 further comprising a fifth primer and a sixth primer, wherein the fifth primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:7 and the sixth primer has a nucleic acid sequence that comprises a sequence of SEQ ID NO:8, wherein said fifth and sixth primers are capable of hybridizing to a perchlorate reductase gene.
Paragraph 13: The composition of any of paragraphs 1 through 12, wherein each of said primers each independently comprise between 20 and 30 nucleotide bases in length.
Paragraph 14: The composition of any of paragraphs 1 through 12, wherein each of said primers each independently comprise between 20 and 40 nucleotide bases in length.
Paragraph 15: The composition of any of paragraphs 1 through 12, wherein each of said primers each independently comprise between 20 and 50 nucleotide bases in length.
Paragraph 16: The composition of paragraph 1 wherein the perchlorate reductase gene is from dissimilatory perchlorate-reducing bacteria (DPRB) species.
Paragraph 17: The composition of paragraph 16, wherein said DPRB is a bacterium from the Dechloromonas spp., Azoarcus spp., Dechlorospirillum spp., Dechloromarinus spp., Ideonella spp., Magnetospirillum spp., Pseudomonas spp., Rhodocyclus spp., Rhodospirillum spp., Azospirillum spp., Wolinella spp., Xanthomonas spp.
Paragraph 18: The composition of paragraph 17, wherein said DPRB is selected from the group consisting of Dechloromonas agitate, Dechloromonas aromatica, Azospira suillum, Dechlorospirillum anomalous, Dechloromarinus chlorophilus, Ideonella dechloratans, and Magnetospirillum magnetotacticum. 
Paragraph 19: The composition of any of paragraphs 1 through 18, wherein the primer is detectably labeled.
Paragraph 20: An oligonucleotide primer pair wherein the first primer of the primer pair comprises a sequence of SEQ ID NO:1 or SEQ ID NO:11 and the second primer of the primer pair comprises a sequence of SEQ ID NO:2 or SEQ ID NO:12.
Paragraph 21: An oligonucleotide primer pair wherein the first primer of the primer pair comprises a sequence of SEQ ID NO:3 and the second primer of the primer pair comprises a sequence of SEQ ID NO:4.
Paragraph 22: An oligonucleotide primer pair wherein the first primer of the primer pair comprises a sequence of SEQ ID NO:5 or SEQ ID NO:13 and the second primer of the primer pair comprises a sequence of SEQ ID NO:6 or SEQ ID NO:14.
Paragraph 23: An oligonucleotide primer pair wherein the first primer of the primer pair comprises a sequence of SEQ ID NO:7 and the second primer of the primer pair comprises a sequence of SEQ ID NO:8.
Paragraph 24: The oligonucleotide primer pair of any of paragraphs 20 through 23 wherein at least one of the primers in the primer pair is detectably labeled.
Paragraph 25: An oligonucleotide primer which has the nucleotide sequence defined in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 11, 12, 13, or 14.
Paragraph 26: The oligonucleotide primer of paragraph 25, wherein said oligonucleotide comprises between 20 and 50 nucleotide bases.
Paragraph 27: The oligonucleotide primer of any of paragraphs 25 through 26 wherein the primer is detectably labeled.
Paragraph 28: The oligonucleotide of paragraph 27, wherein the primer is labeled with an epitope, fluorophore, metal particle, enzyme, carbohydrate, polypeptide, radioactive isotope, dye, biotin, or digitonin.
Paragraph 29: The oligonucleotide primer pair of paragraph 24, wherein the primer is labeled with an epitope, fluorophore, metal particle, enzyme, carbohydrate, polypeptide, radioactive isotope, dye, biotin, or digitonin
Paragraph 30: The composition of paragraph 19, wherein the primer is labeled with an epitope, fluorophore, metal particle, enzyme, carbohydrate, polypeptide, radioactive isotope, dye, biotin, or digitonin.
Paragraph 31: A method of detecting the presence of perchlorate reducing bacteria in a sample comprising: (a) subjecting DNA of bacterial cells in said sample to a first polymerase chain reaction amplification using a pair of primers of paragraph 20, paragraph 21, paragraph 22 or paragraph 23; and (b) detecting the product or products of said first polymerase chain reaction amplification, thereby identifying the presence of said perchlorate-reducing bacteria in said sample.
Paragraph 32: The method of paragraph 31, further comprising subjecting said DNA to a second polymerase chain reaction amplification using a pair of primers of paragraph 20, paragraph 21, paragraph 22 or paragraph 23, that is a different primer set than the primer set that is used in first polymerase chain reaction amplification; and detecting the product or products of said second polymerase chain reaction amplification thereby identifying the presence of said perchlorate-reducing bacteria in said sample.
Paragraph 33: A method of detecting the presence of perchlorate reducing bacteria in a sample comprising: (a) subjecting DNA from said sample to a first polymerase chain reaction amplification using a pair of primers of paragraph 20, paragraph 21, paragraph 22 or paragraph 23; and (b) detecting the product or products of said first polymerase chain reaction amplification, thereby identifying the presence of said perchlorate-reducing bacteria in said sample.
