# Patent

## Title
Methods of detecting sources of microorganism contamination

## Abstract

Methods of detecting a source of a microbial contamination in a suspect sample include detecting at least one member selected from the group consisting of a microbial geosmin synthase, a microbial 2-methylisoborneol synthase and a microbial 2-methylgeranyl diphosphate synthase in the suspect sample. The method can include conducting a nucleic acid amplification assay in the presence of at least one member selected from the group consisting of at least one microbial geosmin primer and at least one microbial 2-methylisoborneol synthase primer on a sample obtained from a suspect source of the microbial contamination.

## Background
RELATED APPLICATIONS
This application is a continuation of International Application No. PCT/US2009/057266, which designated the United States and was filed on Sep. 17, 2009, published in English, which claims the benefit of U.S. Provisional Application No. 61/192,309, filed on Sep. 17, 2008. The entire teachings of the above applications are incorporated herein by reference.


GOVERNMENT SUPPORT
The invention was supported, in whole or in part, by a grant GM30301 from the National Institutes of Health. The Government has certain rights in the invention.


BACKGROUND OF THE INVENTION
Geosmin and 2-methylisoborneol are volatile organic compounds frequently found as contaminants in drinking water and fish raised by aquaculture methods. Although not toxic to humans, geosmin and 2-methylisoborneol are generally associated with an undesirable musty or muddy taste and odor in the sources in which they are present. Geosmin and 2-methylisoborneol are generated by a variety of microorganisms, including several species of cyanobacteria, as products of pathways that include geosmin synthase and 2-methylisoborneol synthase. Currently, geosmin and 2-methylisoborneol are detected after their production by an unpleasant odor in a source. Early detection of sources of microbial contamination, for example, prior to an unpleasant odor, may be important in developing effective remediation plans for reducing or eliminating geosmin and 2-methylisoborneol contamination in natural resources, including water, food sources and other materials. Thus, there is a need to develop new, improved and effective methods for detecting microbial sources of geosmin and 2-methylisoborneol contamination.
SUMMARY OF THE INVENTION
The present invention generally relates to methods and compositions for detecting a source of a microbial contamination in a suspect sample.
In an embodiment, the invention is a method of detecting a source of a microbial contamination in a suspect sample, comprising the step of detecting at least one member selected from the group consisting of a microbial geosmin synthase, a microbial 2-methylisoborneol synthase, and a microbial 2-methylgeranyl diphosphate synthase in the suspect sample.
In another embodiment, the invention is a method of identifying a source of a microbial contamination, comprising the step of conducting a nucleic acid amplification assay in the presence of at least one member selected from the group consisting of at least one microbial geosmin primer, at least one microbial 2-methylisoborneol synthase primer and at least one microbial 2-methylgeranyl diphosphate synthase primer on a sample obtained from a suspect source of the microbial contamination.
In a further embodiment, the invention is a method of detecting at least one member selected from the group consisting of a geosmin producing microorganism and a 2-methylisoborneol producing microorganism in a sample, comprising the steps of amplifying at least one nucleic acid in the sample in the presence of at least one member selected from the group consisting of a geosmin synthase primer, a 2-methylisoborneol synthase primer and a 2-methylgeranyl diphosphate synthase primer to thereby generate amplified products; and detecting at least one member selected from the group consisting of a geosmin synthase nucleic acid, a methylisoborneol synthase nucleic acid, and a 2-methylgeranyl diphosphate synthase nucleic acid in the amplified products.
In an additional embodiment, the invention is an isolated nucleic acid comprising at least one member selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.
In yet another embodiment, the invention is an isolated polypeptide comprising at least one member selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.
The methods and compositions of the invention can be employed to detect sources of microbial contamination. Advantages of the claimed invention include, for example, the ability to more rapidly and reliably predict contamination consequent to microorganisms, such as algal blooms associated with geosmin and 2-methylisoborneol contamination; and the detection of microbial organisms that produce geosmin and/or 2-methylisoborneol before the levels of these compounds become detectable by sensory stimuli, such as smell and taste.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts geosmin synthase (GS)-catalyzed cyclization of farnesyl diphosphate (FPP) to geosmin, germacradienol, germacrene D, and 8,10-dimethyl-1-octalin. Germacradienol and germacrene D are formed by the N-terminal domain of the bifunctional protein, and geosmin is generated from germacradienol via the octalin by the C-terminal domain.
FIGS. 2A and 2B depict an alignment of proteins (SEQ ID NOS.: 15 and 25-28) from Nostoc punctiforme (published ZP—00109187, experimental NJ2, and experimental NPUNMOD), Streptomyces coelicolor A3(2) (SCO6073) and Streptomyces avermitilis (SAV2163). Black boxed residues indicate conserved terpene synthase motifs; the boxed isoleucine residue near the carboxy-terminus of the ZP—00109187 sequence in FIG. 2B identifies the site of a sequencing error (“*” indicates exact sequence match, “:” indicates moderate match, “.” Indicates low match)
FIG. 3 depicts a mass spectrum of geosmin produced in the incubation of FPP with recombinant NPUNMOD protein, the geosmin synthase of Nostoc punctiforme. 
FIG. 4 depicts a GC-MS trace of geosmin produced in the incubation of FPP with recombinant NPUNMOD protein, showing geosmin (1), along with coproducts germacradienol (2), germacrene D (3), octalin (4), and (E)-Nerolidol (5).
FIG. 5 depicts alignments of amino (N)-terminal domains of protein sequences (SEQ ID NOS.: 11-14, 33 and 34) derived from the PCR products obtained from geosmin-producing organisms. Conserved magnesium binding domains, such as DDHFLE (SEQ ID NO.: 37) and NDLFSYQRE (SEQ ID NO.: 44), are indicated in the blacked background. SCO6073 and SAV2163 are protein sequences derived from S. coelicolor and S. avermitilis respectively. (“*” indicates exact sequence match, “:” indicates moderate match, “.” Indicates low match).
FIG. 6 depicts a melting curve of a Phormidium sp. geosmin synthase real time PCR product.
FIG. 7 depicts a melting curve of a Anabaena laxa geosmin synthase real time PCR product.
FIG. 8 depicts a melting curve of a Geitlerinema geosmin synthase real time PCR product.
FIG. 9 depicts a melting curve of a Anabaena circinalis geosmin synthase real time PCR product.
FIG. 10 depicts the biosynthetic pathway for the production of 2-methylisoborneol (MIB) in S. coelicolor (SCO). SCO7701 is an S-adenosyl methionine (SAM)-dependent C-methyl transferase that catalyzes the methylation of geranyl diphosphate (GPP) to produce 2-methyl-GPP. SCO7700 is a terpene synthase that catalyzes the cyclization of 2-methyl-GPP to MIB.
FIG. 11 depicts a schematic of a vectorette approach to determine the sequence of cyanobacterial 2-methylisoborneol synthase genes.
FIGS. 12A, 12B and 12C depict a ClustalW alignment of presumptive germacradienol-geosmin synthase proteins (SEQ ID NOS.: 16-24) with S. coelicolor SCO6073. Q1CYZ, Myxococcus xanthus strain DK 1622; Q09A24, Stigmatella aurantiaca DW4/3-1; Q9X839, S. coelicolor A3(2); Q82L49, S. avermitilis; Q2J565, Frankia sp. Strain Cc13; QORBQ4, Frankia alni ACN14a; Q3WJX6, Frankia sp. EAN1pec; A4FEI8 and A4FGS3, Saccharopolyspora erythraea NRL 2338 (SEQ ID NOS.: 16-24). Conserved Mg2+-binding motifs (DDHFLE (SEQ ID NO: 37); NDLFSYERE (SEQ ID NO: 76); NEVLTSRLQQFE (SEQ ID NO: 77); DDYFP (SEQ ID NO: 45); and NDVFSYQKE (SEQ ID NO: 75) in bold.
FIGS. 13A-13D depict amino acid sequences of geosmin synthases of the microorganisms Streptomyces scabies (SEQ ID NO: 78), Streptomyces peucetius (SEQ ID NO: 79), Saccharopolyspora erythraea (SEQ ID NO: 43) (FIGS. 13A and 13B) and 2-methylisoborneol synthases of the microorganisms Streptomyces sp. (SEQ ID NO: 29), Streptomyces scabies (SEQ ID NO: 30), Streptomyces ambofaciens (SEQ ID NO: 31), and Streptomyces griseus (SEQ ID NO: 32) (FIGS. 13C and 13D).



DETAILED DESCRIPTION OF THE INVENTION
The features and other details of the invention, either as steps of the invention or as combinations of parts of the invention, will now be more particularly described and pointed out in the claims. It will be understood that the particular embodiments of the invention are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention.
In an embodiment, the invention is a method of detecting a source of a microbial contamination in a suspect sample, comprising the step of detecting at least one member selected from the group consisting of a microbial geosmin synthase and a microbial 2-methylisoborneol synthase in the suspect sample.
“Source of a microbial contamination,” as used herein, refers to an origin or point of origin of a microorganism (e.g., fungi, bacteria, algae). The source of the microbial contamination can contain at least one member selected from the group consisting of a geosmin synthase nucleic acid or protein, a 2-methylisoborneol synthase nucleic acid or protein, and a 2-methylgeranyl diphosphate synthase (geranyl diphosphate C-methyl transferase) nucleic acid or protein. Microbial contamination can be consequent to a cyanobacterial contamination, an actinomycete contamination (e.g., actinomycetes, Streptomyces), a myxobacterial contamination and a fungal contamination.
In an embodiment, the source of a microbial contamination is consequent to the presence of a cyanobacterial contamination, for example, a cyanobacterial contamination that includes at least one member selected from the group consisting of Phormidium sp., Phormidium calcicola, Anabaena circinalis, Anabaena laxa, Geitlerinema sp., Nostoc punctiforme, Nostoc sp., Pseudoanabaena limnetica, Pseudanabaena sp., Oscillatoria sp., Lyngbya sp., Planktothrix sp., Tyconema sp., Hyella sp., Anabaena sp. and Aphanizomenon sp.
Microbial contamination can also be consequent to the presence of at least one member selected from the group consisting of Streptomyces coelicolor, Myxococcus xanthus, Stigmatella aurantiaca, Streptomyces avermitilis, Frankia sp., Frankia alni and Saccharopolyspora erythraea. 
Table 1 lists exemplary sources of microbial contamination that may be consequent to bacterial geosmin proteins (SEQ ID NOS.: 16, 17, 19-24, 43, 78 and 79), as shown in FIGS. 12A, 12B and 12C. The microorganisms listed in Table 1 produce geosmin synthase that share identity to the geosmin sythase of SEQ ID NO.: 18. The methods described herein can detect any one or more of the geosmin synthases listed in Table 1 in a suspect sample.







