Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-04-14
2004-09-28
Ketter, James (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S243000, C435S262500
Reexamination Certificate
active
06797817
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the field of molecular biology and microbiology. More specifically, 16S rRNA regions of been identified and isolated from
Dehalococcoides ethenogenes
that enable the identification of dechlorinating bacterial strain. Probes and primers corresponding to the unique regions have been constructed to enable the rapid identification of the dechlorinators.
BACKGROUND
Groundwater pollution by halogenated, and particularly chlorinated solvents is a worldwide problem associated primarily with industrial sites where mishandling or improper disposal has brought these solvents in contact with the soil. The most common and problematic compounds are the chlorinated ethylenes (ethenes) such as tetra- tri- or di-chloroethylene. Carbon tetrachloride, chloroform and methylene chloride are also pervasive pollutants. The reasons for concern are basically threefold. First, most of these solvents are sparingly soluble in water and have the tendency to stick to soil particles. This results in tenacious underground plumes of solvent which cannot readily be removed by standard pump and treat technology (Biswas, N., et al.,
Water Environ. Res
. 64, 170, 10, 1 (1992); Hutter, G. M., et. al.,
Water Environ. Res
. 64, 69, (1992)). Second, the toxicology of many chlorinated solvents suggests that these compounds may be carcinogenic and damaging to specific organs such as the liver and Water, Washington, D.C.(1985); Vogel, T. M.,
Environ. Sci. Technol
., 21, 722, (1987)). Finally, under conditions found in many aquifers and subsurface environments, chlorinated ethylenes and methanes are very slow to be degraded biologically. The result of these factors is that chlorinated solvents are long-lived potentially hazardous groundwater pollutants.
Currently there are two approaches to in situ removal of organohalogen pollutants. The first approach is the standard “pump and treaf” method where groundwater is pumped to the surface for physical stripping of the contaminant from the water. For chlorinated solvents this is more of a containment method than a remediation technology although given sufficient time (typically decades to centuries) this method may capture most of the pollutant. The other approach is biological in nature and utilizes microorganisms for the enzymatic transformation of the halogenated organics. The biological approach may utilize microorganisms indigenous to a particular site where the remediation process consists primarily of making additions to the contaminated site that enhance the growth of the desired microorganism. Alternatively, nonindigenous microorganisms may be introduced to a contaminated site with the necessary amendments needed for growth.
A number of organisms are known to dechlorinate persistent chlorinated pollutants. For example,
Dehalobacter restrictus
and
Dehalospirillium multivorans
, have been shown to partially dechlorinate chlorinated ethenes (Kochian et al.,
Plant Mol Biol
. 46:237 (1995); Delhaize et al.,
Plant Physiol
. 107:315 (1995)). Similarly,
Dehalococcoides ethenogenes
has been shown to effect the complete dechlorination of tetrachloroethene and trichloroethene to ethene [Freedman et al.,
Appl. Environ. Microbiol
. 55:2144 (1989)] and Maymó-Gatell et al. (
Science
, 176:1568 (1997)) have isolated a
D. ethenogenes
strain that is capable of respiratory reductive dechlorination of tetrachloroethene directly to ethene with hydrogen as an electron donor. Analysis of the 16S rRNA of the Maymó-Gatell organism revealed a unique profile that may be used to identify organisms of similar reductive capabilities.
The first step in utilizing the dechlorinating properties of the above identified organisms is rapid and accurate identification. One method of identification involves the use of DNA probes (see for example in WO 89/06704, U.S. Pat. No. 4,851,330, and U.S. Pat. No. 5,574,145). Many such probes derive from the observation (see Woese,
Scientific American
244 (6) 1981 for review) that parts of the 16S and 23s ribosomal RNA (rRNA) sequences vary in different species. This information was used initially for phylogenetic analyses but it has more recently been used for DNA probe-based methods for the identification of organisms. The utility of such a method is based on the conservation of nucleic acid sequence within the rRNA sequences.
Each of the cells of all life forms, except viruses, contains ribosomes and therefore ribosomal RNA. A ribosome contains three separate single strand RNA molecules, namely, a large molecule, a medium sized molecule, and a small molecule. The two larger rRNA molecules vary in size in different organisms. Ribosomal RNA is a direct gene product and is coded for by the rRNA gene. This DNA sequence is used as a template to synthesize rRNA molecules. A separate gene exists for each of the ribosomal RNA sub units. Multiple rRNA genes exist in most organisms, many higher organisms containing both nuclear and mitochondrial rRNA genes. Numerous ribosomes are present in all cells of all life forms. About 85-90 percent of the total RNA in a typical cell is rRNA. A bacteria such as
E. coli
contains about 10
4
ribosomes per cell. Much of the sequences in rRNA highly conserved across broad evolutionary boundaries, however, certain regions are highly variable and may be used to make fine distinctions between species, sub-species and strains (U.S. Pat. No. 5,567,587).
The problem to be overcome therefore is to identify a unique 16S rDNA sequence in a bacteria capable of dechlorination of persistent chlorinated compounds for the identification and ultimate enhancement of that bacteria to remediated a contaminated site. Applicants have solved the stated problem by providing a set of nucleic acid sequences that are unique to various strains of
Dehalococcoides ethenogenes.
SUMMARY OF THE INVENTION
The present invention provides an isolated 16S rDNA sequence indicative of a dechlorinating bacterial strain selected from the group consisting of: (a) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:30 and SEQ ID NO:34; (b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 0.1×SSC, 0.1% SDS at 65° C.; and (c) an isolated nucleic acid molecule that is completely complementary to (a) or (b).
The invention further provides primers and probes useful for the identification of new dechlorinating bacteria selected from the group consisting of: SEQ ID NOs:9-29 and SEQ ID Nos:35-60, and any sequences that hybridize under conditions of 0.1×SSC, 0.1% SDS at 65° C. to those primers and probes.
The invention additionally provides an isolated bacterial strain comprising any one of the sequences of the instant invention as set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 SEQ ID NO:8, SEQ ID NOs:9-29, SEQ ID NO:30, SEQ ID NO:34, and SEQ ID NOs:35-60 wherein said strain has the ability to dechlorinate chlorinated compounds.
The invention further provides a method for identifying a dechlorinating bacterial strain comprising: (i) extracting generic DNA from a cell suspected of being able to dechlorinate chlorinated compounds; (ii) probing the extracted genomic DNA with a probe derived from any one of the sequences instant invention as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 SEQ ID NO:8, SEQ ID NOs:9-29, SEQ ID NO:30, SEQ ID NO:34, and SEQ ID NOs:35-60 under suitable hybridization conditions, wherein the identification of a hybridizable nucleic acid fragment confirms the presence of a bacteria capable of dechlorinating chlorinated compounds.
Similarly the invention provides a method for identifying a dechlorinating bacterial strain comprising (i) extracting genomic DNA from a cell suspected of being able to dechlorinate chlorinated compounds; and (ii) amplifying the extracted genornic DNA with an oligonucleotide primer corresponding to a portion of any one of the sequences instant invention as set fo
Ebersole Richard C.
Hendrickson Edwin R.
E. I. du Pont de Nemours and Company
Ketter James
Lambertson David A.
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