Paragraph 34: A method of detecting the presence of perchlorate-reducing bacteria in a sample comprising: (a) subjecting DNA from said sample to a first polymerase chain reaction amplification using a pair of primers of paragraph 20, paragraph 21, paragraph 22 or paragraph 23; (b) isolating the amplification products from step (a); (c) using the amplification products isolated in step (b) as a template for a second polymerase chain reaction amplification using a second primer pair from paragraph 20, paragraph 21, paragraph 22 or paragraph 23 that is different from the primer pair used in the first polymerase chain reaction; and (d) detecting the product or products of said second polymerase chain reaction amplification, thereby identifying the presence of said perchlorate-reducing bacteria in said sample.
35. A method of detecting the presence of perchlorate-reducing bacteria in a sample comprising: (a) subjecting DNA from said sample to a first polymerase chain reaction amplification using a pair of primers of paragraph 20, paragraph 21, paragraph 22 or paragraph 23; (b) isolating the amplification products from step (a); (c) using the amplification products isolated in step (b) as a template for a second polymerase chain reaction amplification using a second primer pair from paragraph 20, paragraph 21, paragraph 22 or paragraph 23 that is different from the primer pair used in the first polymerase chain reaction; (d) isolating the amplification products from step (c); (e) using the amplification products isolated in step (d) as a template for a third polymerase chain reaction amplification using a third primer pair from paragraph 20, paragraph 21, paragraph 22 or paragraph 23 that is different from the primer pair used in the first or the second polymerase chain reaction; and (f) detecting the product or products of said third polymerase chain reaction amplification, thereby identifying the presence of said perchlorate-reducing bacteria in said sample.
Paragraph 36: A method of detecting the presence of perchlorate-reducing bacteria in a sample comprising: (a) subjecting DNA from said sample to a first polymerase chain reaction amplification using a pair of primers of paragraph 20, paragraph 21, paragraph 22 or paragraph 23; (b) isolating the amplification products from step (a); (c) using the amplification products isolated in step (b) as a template for a second polymerase chain reaction amplification using a second primer pair from paragraph 20, paragraph 21, paragraph 22 or paragraph 23 that is different from the primer pair used in the first polymerase chain reaction; (d) isolating the amplification products from step (c); (e) using the amplification products isolated in step (d) as a template for a third polymerase chain reaction amplification using a third primer pair from paragraph 20, paragraph 21, paragraph 22 or paragraph 23 that is different from the primer pair used in the first or the second polymerase chain reaction; (f) isolating the amplification products from step (e); (g) using the amplification products isolated in step (d) as a template for a fourth polymerase chain reaction amplification using a fourth primer pair from paragraph 20, paragraph 21, paragraph 22 or paragraph 23 that is different from the primer pair used in the first second or the third polymerase chain reaction; and (h) detecting the product or products of said third polymerase chain reaction amplification, thereby identifying the presence of said perchlorate-reducing bacteria in said sample.
Paragraph 37: The method of any one of paragraphs 31 through 36 wherein DNA is isolated from bacterial lysates from said sample prior to step (a).
Paragraph 38: The method of any of paragraphs 31 through 37, wherein said sample is water sample.
Paragraph 39: The method of any of paragraphs 31 through 38, wherein said sample is a soil sample.
Paragraph 40: The method of paragraph 38, wherein said water sample is collected from a water supply that has been contaminated with perchlorate.
Paragraph 41: The method of paragraph 39, wherein said soil sample is collected from land that has been contaminated with perchlorate.
Paragraph 42: The method of paragraph 40 or 41, wherein said perchlorate contamination is a result of waste disposal from paper mill waste, airbag production, firework manufacture and use, fertilizer manufacture and use.
Paragraph 43: A method of determining whether a bioremediation formulation that comprises bacteria will be effective at decreasing perchlorate contamination comprising: (a) subjecting DNA from said bioremediation formulation to a first polymerase chain reaction amplification using a pair of primers of paragraph 20, paragraph 21, paragraph 22 or paragraph 23; and (b) detecting the product or products of said first polymerase chain reaction amplification, wherein the presence of said amplification products indicates the presence of perchlorate reducing bacteria in bioremediation formulation thereby indicating that said formulation is effective at reducing perchlorate contamination.
Paragraph 44: The method of paragraph 43, further comprising subjecting said DNA to a second polymerase chain reaction amplification using a second pair of primers of paragraph 20, paragraph 21, paragraph 22 or paragraph 23 that is different from the first pair of primers; and detecting the presence of perchlorate-reducing bacteria in said bioremediation formulation by visualizing the product or products of said second polymerase chain reaction amplification, wherein the presence of said amplification products indicates that said formulation is effective at reducing perchlorate contamination.