TABLE 1







Amino acid sequence comparison of S. coelicolor


SCO6073 germacradienol-geosmin synthase (Q9X839,


CYC2_STRCO; SEQ ID NO: 18) with bacterial orthologs.












UniProt

Identity
Similarity


Organism
ID
aa
(%)
(%)





Streptomyces scabies
a
738
78
85


(SEQ ID NO: 78)


Streptomyces avermitilis
Q82L49
725
76
85


(SEQ ID NO: 19)


Streptomyces peucetius ATCC
spterp13 b
732
64
74


27952


(SEQ ID NO: 79)


Frankia sp. Strain Cc13
Q2J565
751
60
72


(SEQ ID NO: 20)


Saccharopolyspora erythraea
A4FEI8
758
58
70


NRL 2338


(SEQ ID NO: 23)


Frankia alni ACN14a
Q0RBQ4
758
58
70


(SEQ ID NO: 21)


Frankia sp. EAN1pec
Q3WJX6
750
59
72


(SEQ ID NO: 22)


Myxococcus xanthus strain DK
Q1CYZ7
755
57
72


1622


(SEQ ID NO: 16)


Saccharopolyspora erythraea
A4FJE8
763
56
69


NRL 2338


(SEQ ID NO: 43)


Stigmatella aurantiaca DW4/3-1
Q09A24
704
55
67


(SEQ ID NO: 17)


Saccharopolyspora erythraea
A4FGS3
732
45
57


NRL 2338


(SEQ ID NO: 24)





a S. scabies chromosome, nt 2284449-2282248; Sanger Centre.


b Singh, B., Oh, T. J., and Sohng, J. K. Exploration of geosmin synthase from Streptomyces peucetius ATCC 27952 by deletion of doxorubicin biosynthetic gene cluster, J. Ind. Microbiol. Biotechnol. (2009); 10.1007/s10295-009-0605-0.






Table 2 lists exemplary sources of microbial contamination that may be consequent to bacterial 2-methylisoborneol synthase proteins (SEQ ID NOS.:29-32), as shown in FIGS. 13A, 13B and 13C. The methods described herein can detect any one or more of the geosmin synthases listed in Table 2 in a suspect sample.







TABLE 2







Amino acid sequence comparison of S. coelicolor SCO7700 2-


methylisoborneol synthase (Q9F1Y6) with bacterial orthologs.












UniProt

Identity
Similarity


Organism
ID
aa
(%)
(%)














Streptomyces sp. Mg1
B4VFG0
314
83
89


(SEQ ID NO: 29)


Streptomyces
A3KI17
440
83
88


ambofaciens


(SEQ ID NO: 31)


Streptomyces scabies
a
>367
77
81


(SEQ ID NO: 30)


Streptomyces griseus
B1VVB4
437
69
75


(SEQ ID NO: 32)





a S. scabies chromosome, nt 572020-570920; Sanger Centre.