Paragraph 45: The method of paragraph 44, further comprising subjecting said DNA to a third polymerase chain reaction amplification using a third pair of primers of paragraph 20, paragraph 21, paragraph 22 or paragraph 23 that is different from the first and the second pair of primers; and detecting the presence of perchlorate-reducing bacteria in said bioremediation formulation by visualizing the product or products of said third polymerase chain reaction amplification, wherein the presence of said amplification products indicates that said formulation is effective at reducing perchlorate contamination.
Paragraph 46: The method of paragraph 45, further comprising subjecting said DNA to a fourth polymerase chain reaction amplification using a fourth pair of primers of paragraph 20, paragraph 21, paragraph 22 or paragraph 23 that is different from the first, second and the third pair of primers; and detecting the presence of perchlorate-reducing bacteria in said bioremediation formulation by visualizing the product or products of said fourth polymerase chain reaction amplification, wherein the presence of said amplification products indicates that said formulation is effective at reducing perchlorate contamination.
Paragraph 47: A method of determining whether a bioremediation formulation that comprises bacteria will be effective at decreasing perchlorate contamination comprising (a) subjecting said DNA from said bioremediation formulation to a first polymerase chain reaction amplification using a pair of primers of paragraph 20, paragraph 21, paragraph 22 or paragraph 23; (b) isolating the amplification products from step (a); (c) using the amplification products isolated in step (b) as a template for a second polymerase chain reaction amplification using a primer pair from paragraph 20, paragraph 21, paragraph 22 or paragraph 23 that is different from the pair of primers used in the first polymerase chain reaction amplification; and (d) detecting said perchlorate-reducing bacteria by visualizing the product or products of said second polymerase chain reaction amplification.
Paragraph 48: A method of determining whether a bioremediation formulation that comprises bacteria will be effective at decreasing perchlorate contamination comprising: (a) subjecting said DNA from said bioremediation formulation to a first polymerase chain reaction amplification using a pair of primers of paragraph 20, paragraph 21, paragraph 22 or paragraph 23; (b) isolating the amplification products from step (a); (c) using the amplification products isolated in step (b) as a template for a second polymerase chain reaction amplification using a primer pair from paragraph 20, paragraph 21, paragraph 22 or paragraph 23 that is different from the pair of primers used in the first polymerase chain reaction amplification; (d) isolating the amplification products from step (c); (e) using the amplification products isolated in step (d) as a template for a third polymerase chain reaction amplification using a primer pair from paragraph 20, paragraph 21, paragraph 22 or paragraph 23 that is different from the pair of primers used in the first and second polymerase chain reaction amplification; and (f) detecting said perchlorate-reducing bacteria by visualizing the product or products of said second polymerase chain reaction amplification.
Paragraph 49. A method of determining whether a bioremediation formulation that comprises bacteria will be effective at decreasing perchlorate contamination comprising (a) subjecting said DNA from said bioremediation formulation to a first polymerase chain reaction amplification using a pair of primers of paragraph 20, paragraph 21, paragraph 22 or paragraph 23; (b) isolating the amplification products from step (a); (c) using the amplification products isolated in step (b) as a template for a second polymerase chain reaction amplification using a primer pair from paragraph 20, paragraph 21, paragraph 22 or paragraph 23 that is different from the pair of primers used in the first polymerase chain reaction amplification; (d) isolating the amplification products from step (c); (e) using the amplification products isolated in step (d) as a template for a third polymerase chain reaction amplification using a primer pair from paragraph 20, paragraph 21, paragraph 22 or paragraph 23 that is different from the pair of primers used in the first and second polymerase chain reaction amplification; (f) isolating the amplification products from step (e); (g) using the amplification products isolated in step (f) as a template for a fourth polymerase chain reaction amplification using a primer pair from paragraph 20, paragraph 21, paragraph 22 or paragraph 23 that is different from the pair of primers used in the first second and third polymerase chain reaction amplification; and (h) detecting said perchlorate-reducing bacteria by visualizing the product or products of said second polymerase chain reaction amplification.
Paragraph 50: The method of any of paragraphs 43 through 49, wherein said method further comprises determining the presence of chlorite dismutase in said sample prior to determining the presence of perchlorate reductase in said sample.