“Suspect,” as used herein in reference to a sample, refers to a sample that may be or is known to contain a microbial contamination. Suspect samples can include at least one member selected from the group consisting of a drinking (potable) water sample, an aquaculture sample, a soil sample, a slime sample, a biofilm sample and a substrate sample.
“Substrate sample,” as used herein, refers to a synthetic substance or object, such as construction materials (e.g., drywall, plaster, sheet rock, brick, concrete, stone).
In an embodiment, the suspect sample is a water sample. The water sample can be from a potable water source or a non-potable water source. Water samples can be obtained from at least one member selected from the group consisting of a lake, a pond, a stream, a reservoir and a water-treatment facility.
In another embodiment, the suspect sample is an aquaculture sample. Exemplary aquaculture samples include aquaculture water samples (e.g., water from an aquaculture enclosure), aquaculture biological samples (e.g., tissues or cells of an organism raised by an aquaculture method) and whole organisms, such as fish from an aquaculture farm (e.g., salmon, trout, catfish, tilapia, cobia) and crustaceans from an aquaculture farm (e.g., shrimp, prawns, and lobsters).
The suspect sample can include a level of at least one member selected from the group consisting of geosmin (e.g., geosmin synthase nucleic acid, geosmin synthase protein), and 2-methylisoborneol (e.g., 2-methylisoborneol synthase nucleic acid, 2-methylisoborneol synthase protein, 2-methylgeranyl diphosphate synthase nucleic acid, 2-methylgeranyl diphosphate synthase protein) that is undetectable by the olfactory (odor, smell) and gastric (taste) senses. The concentration of geosmin or 2-methylisoborneol in a suspect sample, such as a suspect sample that does not yet have an unpleasant odor consequent to geosmin and/or 2 methylisoborneol contamination, can be less than about 10.0 ng of protein per liter (ng/L), for example, at least one member selected from the group consisting of about 0.0 ng/L, about 1 ng/L, about 2.0 ng/L, about 3.0 ng/L, about 4.0 ng/L, about 5.0 ng/L, about 6.0 ng/L, about 7.0 ng/L, about 8.0 ng/L, and about 9.0 ng/L. The suspect sample can also include a concentration of geosmin and/or 2-methylisoborneol that produces an unpleasant odor, such as a level of geosmin and/or 2-methylisoborneol that is greater about 10 ng/L (e.g., at least one member selected from the group consisting of about 15 ng/L, about 20 ng/L, about 25 ng/L and about 30 ng/L). In suspect samples that have unpleasant odors, the source of the unpleasant odor in the suspect sample can rapidly and reliably be determined to be a consequence of geosmin and/or 2-methylisoborneol contamination by employing the methods of the invention.
In an embodiment, a microbial geosmin synthase (e.g., a cyanobacterial geosmin synthase) is detected. The microbial geosmin synthase that is detected can be at least one member selected from the group consisting of at least a portion of a microbial geosmin synthase nucleic acid and at least a portion of a microbial geosmin synthase protein. “At least a portion,” as used herein, means any part or the entirety of an amino acid sequence or nucleic acid sequence. For example, at least a portion of a geosmin sythase can include DDHFLE (SEQ ID NO: 37), NDLFSYERE (SEQ ID NO: 76) and NEVLTSRLQQFE (SEQ ID NO: 77).
Exemplary microbial geosmin synthase nucleic acids that can be detected include at least a portion of at least one member selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. Exemplary microbial geosmin synthase proteins include at least a portion of at least one member selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 43, SEQ ID NO: 78 and SEQ ID NO: 79.
In another embodiment, a microbial 2-methylisoborneol synthase (e.g., a cyanobacterial 2-methylisoborneol synthase) is detected. The microbial 2-methylisoborneol synthase can be at least a portion of at least one member selected from the group consisting of a microbial 2-methylisoborneol synthase nucleic acid and a microbial 2-methylisoborneol synthase protein.
In another embodiment, a microbial 2-methylgeranyl diphosphate synthase (e.g., a cyanobacterial 2-methylgeranyl diphosphate synthase) is detected. The microbial 2-methylgeranyl diphosphate synthase can be at least a portion of at least one member selected from the group consisting of a microbial 2-methylgeranyl diphosphate synthase nucleic acid and a microbial 2-methylgeranyl diphosphate synthase protein.
Microbial geosmin synthase nucleic acids, microbial 2-methylgeranyl diphosphate synthase nucleic acids, and microbial 2-methylisoborneol synthase nucleic acids can be detected in the suspect sample by nucleic acid amplification employing established techniques. Exemplary techniques for detecting nucleic acids in a sample are well-known in the art and include, for example, nucleic acid amplification techniques (e.g., polymerase chain reaction), in situ hybridization, Northern blot hybridization, and Southern blot hybridization (see, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994)).
The nucleic acid amplification employed to detect a geosmin synthase nucleic acid can include the use of at least one primer selected from the group consisting of TTCTTCGACGAYCACTTCC (SEQ ID NO: 1), CCCTYGTTCATGTARCGGC (SEQ ID NO: 2), AACGACCTGTTCTCCA (SEQ ID NO: 3), GCTCGATCTCATGTGCC (SEQ ID NO: 4), CTACTATTGTSAAYGAYCTVTATTC (SEQ ID NO: 5) and ATDAGSACYTATTGCAARCRGCCG (SEQ ID NO: 6). The nucleic acid amplification employed to detect a 2-methylisoborneol synthase can include use of at least one primer selected from the group consisting of SEQ ID NOS: 5, 6, 46-68 and 69.
Detection of the microbial geosmin synthase nucleic acid and/or the microbial 2-methylisoborneol synthase nucleic acid in the suspect sample by nucleic acid amplification can further include the step of sequencing the microbial geosmin synthase nucleic acid and microbial 2-methylisoborneol synthase nucleic acid. Techniques to sequence nucleic acids are known to one of skill in the art, and include techniques described herein.
A microbial geosmin synthase nucleic acid or a microbial 2-methylisoborneol synthase nucleic acid can be a deoxyribonucleic acid (e.g., genomic DNA, cDNA) or a ribonucleic acid (e.g., mRNA) that encodes a complete (e.g., full-length) microbial geosmin synthase protein or at least a portion of a microbial geosmin synthase protein, or a complete (e.g., full-length) microbial 2-methylisoborneol synthase or at least a portion of a microbial 2-methylisoborneol synthase protein. For example, a microbial geosmin synthase nucleic acid or a microbial 2-methylisoborneol synthase nucleic acid can generally be between about 50 and about 2000 nucleotides (also referred to herein as “base pairs”) in length.
The microbial geosmin synthase nucleic acid that is detected in the suspect sample by nucleic acid amplification can be between about 700 nucleotides and about 750 nucleotides in length. The microbial 2-methylisoborneol synthase nucleic acid that is detected in the suspect sample by nucleic acid amplification can be between about 1100 nucleotides to about 1300 nucleotides in length.
In an embodiment, a microbial 2-methylisoborneol synthase nucleic acid includes at least a portion of a S-adenosylmethionine (SAM)-dependent C-methyltransferase (2-methylgeranyl diphosphate synthase) nucleic acid and at least a portion of a 2-methylisoborneol synthase nucleic acid. As depicted in FIG. 10, genes encoding a SAM-dependent C-methyltransferase and a 2-methylisoborneol terpene synthase may be contiguous as a component of a two-gene operon in certain 2-methylisoborneol-producing microorganisms.
At least a portion of a microbial geosmin synthase protein or at least a portion of a microbial 2-methylisoborneol synthase protein can be detected in the methods described herein.
Suitable techniques for detecting the presence or amount of proteins in a sample are also well-known in the art and include immunological and immunochemical methods such as flow cytometry (e.g., FACS analysis), enzyme-linked immunosorbent assays (ELISA), including chemiluminescence assays, radioimmunoassay, immunoblot (e.g., Western blot) assays, and immunohistochemistry (IHC) techniques, or other suitable methods, such as mass spectroscopy. For example, antibodies to a geosmin synthase protein or a microbial 2-methylisoborneol synthase protein can be generated and used to determine the presence and/or level of these proteins in a sample, either directly or indirectly using, e.g., immunohistochemistry (IHC). Methods of producing antibodies are well-known in the art (see, e.g., Harlow et al., Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). The microbial geosmin synthase protein detected by the method can include Mg+2 binding motifs, such as at least one member selected from the group consisting of DDHFLE (SEQ ID NO: 37), NDLFSYERE (SEQ ID NO: 76), NEVLTSRLQQFE (SEQ ID NO: 77), DDYFP (SEQ ID NO: 45) and NDVFSYQKE (SEQ ID NO: 75).
In another embodiment, the invention is a method of identifying a source of a microbial contamination, comprising the step of conducting a nucleic acid amplification assay in the presence of at least one member selected from the group consisting of at least one microbial geosmin primer and at least one microbial 2-methylisoborneol synthase primer on a sample obtained from a suspect source of the microbial contamination.
Exemplary nucleic acid amplification assays include various polymerase chain reaction based assays (e.g., real-time, quantitative real-time, multiplex, reverse transcriptase polymerase chain reaction assays), ligase chain reaction, self sustained sequence replication, transcriptional amplification system and Q-Beta Replicase. Products of nucleic acid amplification can be detected, for example, by labeling of the nucleic acid product during amplification, or by exposure of the product to intercalating compounds/dyes, or probes and by sequencing the nucleic acid sequences.
Suitable primers (e.g., oligonucleotide primers) for amplification of a microbial geosmin synthase nucleic acid include at least one member selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.
In an embodiment, degenerate primers can be employed in the methods described herein. Degenerate primers can include mixtures of similar, not identical, primers. Exemplary degenerate primers for nucleic acid amplification of a microbial geosmin synthase nucleic acid include TTCTTCGACGAYCACTTCC (SEQ ID NO: 1) and CCCTYGTTCATGTARCGGC (SEQ ID NO: 2). Exemplary degenerate primers for nucleic acid amplification of a microbial 2-methylisoborneol synthase nucleic acid include CTACTATTGTSAAYGAYCTVTATTC (SEQ ID NO: 5) and ATDAGSACYTATTGCAARCRGCCG (SEQ ID NO:6).
In another embodiment, non-degenerate primers are used for nucleic acid amplification of a microbial geosmin synthase nucleic acid or a microbial 2-methylisoborneol synthase nucleic acid. Exemplary non-degenerate primers for nucleic acid amplification of a microbial geosmin synthase nucleic acid include AACGACCTGTTCTCCTA (SEQ ID NO: 3) and GCTCGATCTCATGTGCC (SEQ ID NO: 4).
“Microbial geosmin primer,” as used herein, refers to nucleic acid sequences that result in the generation of at least a portion of a microbial (e.g., fungal, bacterial, algal) geosmin nucleic acid (also referred to herein as “a nucleic acid sequence encoding a geosmin synthase protein” or “a nucleic acid sequence encoding at least a portion of a geosmin synthase protein). Likewise, “Microbial 2-methyliseborneol synthase primer,” as used herein, refers to nucleic acid sequences that result in the generation of at least a portion of a microbial (e.g., fungal, bacterial, algal) 2-methylisoborneol synthase nucleic acid (also referred to herein as “a nucleic acid sequence encoding a 2-methylsoborneol synthase protein” or “a nucleic acid sequence encoding at least a portion of a 2-methylisoborneol synthase protein”).
Optionally, following the detection of a nucleic acid amplification product, the product can be sequenced using one of several well-known sequencing techniques (e.g., Maxam Gilbert sequencing, Sanger sequencing, dye terminator sequencing, sequencing by ligation, parallelized sequencing, sequencing by hybridization) to determine the sequence of the microbial geosmin synthase, the microbial 2-methylgeranyl diphosphate synthase, or the microbial 2-methylisoborneol synthase in the suspect sample.
In an embodiment, the method further includes the step of generating a melting curve in conjunction with the nucleic acid amplification assay to thereby produce at least one member selected from the group consisting of a microbial geosmin synthase melting curve and a microbial 2-methylisoborneol synthase melting curve.
Methods of generating nucleic acid melting curves are known in the art. For example, real time nucleic acid amplification can be performed in the presence of at least one member selected from the group consisting of at least one microbial geosmin primer and at least one microbial 2-methylisoborneol synthase primer, and, in addition, at least one nucleic acid-binding dye (e.g., SYTO9® fluorescent dye (Invitrogen)) to generate a labeled amplified product. Towards the conclusion of the nucleic acid amplification assay, a final amplification cycle can be replaced with a nucleic acid (e.g., DNA) melting analysis by subjecting the amplified product(s) to increasing temperatures (e.g., between about 75° C. and about 95° C.), with a hold step at regular temperature intervals (e.g., about 5 seconds at every whole degree Celsius) and data can be obtained at each interval.
In a particular embodiment, the nucleic acid amplification is real-time nucleic acid amplification. Real time nucleic acid amplification, also referred to a quantitative real time nucleic acid amplification, amplifies and simultaneously quantifies a microbial geosmin synthase nucleic acid and/or a microbial 2-methylisoborneol synthase nucleic acid. Such a method will permit both detection and amplification (e.g., as absolute members of copies or relative amounts when normalized to a nucleic acid standard) of a geosmin synthase nucleic acid and/or 2-methylisoborneol synthase nucleic acid in the suspect sample. In this method, the amplified microbial geosmin synthase nucleic acid and/or microbial 2-methylisoborneol synthase nucleic acid is quantified as it accumulates in the nucleic acid amplification reaction in real time after each amplification cycle.
Quantification of the microbial geosmin synthase nucleic acid or the microbial 2-methylisoborneol synthase nucleic acid can be achieved by the use of fluorescent dyes, such as SYTO9® fluorescent dye, SYBR Green®, EvaGreen®, LC Green®, BEBO®, YOYO®, YOPRO® that intercalate with the double-stranded amplified nucleic acid and/or modifying the microbial goesmin synthase nucleic acid and/or 2-methylisoborneol synthase nucleic acid probes so they fluoresce when hybridized to complementary nucleic acids. Real-time nucleic acid amplification may be combined with reverse transcription to quantify the mRNA of microbial geosmin synthase and/or 2-methylisoborneol synthase in the suspect sample.
Exemplary microbial geosmin synthase melting curves, which are indicative of contamination by geosmin-producing microorganisms (e.g., cyanobacteria), include at least one melting curve that is substantially similar to at least one member selected from the group consisting of in FIG. 6, FIG. 7, FIG. 8 and FIG. 9.
A geosmin synthase melting curve can include at least one fluorescence intensity peak between about 0.80 dF/dT to about 9.00 dF/dT in a temperature range of between about 78° C. to about 88° C., which is indicative of microbial (e.g., cyanobacterial) contamination.
In an embodiment, a geosmin synthase melting curve can include a fluorescence intensity peaks of about 3.50 dF/dT at a temperature range between about 84° C. to about 85° C. and about 4.75 dF/dT at a temperature range between about 87° C. to about 88° C., which is indicative of a Phormidium sp. cyanobacterial contamination.
In another embodiment, a geosmin synthase melting curve can include a fluorescence intensity peak of about 6.5 dF/dT at a temperature range between about 84° C. to about 85° C., which is indicative of a Anabaena circinalis cyanobacterial contamination.
In an additional embodiment, a geosmin synthase melting curve can include fluorescence intensity peaks of about 3.75 dF/dT at a temperature range between about 82° C. to about 83° C. and about 9 dF/dT at a temperature range between about 84° C. to about 86° C., which is indicative of a Anabaena laxa cyanobacterial contamination.
In another embodiment, a geosmin synthase melting curve can include fluorescence intensity peaks of about 0.90 dF/dT at a temperature of about 78° C., about 0.80 dF/dT at a temperature between about 84° C. to about 85° C. and about 1.2 dF/dT at a temperature between about 86° C. to about 87° C., which is indicative of a Geitlerinema sp. cyanobacterial contamination.
In yet another embodiment, the invention is an isolated acid comprising at least one member from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. At least portions of SEQ ID NOS: 7-10 can be employed in the methods described herein.
In a further embodiment, the invention is an isolated polypeptide comprising at least one member selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.
An additional embodiment of the invention is a method of detecting at least one member selected from the group consisting of a geosmin producing microorganism (e.g., cyanobacteria, fungi, algae) and a 2-methylisoborneol producing microorganism in a sample, comprising the steps of amplifying at least one nucleic acid in the sample in the presence of at least one member selected from the group consisting of a geosmin synthase primer (e.g., a microbial geosmin synthase primer that includes at least one primer selected from the group consisting of TTCTTCGACGAYCACTTCC (SEQ ID NO: 1), CCCTYGTTCATGTARCGGC (SEQ ID NO: 2), AACGACCTGTTCTCCTA (SEQ ID NO: 3), and GCTCGATCTCATGTGCC (SEQ ID NO: 4)) and a 2-methylisoborneol synthase primer to (e.g., a microbial 2-methylisoborneol synthase primer that includes at least one member selected from the group consisting of CTACTATTGTSAAYGAYCTVTATTC (SEQ ID NO: 5) and ATDAGSACYTATTGCAARCRGCCG (SEQ ID NO: 6)), thereby generating amplified products; and detecting at least one member selected from the group consisting of a geosmin synthase nucleic acid and a methylisoborneol synthase nucleic acid in the amplified products.
The presence of a geosmin synthase nucleic acid is indicative of a geosmin producing microorganism (e.g., cyanobacteria, fungus) in the sample and the presence of a 2-methylisoborneol synthase nucleic acid is indicative of a 2-methylisoborneol producing microorganism in the sample. Likewise, the absence of a geosmin synthase nucleic acid indicates that the sample does not include a geosmin producing microorganism and the absence of a 2-methylisoborneol synthase nucleic acid indicates that the sample does not include a 2-methylisoborneol producing microorganism.
Taste and odor episodes continue to trouble water utilities worldwide on several fronts, including management of reservoirs and consumer complaints. One of the most common consumer complaints is the detection of earthy/musty compounds, namely geosmin and/or 2-methylisoborneol (MIB). The methods and compositions described herein can be employed to rapidly improve the quality of water utilities. Humans are able to detect these compounds at extremely low levels, and detection of these compounds is often associated with a failed disinfection regime and that the water may be unsafe to drink as it is perceived to be “dirty.”
Determination, at the molecular level, of the genes responsible for the production of these taste and odor compounds will provide tools to identify and better manage potential taste and odor episodes. Little is known regarding the cyanobacterial mechanisms of geosmin and MIB production and only recently has the mystery of the biosynthetic production of geosmin been unraveled in Streptomyces sp, with the discovery of a geosmin synthase gene. Even more recently, the proponents have successfully confirmed geosmin synthase functionality in cyanobacteria.
The methods described herein can combine the two assays (geosmin and MIB detection) (multiplex) as a screening tool for taste and odor nuisance organisms. At any given water storage the developed technology can be used, for example, to predict what preventative measures may be necessary in the short term; track the emergence of taste and odor producers in the longer term; and anticipate what trends may occur in the future.
The methods described herein provide a new nucleic acid-based detection method (microarrays, oligoprobe-based nanotechnology etc) and alternative biochemical test, such as an ELISA, to detect microbial contamination.
The detection and remediation of methylisoborneol (MIB) presents similar challenges to the water industry. This compound, which has a characteristic muddy-odor and taste, is found in a variety of actinomycete, myxobacterial, and cyanobacterial organisms, as is the case with geosmin. Much of the effort to control MIB, like geosmin, has concentrated solely on the dealing with the problem when it arises. Management options are currently limited to using activated carbon, biological filtration, oxidation, and ozonation (Ho et al., 2007, Park et al., 2007, Persson et al., 2007), with no foreseeable changes in practice.
Unlike geosmin, the mechanism of MIB production by actinomycetes and cyanobacteria has largely remained unstudied. Recently, in the myxobacterium Nannocystis exedens, Dickschat et al (Dickschat et al., 2007) reported incorporation of mevalonate precursors as well as methyl-labeled S-adenosylmethionine (SAM) into MIB and postulated a biosynthetic pathway in which the universal monoterpene precursor geranyl diphosphate (GPP) would undergo SAM-dependent methylation, with generation of the novel intermediate 2-methyl-GPP, which may undergo direct cyclization to MIB. Detection of 2-methylgeraniol in the volatile extract of N. exedens, as well as the incorporation of labeled 2-methylgeraniol into MIB, by cultures of this organism was reported.
The SCO7700 protein catalyzes the cyclization of 2-methyl-GPP to MIB (FIG. 10). The SCO7701 protein encoded by the immediately downstream gene catalyzes the SAM-dependent methylation of geranyl diphosphate to generate 2-methyl-GPP. The steady kinetic parameters (kcat and Km) of both reactions have been determined and it has been demonstrated that incubation of GPP and SAM with both recombinant SCO7701 and SCO7700 proteins results directly in the formation of MIB. The stop codon at the 3′ end of the SCO7700 gene is only 16 nt upstream of the corresponding start codon for the SCO7701 gene, with putative Streptomyces ribosome binding sites immediately upstream of each open reading frame.
BLAST searches have revealed the presence of homologous two-gene MIB operons in several other actinomycete species (Table 2). Highly similar terpene synthase/C-methyl transferase open reading frames can be found in MIB producing organisms including Streptomyces griseus, Streptomyces ambofaciens, Streptomyces scabies, and Streptomyces sp. Mg1. A homologous two-gene cluster may also be responsible for MIB biosynthesis in cyanobacteria.
In yet another embodiment, the invention is a kit for use in performing the methods described herein. Kits of the invention may be employed in field testing of suspect samples, such as water treatment facility samples or aquaculture samples.
Elucidation of the MIB Synthase Gene Cluster in Pseudanabaena Limnetica (MIB-Producer)
Degenerate primers for the Streptomyces MIB synthase gene cluster (e.g., see Table 3) are designed taking into account differential codon usage between Streptomyces and cyanobacteria. Streptomyces DNA has a higher GC-content (73%) compared to most other organisms and introducing redundancies at the third nucleotide in the codon triplicate will minimize issues with too many redundancy factors associated with primer design. Primers will be designed individually for SCO7700 and SCO7701 homologues, and for the entire SCO7700/SCO7701 cluster, as well as several internal gene sequences. In Streptomyces, the MIB synthase genes are ordered proximally, which may not be different in cyanobacteria.







TABLE 3







Sequences of degenerate primers for amplifying cyanobacterial MIB synthase


genes








Primer
Sequence (5′ to 3′)












MIB7700_NSE_F3
CGACCATCGTCAACGACCTCTACTC
(SEQ ID NO.: 46)


 


MIB7700_NSE_F3_MOD
CTACTATTGTSAAYGAYCTVTATTC
(SEQ ID NO.: 5)


 


MIB7700_F1
CGGCTGATGGTCGCGGAGAA
(SEQ ID NO.: 47)


 


MIB7700_F1_MOD
CGACTVATGGTSGCWGAGAA
(SEQ ID NO.: 48)


 


MIB7700_R1
TGCGGTGCCAGTCGTGGTTG
(SEQ ID NO.: 49)


 


MIB7700_R1_MOD
TTCGRTGCCARTCRTGGTTG
(SEQ ID NO.: 50)


 


MIB7700_F2
TTCGACGGCTTCTCGGTGG
(SEQ ID NO.: 51)


 


MIB7700_F2_MOD
TTYGAYGGYTTCTTWGTGGG
(SEQ ID NO.: 52)


 


MIB7700_DDCYCED_F4
GTGGACGACTGCTACTGCGAGGACC
(SEQ ID NO.: 53)


 


MIB7700_DDCYCED_F4_M
GTSGAYGAYTGYTARTGYGAAGATC
(SEQ ID NO.: 54)


 


MIB7700_NSE_R2
GAGTTCCTTGGTGTAGGAGTAGAGG
(SEQ ID NO.: 55)


 


MIB7700_NSE_R2_MOD
BAGWTCTTTGTKTAWAASTAGAGG
(SEQ ID NO.: 56)


 


MIB7700_R3
GGCAGGCTGTAGCGGTAGGTGT
(SEQ ID NO.: 57)


 


MIB7700_R3_MOD
GGBAGWCTKTATCGVTAGGTGT
(SEQ ID NO.: 58)


 


MIB7701_PCR_F1
GAGGGAGTGACCCTGTCCG
(SEQ ID NO.: 59)


 


MIB7701_PCR_F1_MOD
GAAGGTGTCACTCTBTCCG
(SEQ ID NO.: 60)


 


MIB7701_PCR_R3
GAAGTGGGCGTTGATCTGG
(SEQ ID NO.: 61)


 


MIB7701_PCR_R3_MOD
AAARTGWGCRTTTATCTGG
(SEQ ID NO.: 62)


 


MIB7701_GGGFOLD_R1_M
TCACCATWAANCCNCCTCGAGGGCA
(SEQ ID NO.: 63)


 


MIB7701_PCR_F2
GACGCCGTACCAGGAGG
(SEQ ID NO.: 64)


 


MIB7701_PCR_F2_MOD
GACTCCGTAYCAYGAGG
(SEQ ID NO.: 65)


 


MIB7701_PCR_R4
CCGGGAGTGGATGTTGC
(SEQ ID NO.: 66)


 


MIB7701_PCR_R4_MOD
YCCNGARTGRATGTTGC
(SEQ ID NO.: 67)


 


MIB7701_R2
ATCAGGACGTACTGGAAGGAGCCGT
(SEQ ID NO.: 68)


 


MIB7701_R2_MOD
ATDAGSACYTATTGCAARCRGCCG
(SEQ ID NO.: 6)


 


MIB7701_GGGFOLD_R1
TGACCATGGAACCGCCGCGTCCGCA
(SEQ ID NO.: 69)