Paragraph 51: The method of paragraph 50, wherein said determining the presence of chlorite dismutase in said sample comprises performing polymerase chain reactions using primers selected from the group consisting of SEQ ID NO:15 through SEQ ID NO:24.
Paragraph 52: The method of any of paragraphs 43 through 51 wherein DNA is isolated from a bacterial lysates from said bioremediation formulation prior to step (a).
Paragraph 53: The method of any of paragraphs 43 through 51 wherein said bioremediation formulation is a cocktail of microorganisms that are used to remove contaminants from a sample of soil or water in need of decontamination, wherein said cocktail of microorganisms comprises a mixture of DPRBs.
Paragraph 54: The method of paragraph 53, wherein said cocktail of microorganisms further microorganisms that are denitrifiers.
Paragraph 55: The method of paragraph 53, wherein said cocktail of microorganisms further comprises microorganisms that can degrade toluene, xylene, benzene, petroleum, and creosote.
Paragraph 56: A method of determining whether a sample contains bacteria that is reducing perchlorate in said sample comprising: (a) isolating nucleic acid from said sample; (b) incubating said nucleic acid with a DNase to isolate RNA; (c) performing a reverse transcription reaction on said RNA using one or more of the primers selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 11, 12, 13 and 14; and (d) detecting the product or products of said reverse transcription reaction that are expressing perchlorate reductase, thereby identifying the presence of bacteria in said sample that are expressing perchlorate reductase for reducing the perchlorate content of said sample.
Paragraph 57: A method of determining whether a sample contains bacteria that is reducing perchlorate in said sample comprising: (a) isolating nucleic acid from said sample; (b) incubating said nucleic acid with a DNase to isolate RNA; (c) performing a reverse transcriptase reaction on said RNA using one or more of the primers selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 11, 12, 13 and 14; (d) isolating the reaction products from step (c); (e) using the reaction products isolated in step (d) as a template for a polymerase chain reaction amplification using a primer pair from paragraph 20, paragraph 21, paragraph 22 or paragraph 23; and (f) detecting the product or products of said polymerase chain reaction amplification of step (e), thereby identifying the presence of bacteria in said sample that are expressing perchlorate reductase for reducing the perchlorate content of said sample.
Paragraph 58: The method of any of paragraphs 56 or 57, wherein said method further comprises determining the presence of chlorite dismutase in said sample prior to determining the presence of perchlorate reductase in said sample.
Paragraph 59: The method of paragraph 58, wherein said determining the presence of chlorite dismutase in said sample comprises performing polymerase chain reactions using primers selected from the group consisting of SEQ ID NO:15 through SEQ ID NO:24.
Paragraph 60: The method of paragraph 59, further comprising isolating the amplification products of step (e) prior to performing step (f) and using said isolated amplification products as a template for a second polymerase chain reaction amplification using a primer pair of paragraph 20, paragraph 21, paragraph 22 or paragraph 23 that is different from the primer pair used in step (e).
Paragraph 61: The method of paragraph 60 further comprising performing a third or fourth polymerase chain reaction amplification using a primer pair of paragraph 20, paragraph 21, paragraph 22 or paragraph 23 that is different from the primer pairs used in the first and second polymerase chain reaction amplifications.
Paragraph 62: A kit for amplifying perchlorate reductase polynucleotide, said kit comprising: composition of any of paragraphs 1 through 19, or an oligonucleotide pair of any of any of paragraphs 20 through 24, or an oligonucleotide of any of paragraphs 25 through 28; and instructions for carrying out the method according to any of paragraphs 31 through 61.
Paragraph 63: The kit of paragraph 62, which further comprises enzymes and nucleotide components of a PCR reaction.
Paragraph 64: The kit of paragraph 62, wherein said kit further comprises a solid support.
Paragraph 65: The kit of paragraph 62, wherein each of said primers is arrayed on said solid support.
Paragraph 66: The kit of paragraph 62, wherein each of the primers are in separate containers.
Paragraph 67: The kit of any one of paragraphs 62 through 66 wherein said kit further comprises an oligonucleotide primer or primer pair for amplifying chlorite dismutase.
Paragraph 68: The kit of paragraph 67 wherein said oligonucleotide primer is selected from the group consisting of SEQ ID NO:15 through SEQ ID NO:24.
Paragraph 69: A library of primers for the detection of a perchlorate reductase gene from DPRB, said library comprising at least 6 primers derived from the sequences set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 11, 12 13, or 14.
Paragraph 70: A library of primers for the detection of DPRB, said library comprising at least 6 primers derived from the sequences set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 11, 12, 13, or 14 for detecting perchlorate reductase and the sequences set forth in SEQ ID NO:15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 for detecting chlorite dismutase.