A vectorette approach of genome walking (FIG. 11) will be run after initial attempts of degenerate PCR. First described in 1991 (Arnold and Hodgson, 1991), it offers advantages over conventional DNA library construction in terms of time, efficiency, and screening options. Unlike conventional screening of libraries using Southern Hybridization probes to detect similar sequences, the vectorette approach involves:
1. Digesting DNA with several restriction enzymes
2. Using a primer anchored on a known sequence within the target gene
3. Ligation of the digested product with a “vectorette” of known sequence
4. Performing PCR
5. Sequencing the product
Degenerate PCR can be employed to generate sequence data that can be used to design the specific internal anchoring primers, or primers can be designed for the SCO7700 terpene synthase gene around the DDCYCED (SEQ ID NO: 35) and NSE motifs or the characteristic GXGXG (SEQ ID NO: 36)-Rossmann fold of the SCO7701 C-methyl transferase gene. Several other highly conserved regions within these genes also occur, and provide several other options. Selected PCR products from either degenerate PCR or the vectorette approach will be sequenced by conventional methods to confirm identity before being used for further work described herein.
In addition, a cosmid library for use in Southern Hybridization can be employed, to yield suitable leads for the MIB synthase gene cluster. Methods for cosmid library preparation are well established but require considerable time for preparation of cultures and the extraction of good quality DNA.
The library will be screened for suitable clones and constructs using probes generated from the SCO7700, SCO7701, and SCO7701+7700 genes using standard methods, as described herein. Positive “hits” of clones will be further investigated by restriction enzyme digests, agarose gel electrophoresis, and Southern hybridization with subsequent sequencing of positive bands.
Once the gene sequences are confirmed, each gene will be ligated into a protein expression vector (e.g. pEt 21d(+) or pEt-28) using standard methods. The ligated insert/vector will be transformed into E. coli XL1 Blue competent cells and the insert confirmed again by sequencing for sequence fidelity and orientation. The expression vector will then be transformed into the tightly regulated E. coli BL21 (DE3) pLysS protein expression host system. Each gene will be induced by standard methods to generate the corresponding recombinant protein carrying, as appropriate, N- or C-terminal His6-Tag affinity sequences, and the protein sizes will be confirmed by SDS-PAGE against standard protein markers.
Both the cyanobacterial C-methyltransferase and the terpene synthase will be purified to homogeneity using standard methods developed for the corresponding S. coelicolor proteins (for example, immobilized metal-affinity chromatography using Ni(2+) or Co(3+) and His6-Tag proteins, followed, as necessary, by ion exchange on Q-Sepaharose, hydrophobic interaction chromatography on butyl-Sepharose, and/or gel filtration on an ÅKTA Rapid Protein Purification System. The actual molecular mass of each protein will be determined directly by Matrix-Assisted Laser Desorption/Ionization, Time-of-Flight (MALDI-TOF) mass spectrometry (Brown University, Department of Chemistry). The expected size of the cyanobacterial ortholog of the Streptomyces C-methyltransferase SCO7701 is about 33 KDa, while the MIB synthase (terpene cyclase) corresponding to SCO7700 will be about 48 KDa.
The enzyme activity of each of the purified recombinant P. limnetica proteins will be established using the assays already developed for the corresponding S. coelicolor enzymes. Thus, the SCO7701 homolog (C-methyltransferase) will be incubated with GPP and SAM. The resultant product will be treated with a mixture of a pyrase and alkaline phosphatase and the liberated 2-methylgeraniol will be directly identified by capillary GC-MS comparison with an authentic sample. Similarly, synthetic 2-methylgeranyl diphosphate (2-MeGPP) will be incubated with the recombinant terpene P. limnetica homolog of SCO7700 and formation of the resultant methylisoborneol (MIB) confirmed by direct GC-MS comparison with an authentic sample. These same assays can be used to verify the predicted biochemical activity of homologous 2-MeGPP synthase and MIB synthase cloned from additional cyanobacterial species. A combined incubation of GPP and SAM will be carried out with both recombinant P. limnetica proteins, with GC-MS analysis of the pentane-soluble extract to confirm the formation of MIB.
Using established methods, the steady-state kinetic parameters, kcat and Km will be determined for both P. limnetica proteins. For the C-methyltransferase, the recently developed coupled UV-assay reported by Dorgan et al (Dorgan et al., 2006) will be used, in which the initially generated co-product, S-adenosylhomocysteine, normally a potent inhibitor of SAM-dependent methyl transferases, is converted in situ to S-ribosylhomocysteine and adenine by recombinant S-adenosylhomocysteine/5′-methylthioadenosine nucleosidase (SAHN/MTAN, EC 3.2.2.9). Subsequently, adenine generated from AdoHcy is further hydrolyzed to hypoxanthine and ammonia by recombinant adenine deaminase (EC 3.5.4.2). This deamination is associated with a decrease in absorbance at 265 nm that can be monitored continuously. For the MIB synthase, [1-3H]2-MeGPP can be used as a substrate and the standard terpene synthase assay liquid scintillation assay of pentane soluble [3H]methylisoborneol. Alternatively, using unlabeled 2-MeGPP, formation of inorganic pyrophosphate can be directly monitored using the coupled chemiluminescence assay originally developed by Lahser et al for RNA and DNA polymerases (Lahser and Malcolm, 2004).
Nucleic acid amplification will be designed using sequences aligned from Pseudanabaena limnetica and other MIB-producing Streptomyces. Multiple strategies will be employed:
1. PCRs for C-methyl transferase (entire gene and smaller internal fragments)
2. PCRs for terpene synthase (entire gene and smaller internal fragments)
3. PCRs for combined C-methyl transferase/terpene synthase
Methyl transferases are relatively common in cyanobacteria and may differ between the methyl transferases involved in the MIB synthase cluster and other methyl transferases, and similarly for terpene synthases. Given that the two genes of the cyanobacterial MIB operon, the C-methyl transferase and the terpene synthase, may be adjacent to one another as they are in the various Actinomyces species.
The Australia Water Quality Centre has a large cyanobacterial culture collection obtained from a wide geographical distribution that will provide numerous isolates to challenge the specificity of the PCRs. PCRs will be run and electrophoretic bands of the expected size will be excised, purified, reamplified, and cloned into TOPO® cloning system for sequencing. Sequence data will be collated, aligned and suitable real-time PCR assays will be designed.
Having verified both the MIB and geosmin synthase PCRs, they will be combined into a multiplex format to create a quantitative “taste and odor real-time PCR,” that will form the basis for tests described herein. The multiplex PCR will be designed for both a lab based diagnostic test and for a portable PCR platform that will enable this technology to be used in the field.
The taste and odor PCR will be tested using environmental samples in the following ways:
1. screening of implicated taste and odor isolates
2. monitoring taste and odor episodes in nuisance reservoirs
The mechanisms involved in the production of these taste and odor compounds are not well understood. Globally, there has been increased production of these compounds by blooms in reservoirs and the incidences of such events appears to be on the increase, possibly due to global warming and increased human impacts on water quality. Understanding the mechanisms behind this phenomenon would provide information for managing these severe water quality incidents in water resources.
The invention described herein develops and applies new tools to track the growth and decline of natural cyanobacterial bloom populations in reservoirs in order to develop an understanding of the regulation of production of geosmin and/or MIB in waters, which will increase an understanding of genetic and physiological control of geosmin production in the longer term to develop predictive models of the production of these problem contaminants in drinking water.
The identification of the genetic machinery for taste and odor producing cyanobacteria will provide a fundamental step forward. Gene sequences will also provide the starting point for the understanding of the expression and regulation of these genes in the natural environment. In addition, expression of the proteins involved would allow the future development of antibodies directed against these enzymes that could be used in an alternative biochemical test, such as an ELISA.
The invention described herein will also be of value to the freshwater aquaculture industry, which currently experiences loss of farmed fish due to tainting of flesh with geosmin and/or MIB (Martin et al., 1991, Van der Ploeg and Boyd, 1991, Schrader and Dennis, 2005). Estimated losses to U.S. farmers are in the vicinity of about $60 million per annum (Schrader et al., 2003). The potential for screening of fish ponds for nuisance algae; therefore, would be a valuable monitoring and management tool for this industry also.
REFERENCES


    ARNOLD, C. & HODGSON, I. J. (1991) Vectorette PCR: a novel approach to genomic walking PCR Methods Appl, 1, 39-42.
    BENTLEY, R. & MEGANATHAN, R. (1981) Geosmin and methylisoborneol biosynthesis in streptomycetes. Evidence for an isoprenoid pathway and its absence in non-differentiating isolates. FEBS Lett, 125, 220-2.
    BOWMER, K. H., PADOVAN, A., OLIVER, R. L., KORTH, W. & GANF, G. G. (1992) Physiology of Geosmin production by Anabaena circinalis isolated from the Murrumbidgee river, Australia. Wat Sci Tech, 25, 259-267.
    CANE, D. E., HE, X., KOBAYASHI, S., OMURA, S. & IKEDA, H. (2006) Geosmin biosynthesis in Streptomyces avermitilis. Molecular cloning, expression, and mechanistic study of the germacradienol/geosmin synthase. J Antibiot (Tokyo), 59, 471-9.
    CANE, D. E. & WATT, R. M. (2003) Expression and mechanistic analysis of a germacradienol synthase from Streptomyces coelicolor implicated in geosmin biosynthesis. Proc Natl Acad Sci USA, 100, 1547-51.
    COOK, D., NEWCOMBE, G. & SZTAJNBOK, P. (2001) The application of powdered activated carbon for MIB and geosmin removal: predicting PAC doses in four raw waters. Water Res, 35, 1325-33.
    DICKSCHAT, J. S., BODE, H. B., MAHMUD, T., MULLER, R. & SCHULZ, S. (2005) A novel type of geosmin biosynthesis in myxobacteria. J Org Chem, 70, 5174-82.
    DICKSCHAT, J. S., NAWRATH, T., THIEL, V., KUNZE, B., MULLER, R. & SCHULZ, S. (2007) Biosynthesis of the off-flavor 2-methylisoborneol by the myxobacterium Nannocystis exedens. Angew Chem Int Ed Engl, 46, 8287-90.
    DORGAN, K. M., WOODERCHAK, W. L., WYNN, D. P., KARSCHNER, E. L., ALFARO, J. F., CUI, Y., ZHOU, Z. S. & HEVEL, J. M. (2006) An enzyme-coupled continuous spectrophotometric assay for S-adenosylmethionine-dependent methyltransferases. Anal Biochem, 350, 249-55.
    GAGNE, F., RIDAL, J., BLAISE, C. & BROWNLEE, B. (1999) Toxicological effects of geosmin and 2-methylisoborneol on rainbow trout hepatocytes. Bulletin of Environmental Contamination and Toxicology, 63, 174-180.
    GERBER, N. N. (1979) Volatile substances from actinomycetes: their role in the odor pollution of water. CRC Crit Rev Microbiol, 7, 191-214.
    GERBER, N. N. & LECHEVALIER, H. A. (1965) Geosmin, an earthly-smelling substance isolated from actinomycetes. Appl Microbiol, 13, 935-8.
    GIGLIO, S., JIANG, J., SAINT, C. P., CANE, D. E. & MONIS, P. T. (2008) Isolation and characterization of the gene associated with geosmin production in cyanobacteria. Environ Sci Technol, 42, 8027-32.
    GIGLIO, S., MONIS, P. T. & SAINT, C. P. (2005) Legionella confirmation using real-time PCR and SYTO9 is an alternative to current methodology. Appl Environ Microbiol, 71, 8944-8.
    GUST, B., CHALLIS, G. L., FOWLER, K., KIESER, T. & CHATER, K. F. (2003) PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc Natl Acad Sci USA, 100, 1541-6.
    HAYES, K. & BURCH, M. (1989) Odorous compounds associated with algal blooms in South Australian waters. Wat Res, 23, 115-121.
    HAYES, S. J., HAYES, K. & ROBINSON, B. S. (1991) Geosmin as an odorous metabolite in cultures of a free-living amoeba, Vannella species (Gymnamoebia, Vannellidae). J Protozool, 38, 44-47.
    HO, L., HOEFEL, D., BOCK, F., SAINT, C. P. & NEWCOMBE, G. (2007) Biodegradation rates of 2-methylisoborneol (MIB) and geosmin through sand filters and in bioreactors. Chemosphere, 66, 2210-8.
    IZAGUIRRE, G., HWANG, C. J., KRASNER, S. W. & MCGUIRE, M. J. (1982) Geosmin and 2-methylisoborneol from Cyanobacteria in three water supply systems. Appl Environ Microbiol, 43, 708-714.
    IZAGUIRRE, G., TAYLOR, W. D. & PASEK, J. (1999) Off-flavor problems in two reservoirs, associated with planktonic Pseudanabaena species. Water Science and Technology, 40, 85-90.
    JIANG, J. & CANE, D. E. (2008) Geosmin biosynthesis. Mechanism of the fragmentation-rearrangement in the conversion of germacradienol to geosmin. J Am Chem Soc, 130, 428-9.
    JIANG, J., HE, X. & CANE, D. E. (2006) Geosmin Biosynthesis. Streptomyces coelicolor Germacradienol/Germacrene D Synthase Converts Farnesyl Diphosphate to Geosmin. J Am Chem Soc, 128, 8128-9.
    JIANG, J., HE, X. & CANE, D. E. (2007) Biosynthesis of the earthy odorant geosmin by a bifunctional Streptomyces coelicolor enzyme. Nat Chem Biol, 3, 711-5.
    KEEGAN, A. R., FANOK, S., MONIS, P. T. & SAINT, C. P. (2003) Cell culture-Taqman PCR assay for evaluation of Cryptosporidium parvum disinfection. Appl Environ Microbiol, 69, 2505-11.
    KOMATSU, M., TSUDA, M., OMURA, S., OIKAWA, H. & IKEDA, H. (2008) Identification and functional analysis of genes controlling biosynthesis of 2-methylisoborneol. Proc Natl Acad Sci USA, 105, 7422-7.
    LAHSER, F. C. & MALCOLM, B. A. (2004) A continuous nonradioactive assay for RNA-dependent RNA polymerase activity. Anal Biochem, 325, 247-54.
    LLOYD, S. W. & GRIMM, C. C. (1999) Analysis of 2-methylisoborneol and geosmin in catfish by microwave distillation—solid-phase microextraction. J Agric Food Chem, 47, 164-9.
    MARTIN, J. F., IZAGUIRRE, G. & WATERSTRAT, P. (1991) A planktonic Oscillatoria species from Mississippi catfish ponds that produces the off-flavour compound 2-methylisoborneol. Water Res, 25, 1447-1451.
    MCGUIRE, M. J. (1995) Off-flavour as the consumers measure of drinking water safety. Wat Sci Tech, 31, 1-8.
    NAES, H., AARNES, H., UTKILEN, H. C., NILSEN, S. & SKULBERG, O. M. (1985) Effect of photon fluence rate and specific growth rate on geosmin production of the cyanobacterium Oscillatoria brevis (Kutz.) Gom. Appl Environ Microbiol, 49, 1538-1540.
    NAES, H. & POST, A. F. (1988) Transient stages of geosmin, pigments, carbohydrates and proteins in continous cultures cultures if Oscillatoria brevis induced by changes in notrogen supply. Arch Microbiol, 150, 333-337.
    PARK, G., YU, M., GO, J., KIM, E. & KIM, H. (2007) Comparison between ozone and ferrate in oxidising geosmin and 2-MIB in water. Water Sci Technol, 55, 117-25.
    PERSSON, F., HEINICKE, G., HEDBERG, T., HERMANSSON, M. & UHL, W. (2007) Removal of geosmin and MIB by biofiltration—an investigation discriminating between adsorption and biodegradation. Environ Technol, 28, 95-104.
    RASMUSSEN, J. P., SAINT, C. P. & MONIS, P. T. (2007) Use of DNA melting simulation software for in silico diagnostic assay design: targeting regions with complex melting curves and confirmation by real-time PCR using intercalating dyes. BMC Bioinformatics, 8, 107.
    ROBINSON, B. S., MONIS, P. T. & DOBSON, P. J. (2006) Rapid, sensitive, and discriminating identification of Naegleria spp. by real-time PCR and melting-curve analysis. Appl Environ Microbiol, 72, 5857-63.
    SAADOUN, I. M., SCHRADER, K. K. & BLEVINS, W. T. (2001) Environmental and nutritional factors affecting geosmin synthesis by Anabaena sp. Water Res, 35, 1209-18.
    SCHNURER, J., OLSSON, J. & BORJESSON, T. (1999) Fungal volatiles as indicators of food and feeds spoilage. Fungal Genet Biol, 27, 209-17.
    SCHRADER, K. K. & DENNIS, M. E. (2005) Cyanobacteria and earthy/musty compounds found in commercial catfish (Ictalurus punctatus) ponds in the Mississippi Delta and Mississippi—Alabama Blackland Prairie. Water Res, 39, 2807-14.
    SCHRADER, K. K., NANAYAKKARA, N. P., TUCKER, C. S., RIMANDO, A. M., GANZERA, M. & SCHANEBERG, B. T. (2003) Novel derivatives of 9,10-anthraquinone are selective algicides against the musty-odor cyanobacterium Oscillatoria perornata. Appl Environ Microbiol, 69, 5319-27.
    SCHULZ, S., FUHLENDORFF, J. & REICHENBACH, H. (2004) Identification and synthesis of volatiles released by the myxobacterium Chondromyces crocatus. Tetrahedron, 60, 3863-3872.
    SUGIURA, N., IWAMI, N., INAMORI, Y., NISHIMURA, O. & SUDO, R. (1998) Significance of attached cyanobacteria relevant to the occurrence of musty odor in Lake Kasumigaura. Water Research, 32, 3549-3554.
    UTKILEN, H. C. & FROSHAUG, M. (1992) Geosmin production and excretion in a planktonic and benthic Oscillatoria. Wat Sci Tech, 25, 199-206.
    VAN BREEMEN, L. W. C. A., DITS, J. S. & KETELAARS, H. A. M. (1992) Production and reduction of geosmin and 2-methylisoborneol during storage of river water in deep reservoirs. Wat Sci Tech, 25, 233-240.
    VAN DER PLOEG, M. & BOYD, C. E. (1991) Gesomin production by Cyanobacteria (Blue-green algae) in fish ponds at Auburn, Ala. Journal of the World Aquaculture Society, 22, 207-216.
    VAN DER PLOEG, M., TUCKER, C. S. & BOYD, C. E. (1992) Geosmin and 2-methylisoborneol production by cyanobacteria in fish ponds in the southeastern united states. Wat Sci Tech, 25, 283-290.
    WANG, C. M. & CANE, D. E. (2008) Biochemistry and molecular genetics of the biosynthesis of the earthy odorant methylisoborneol in Streptomyces coelicolor. J Am Chem Soc, 130, 8908-9.
    WATSON, S. B. (2003) Cyanobacterial and eukaryotic algal odour compounds: signals or by-products? A review of their biological activity. Phycologia, 42, 332-350.