## Claims

1. A composition comprising a first primer and a second primer, wherein the first primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:1 or SEQ ID NO:11 and the second primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:2 or SEQ ID NO:12, wherein said first and second primers are capable of hybridizing to a perchlorate reductase gene.

2. The composition of claim 1, further comprising a third primer and a fourth primer, wherein the third primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:3 and the fourth primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:4, wherein said third and fourth primers are capable of hybridizing to a perchlorate reductase gene.

3. The composition of claim 2, further comprising a fifth primer and a sixth primer, wherein the fifth primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:5 or SEQ ID NO:13 and the sixth primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:6 or SEQ ID NO:14, wherein said fifth and sixth primers are capable of hybridizing to a perchlorate reductase gene.

4. The composition of claim 3, further comprising a seventh primer and an eighth primer, wherein the seventh primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:7 and the eighth primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:8, wherein said seventh and eighth primers are capable of hybridizing to a perchlorate reductase gene.

5. The composition of claim 1, further comprising a third primer and a fourth primer, wherein the third primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:5 or SEQ ID NO:13 and the fourth primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:6 or SEQ ID NO:14, wherein said third and fourth primers are capable of hybridizing to a perchlorate reductase gene.

6. The composition of claim 1, further comprising a third primer and a fourth primer wherein the third primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:7 and the fourth primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:8 wherein said third and fourth primers are capable of hybridizing to a perchlorate reductase gene.

7. The composition of claim 1, wherein each of said primers each independently comprise between 20 and 50 nucleotide bases in length.

8. The composition of claim 1 wherein the perchlorate reductase gene is from dissimilatory perchlorate-reducing bacteria (DPRB) species.

9. The composition of claim 8, wherein said DPRB is a bacterium from the Dechloromonas spp., Azoarcus spp., Dechlorospirillum spp., Dechlorornarinus spp., Ideonella spp., Magnetospirillum spp., Pseudomonas spp., Rhodacyclus spp., Rhodospirdlum spp., Azospirillum spp., Wolinella spp., and Xanthomonas spp.

10. The composition of claim 9, wherein said DPRB is selected from the group consisting of Dechloromonas agitate, Dechloromonas aromatica, Azospira suillum, Dechlorospirillum anomalous, Dechloromarinus chlorophulus, Ideonella dechloratans, and Magnetospirillum magnetotacticum.

11. The composition of claim 1, wherein the primer is detectably labeled.

12. A composition comprising a first primer and a second primer, wherein the first primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:3 and the second primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:4, wherein said first and second primers are capable of hybridizing to a perchlorate reductase gene.

13. The composition of claim 12, further comprising a third primer and a fourth primer, wherein the third primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:5 or SEQ ID NO:13 and the fourth primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:6 or SEQ ID NO:14, wherein said third and fourth primers are capable of hybridizing to a perchlorate reductase gene.

14. The composition of claim of claim 12, further comprising a third primer and a fourth primer, wherein the third primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:7 and the fourth primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:8 wherein said third and fourth primers are capable of hybridizing to a perchlorate reductase gene.

15. A composition comprising a first primer and a second primer, wherein the first primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:5 or SEQ ID NO:13 and the second primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:6 or SEQ ID NO: 14, wherein said first and second primers are capable of hybridizing to a perchlorate reductase gene.

16. The composition of claim of claim 15, further comprising a third primer and a fourth primer, wherein the third primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:7 and the fourth primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:8 wherein said third and fourth primers are capable of hybridizing to a perchlorate reductase gene.

17. The composition of claim 16 further comprising a fifth primer and a sixth primer, wherein the fifth primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:7 and the sixth primer has a nucleic acid sequence that comprise the sequence of SEQ ID NO:8, wherein said fifth and sixth primers are capable of hybridizing to a perchlorate reductase gene.

18. An oligonucleotide primer pair wherein:
the first primer of the primer pair comprise the sequence of SEQ ID NO:1 or SEQ ID NO:11 and the second primer of the primer pair comprise the sequence of SEQ ID NO:2 or SEQ ID NO:12.

19. The oligonucleotide primer pair of claim 18 wherein at least one of the primers in the primer pair is detectably labeled.