The teachings of all of the references cited herein are hereby incorporated by reference in their entirety.
EXEMPLIFICATION
Geosmin is a secondary metabolite responsible for earthy tastes and odors in potable water supplies. Geosmin continues to be a challenge to water utility management regimes and remains one of the most common causes of consumer complaints, as the taste of “dirty” water may suggest a failed disinfection regime and that the water may be unsafe to drink. Although cyanobacteria have been reported to be largely responsible for these taste and odor events, the answer as to how or why geosmin is produced has been elusive. Described herein is the mechanism by which geosmin is produced in a model cyanobacterium, Nostoc punctiforme PCC 73102 (ATCC 29133), in which it has been demonstrated that a single enzyme is utilized to catalyze the cyclization of farnesyl diphosphate to geosmin. Using this information, a PCR-based assay has been developed that allows the rapid detection of geosmin-producing cyanobacteria. This test may be utilized to confirm and track the emergence of taste and odor-producing cyanobacteria in any given water body and thus can be used as an early warning system by managers of water bodies that may suffer from adverse taste and odor episodes.
Globally, cyanobacteria are nuisance organisms in fresh water supplies and have been primarily responsible for taste and odor episodes as well as toxic incidents (1-4). Consumer taste and odor complaints to water utilities are high, and are second only to those concerning chlorinous taints. One of the commonest complaints, that of an “earthy” or musty odor, is largely a result of geosmin and/or 2 methylisoborneol (MIB) produced by cyanobacteria. The human taste and odor sensitivity threshold for geosmin is an extraordinarily low about 10 ng/L (10 ppt) (5) and, although geosmin has no known adverse side effects, consumers associate the detection of this compound with poorly treated water that might be unsafe to drink. The detection and management of these geosmin-producing cyanobacteria is therefore of paramount importance to water utilities. Currently, the only options to deal with this issue are dosing water bodies with environmentally unfriendly chemicals such as copper sulfate in order to control algal blooms, as well as the use of powdered activated carbon in water treatment plants to remove the responsible taste and odor compounds. Biological filtration using aged sand filters may represent a suitable alternative to remove these undesired compounds (6).
The volatile metabolite geosmin is produced by a wide range of microorganisms, including most species of Streptomyces and a variety of other actinomycetes, myxobacteria and cyanobacteria, as well as certain fungi and selected higher plants such as liverworts and beets. Although geosmin was first identified by Gerber in 1965 (7), the biochemical pathway of geosmin production remained obscure for many years. In 1981, Bentley first proposed that geosmin was likely a degraded sesquiterpene, based on the observed incorporation of labeled acetate by cultures of Streptomyces antibioticus (8). Within the last several years, however, the mechanism of geosmin biosynthesis in Streptomyces and myxobacteria (9-11) has been elucidated in detail.
Despite earlier confusion as to how many enzymes were involved, and indeed if the mechanism of geosmin production was different in different organisms, it is now known that in S. coelicolor A3(2) a single 726-amino acid protein, encoded by the 2181-bp SCO6073 gene (cyc2) (12,13), catalyzes the Mg2+-dependent cyclization of farnesyl diphosphate (FPP), the universal acyclic precursor of all sesquiterpenes, to a mixture of geosmin and the sesquiterpene alcohol germacradienol, accompanied by smaller amounts of the bicyclic hydrocarbons germacrene D and 8,10-dimethyl-1-octalin (14-17) (FIG. 1). The S. coelicolor geosmin/germacradienol synthase is in fact a bifunctional enzyme in which both the N-terminal and C-terminal halves show a high degree of sequence similarity to the well-studied 336-amino acid sesquiterpene synthase, pentalenene synthase (16). Experiments with individually expressed recombinant proteins corresponding to the N-terminal and C-terminal domains have shown that the N-terminal half of the synthase catalyzes the cyclization of FPP to a 85:15 mixture of germacradienol and germacrene D, accompanied by traces of the octalin, while the C-terminal domain catalyzes the Mg2+-dependent cyclization—fragmentation of germacradienol to geosmin, with release of the 2-propanol side chain as acetone (14,16). Site-directed mutagenesis experiments have confirmed that the N- and C-terminal domains each harbor catalytically independent active sites (16).
It has also been shown that the closely related GeoA (SAV2163) protein of S. avermitilis (about 78% sequence identity and about 85% similarity) catalyzes the same biochemical reaction (18), while more than a dozen known or presumed geosmin synthases deduced from a variety of Streptomyces, Frankia, Saccharopolyspora, and myxobacterial genome sequences share correspondingly high levels of sequence conservation over all about 730 to about 740 amino acids (about 45 to about 75% identity, about 57 to about 85% similarity). In all these proteins, the N-terminal region contains two strictly conserved motifs, a, DDHFLE (SEQ ID NO: 37) sequence, typically about 80 to about 100 amino acids, from the N-terminus and a ND(L/I)FSY(Q/E)RE (SEQ ID NO: 38) motif about 140-amino acids downstream, corresponding to the universally conserved aspartate-rich DDXXD (SEQ ID NO: 39) motif and NSE triad (N/D)DXX(S/T)XXXE (SEQ ID NO: 40), respectively, that are found in all sesquiterpene synthases and that are known to be involved in the binding of the essential cofactor Mg2+ (19,20). Similarly the C-terminal half of geosmin synthase has a canonical variant of the aspartate rich motif, DDYYP (SEQ ID NO: 41), as well as a downstream ND(V/I/L)FSYQKE (SEQ ID NO: 42) variant of the NSE triad.
Following the initial biochemical characterization of the SCO6073 gene (13) and its implication in geosmin biosynthesis (12), Ludwig et al (21) reported the use of PCR to isolate homologous genes from an environmental geosmin-producing Phormidium sp. that were similar in sequence both SCO6073 and SAV2163. Although they demonstrated that these genes were expressed in the parent cyanobacteria and speculated on the possibility of a “geosmin operon”, they did not directly correlate the expression with geosmin production nor did they explicitly demonstrate that these genes were functionally responsible for geosmin production in the Phormidium isolate examined.
The npun02003620 gene in the reported genome sequence of the cyanobacterium Nostoc punctiforme PCC 73102 (ATCC 29133), encodes a hypothetical protein ZP—00109187 with about 55% amino acid sequence similarity to the N-terminal region of SCO6073, including the presence of the universally conserved DDHFLE (SEQ ID NO: 37) and NDLFSYQRE (SEQ ID NO: 44) motifs characteristic of this class of enzyme. The predicted protein, however, consists of only 630 amino acids, about 100 amino acids shorter than the SCO6073 protein or any other known or predicted geosmin synthase. Equally importantly, although the C-terminal half of ZP—00109187 also is predicted to harbour a typical DDYFP (SEQ ID NO: 45) motif, the essential NSE triad is apparently absent.
The model cyanobacterium Nostoc punctiforme PCC 73102 (ATCC 29133) was examined first and the hypothetical protein ZP—00109187 encoded by npun02003620 was demonstrated to be, in fact, a truncated protein that, while catalyzing the conversion of FPP to germacradienol, is incapable of supporting geosmin formation. The apparent truncation has been shown to be the result of a single, but critical, sequencing error in the published DNA sequence and that the corrected open reading frame corresponds to a fully functional geosmin synthase, dubbed NPUNMOD, that is of similar length and sequence to the S. coelicolor SCO6073 enzyme and all other geosmin synthase proteins. Having established the identity and biochemical function of the Nostoc geosmin synthase gene, this information was utilized to design a PCR-based diagnostic tool for the detection of geosmin-producing cyanobacteria. Collectively, these results provide the fundamental step forward for understanding taste and odor episodes and provide a powerful tool that can be used to predict whether an emerging cyanobacterial bloom will be a geosmin producer and assist in designing strategies to limit its effects in the short term; track the emergence of taste and odor producers in the longer term; and anticipate what trends may occur in the future.
Culture of Cyanobacteria and DNA Extraction
Nostoc punctiforme PCC 73102 (ATCC 29133), a known geosmin producer, was maintained in ATCC medium #819 according to the provided product information sheet. The following geosmin- and 2-methylisoborneol (MIB)-producing isolates, confirmed by GC-MS, were a generous gift from G. Izaguirre: Pseudoanabaena limnetica (MIB producer), Anabaena laxa (geosmin producer), Nostoc sp. UTAH12-18b (geosmin producer), and Phormidium calcicola (geosmin and MIB producer). GC-MS of additional environmental isolates used in this study were performed using fresh cultures of cyanobacteria (not normalized for cell number). The above isolates were grown in BG-11 medium and maintained under standard conditions at 25° C. Before DNA extraction, cultures were subcultured on starch-casein agar for the detection of possible contaminating geosmin-producing actinomycete bacteria. All subcultures were negative for actinomycete organisms. DNA was extracted using the Qiagen DNA Mini Spin kit according to manufacturer's instructions, using 1 ml of cyanobacterial culture, with the addition of an overnight proteinase K incubation at about 56° C.
Cloning and Expression of npun020003620 and NPUNMOD Proteins, Incubation with Farnesyl Pyrophosphate, and GC-MS Analysis
The DNA sequence corresponding to npun020003620 was amplified using the primers npunstart1 (5′-ATTTTATCCATGGTTATGCAACCCTTTGAACTGCCAGAA-3′) (SEQ ID NO.:70) and npunhalt1 (5′-TAATAACTCGAGTTATGGATTTCGCCCTCG-3′) (SEQ ID NO.:71), while the full length natural NPUNMOD gene was amplified with primers npunstart1 and npunhalt4 (5′-TAATAACTCGAGTAATTGACCGAGTAATGAC-3′) (SEQ ID NO.:72), inserting NcoI and XhoI restriction sites (underlined) for the npunstart1 and npunhalt primers respectively. Fragments were PCR-amplified using proofreading Elongase Taq polymerase (Invitrogen) as described by the manufacturer, using 35 cycles consisting of 94° C. for 30 s, 55° C. for 30 s, and 68° C. for 120 s, with a final hold at 4° C. until needed. The products were digested with NcoI and XhoI (Promega, USA) and ligated into a similarly digested pET21d(+) expression vector (Novagen). The previously described protocols for propagation, expression, and purification of recombinant S. coelicolor geosmin synthase were followed for the ZP—00109187 and NPUNMOD proteins (12, 14), except that transformants were induced with 1 mM IPTG for 2 h at 35° C.
Successful transformants were sequenced using Big Dye Terminator sequencing. Recombinant ZP—00109187 protein, which did not carry a His6-tag, was obtained in soluble form, while the initially generated recombinant NPUNMOD protein, carrying a C-terminal His6-tag, was obtained as insoluble inclusion bodies that could not be resolubilized in active form despite the use of a wide variety of re-folding conditions. To remove the His6-tag, a stop codon was introduced by site directed mutagenesis using the QuikChange® Site-Directed Mutagenesis Kit (Stratagene, USA) using the mutagenic primers NPM2—58-notag (5′-TTACTCGGTCAATGATTACTCGAGCAC-3′) (SEQ ID NO.:73) and NPM2—58-notaga (5′-GTGCTCGAGTAATCATTGACCGAGTAA-3′) (SEQ ID NO.:74) (stop codon in bold, XhoI restriction sites underlined). The sequence of NPM2-58-pB32 was confirmed by resequencing (University of California, Davis Sequencing Facility, Davis, Calif., USA).
Protein expression from E. coli BL21(DE3)pLysS once again gave insoluble inclusion bodies that could not be properly solubilized under a variety of denaturation—refolding conditions. Therefore, the NPM2-58 protein was co-expressed along with the chaperone protein combination GroES/GroEL by co-transformation of the two plasmids, NPM2-58-pJJ32 (ampicillin-resistant) and pGro12 (kanamycin-resistant) into E. coli BL21(DE3)pLysS, selecting a single colony displaying ampicillin-kanamycin-chloramphenicol multiresistance. Cells from this colony were used to inoculate LB broth containing 100 μg/ml ampicillin, 50 μg/mlkanamycin and 34 μg/ml chloramphenicol (LBAKC) and the overnight culture was transferred to 500 ml of LBAKC broth and incubated at 37° C. and 250 rpm for 2 h. Chaperone expression was induced by addition of arabinose to a final concentration of 4 mg/ml and the desired protein expression was then induced by addition of IPTG to a final concentration of 0.4 mM. Incubation was continued for an additional 18 h at 18° C., 250 rpm. The cells were harvested by centrifugation and resuspended in 30 ml of lysis buffer (20 mM Tris-HCl, 10% glycerol, 0.1 mM DTT, pH 8.0). The cells were disrupted by sonication and the cell lysate was clarified by centrifugation.
The supernatant was purified by 12% ammonium sulfate precipitation followed by purification on a 25-ml n-butyl-Sepharose column that had been pre-equilibrated in buffer A (0.5 M ammonium sulfate, 50 mM NaH2PO4, 0.1 mM DTT, pH 7.0). After loading of the supernatant onto the column, the resin was washed with 60 ml of buffer A followed by a 180-ml linear gradient from buffer A to buffer B (50 mM NaH2PO4, 0.1 mM DTT, pH 7.0). The purified protein eluted in buffer B as a 1:1 mixture with chaperone protein, as determined by SDS-PAGE. The apparent molecular weight (Mr) of 82 kDa of the desired protein is very close to the theoretical MW 85 kDa. Incubation of the purified protein, free of all contaminating proteins, with FPP and subsequent GC-MS analysis were performed using the procedures described by Jiang et al (15).
Geosmin Synthase PCR, Cloning of PCR Products and Sequencing
Geosmin synthase PCR (G-PCR1) mastermix consisted of 2.5 mM MgCl2, 1×PCR buffer, 200 μM dNTP, 300 μM each of forward primer 250F (5′-TTCTTCGACGAYCACTTCC-3′) (SEQ ID NO.:1) and reverse primer 971R (5′-CCCTYGTTCATGTARCGGC-3′) (SEQ ID NO.:2), 5% dimethyl sulphoxide, 1 U of Platinum Taq DNA polymerase (Invitrogen), and 2 μl of extracted cyanobacterial DNA. Reactions were run on a Perkin Elmer 2400 thermal cycler with an initial denaturation step of about 95° C. for 5 min, followed by 55 cycles of about 95° C. for 30 s, about 55° C. for 30 s, and about 72° C. for 120 s, with a final extension step of 10 min at about 72° C. Samples were then run on a 1% agarose gel with 10 μl of SYBR Safe (Invitrogen) for 45 min at 80V and bands were visualized using a Dark reader transilluminator (Clare Chemical Research, Dolores, Colo., USA). For selected sample bands of expected size (743 bp), DNA was extracted using a Qiagen Qiaquick Gel extraction kit according to the manufacturer's instructions. The purified PCR product was then cloned into vector PCR 2.1 TOPO® according to the manufacturer's instructions (TOPO cloning kit, Invitrogen), and subsequently sequenced using Big Dye Terminator sequencing reactions.
A second geosmin synthase PCR (G-PCR2) was developed for real time PCR screening of other cyanobacterial DNA samples. Reactions were first optimized by standard thermal cycling as described above, and then 2.5 μM SYTO9 (Invitrogen) was incorporated into the mastermix. Primers used for G-PCR2 were: 288AF (5′-AACGACCTGTTCTCCTA-3′) (SEQ ID NO.:3), and 288AR (5′-GCTCGATCTCATGTGCC-3′) (SEQ ID NO.:4), generating an amplicon of 288 bp. Real time PCR was performed using a Corbett Research Rotorgene 6000HRM (Corbett Research, Australia) under the same conditions as above, except that the final extension step was replaced with a DNA melting analysis from about 75 to about 95° C., with data being acquired every degree with a 5 s hold at each step. All data were acquired on the “Green” channel, with excitation at 470 nm and emission at 510 nm.
Results and Discussion
Demonstration of Geosmin Synthase in the Model Cyanobacterium Nostoc Punctiforme 
The reported 1893-bp npun02003620 gene of Nostoc punctiforme PCC 73102 (ATCC 29133), currently annotated as encoding a hypothetical protein (ZP—00109187), has 55% amino acid sequence similarity to the N-terminal region of both known geosmin synthases, S. coelicolor A3(2) SCO6073 and S. avermitilis SAV2163, thereby suggesting the likely biochemical function of this cyanobacterial gene may be similar to the N-terminal mediated reactions of the reported Streptomyces. The reported open reading frame is, however, about 300 bp shorter than either the SCO6073 or SAV2163 gene or any of the homologous Actinomycete and myxobacterial geosmin synthase genes. The predicted ZP—00109187 protein also lacks the essential NSE triad in the C-terminal domain that is found in all other geosmin synthases as well as in all other known terpene synthases. To assess the biochemical function of the reported npun020003620 open reading frame, recombinant ZP—00109187 protein was generated based on the start and stop codons predicted by the deposited sequence, resulting in a 71 KDa protein of the expected size as determined by SDS-PAGE, which was named NJ2 (GenBank Accession No. FJ010202).
Incubation of recombinant NJ2 with FPP yielded a mixture of germacradienol (94%) and germacrene D (6%) accompanied by a trace of the octalin, but without any detectable geosmin according to the standard GC-MS analysis. The generation of germacradienol indicates that the N-terminal half of the NJ2 protein has been properly folded and has the expected germacradienol/germacrene D synthase activity, consistent with the demonstrated properties of the homologous N-terminal domain of S. coelicolor SCO6073 geosmin synthase. On the other hand, the absence of geosmin production is consistent with the apparent truncation of the C-terminal domain and the absence of a functional active site for geosmin formation. Indeed the biochemical properties of the recombinant Nostoc NJ2 protein are similar to those of the previously reported truncated mutant derived from the N-terminal half of S. coelicolor geosmin synthase, as well as variants of full-length SCO6073 protein carrying mutations in the essential C-terminal DDYYP or NSE triad regions, all of which could convert FPP to germacradienol and germacrene D but were completely defective in geosmin formation.
Comparison of the experimentally determined DNA sequence of the 3′-region of the PCR-amplified NJ2 construct with the published npun02003620 sequence revealed that the sequence recorded in the Nostoc punctiforme genome database contains an extra “T” nucleotide at position 33677, resulting in a false frame shift with the predicted insertion of the isoleucine at amino acid position 616, as well as a premature stop codon after amino acid 630 of the deduced protein ZP—00109187. Excluding this extraneous T nucleotide relieves the implied frame shift and eliminates the erroneous stop codon. The corrected open reading frame encodes an additional 126 amino acids, corresponding to a predicted length of 756 amino acids (85 kDa) (GenBank Accession No. FJ010203). Importantly, the full-length C-terminal domain of the corrected protein sequence, termed NPUNMOD, now has the universally conserved DDYFP (SEQ ID NO.:45) and NDVFSYQKE (SEQ ID NO.:75) motifs that are found in the Streptomyces geosmin synthases SCO6073 and SAV2163 (FIG. 2) as well as all other Actinomycete and myxobacterial orthologs (GenBank accession FJ010202 and FJ010203 for NJ2 and NPUNMOD). Although the PCR-amplified DNA sequence encoding recombinant NJ2 does retain the C-terminal NSE triad just before the C-terminus, the premature truncation presumably prevents proper assembly of the active site of the C-terminal half of the geosmin synthase
Recombinant full-length NPUNMOD protein was shown to be a fully functional geosmin synthase by incubation with FPP under the standard conditions. GC-MS analysis of the hexane-soluble extract confirmed the formation of a typical product mixture consisting of geosmin (4%), germacradienol (78%), germacrene D (8%), and octalin (1%) as well (E)-nerolidol (9%) (FIGS. 3A and 3B). The formation of nerolidol is unusual and may result from some degree of improper folding of the recombinant geosmin synthase or by chaperone-mediated solvolysis of FPP.
Development of a Geosmin Synthase Screening Tool
The corrected, full-length geosmin synthase gene of Nostoc punctiforme, was employed to develop a PCR-based screening procedure to evaluate the presence of geosmin synthase genes in other cyanobacterial strains. A pair of partially degenerate primers and G-PCR1 were used to amplify the target geosmin synthase genes of several cyanobacterial isolates that were directly confirmed as geosmin producers by GC-MS. The PCR primers were designed so that the forward 250F primer sequence would be anchored on the conserved aspartate-rich motif of the N-terminal domain while the 971R reverse primer flanked the NSE triad region normally found 140 amino acids downstream of the aspartate-rich motif. Using this procedure, 743-bp DNA fragments were amplified from geosmin-producing Anabaena laxa, Nostoc sp. UTAH12-18b, and Phormidium calcicola, while the diagnostic fragment was absent in the geosmin non-producing organism Pseudoanabaena limnetica. Positive PCR products were cloned and sequenced as described earlier. All positive amplicons encoded the strictly conserved Mg2+-binding motifs, DDHFLE (SEQ ID NO: 37 and NDLFSYQRE (SEQ ID NO: 44, that are found in the N-terminal domain of all geosmin synthases (FIG. 5).
A rapid real-time PCR (G-PCR2) for screening cyanobacterial isolates was developed, using primers internal to the G-PCR1 products. Seventeen isolates were screened using the PCR assay described herein, which was run in parallel with GC-MS analyses of isolate suspensions. Table 4 demonstrates that G-PCR2 is able to detect additional isolates of Phormidium, Anabaena, and Geitlerinema sp, all of which were GC-MS positive for geosmin. Moreover, the real-time G-PCR2 protocol did not amplify DNA from any GC-MS geosmin-negative isolates such as Planktothrix, Oscillatoria, and Pseudoanabaena sp. indicating that the presence of a geosmin synthase gene is invariably associated with geosmin production by the host, thereby validating the utility of the real-time PCR gene detection as a reliable diagnostic assay for cyanobacterial geosmin producers.







TABLE 4







Comparison of a geosmin synthase PCR assay and GC-MS


analysis using environmental cyanobacterial isolates












GEOSMIN



Isolate
Origin
(ng/L)
G-PCR2













Oscillatoria LM603
Lake Matthews,
<2
−



USA


Oscillatoria
Hope Valley, SA
<2
−


Oscillatoria
Hope Valley, SA
<2
−


Phormidium sp
Happy Valley, SA
112
+


Phormidium sp
Happy Valley, SA
21
+


Phormidium sp
Happy Valley, SA
4
+


Phormidium sp
Happy Valley BR,
47
+



SA


Phormidium sp
Happy Valley, SA
67
+


Phormidium sp
Happy Valley, SA
553
+


Planktothrix sp
Diamond Valley,
<2
−


DVL1003C
USA


Anabaena circinalis
Myponga, SA
152
+


ANA346B


Pseudoanabaena sp
Happy Valley, SA
<2
−


Pseudoanabaena sp
Happy Valley, SA
<2
−


Geitlerinema
Little Para, SA
3113
+


Phormidium 007D
Hope Valley, SA
198
+


Phormidium 005E
Hope Valley, SA
60
+


Phormidium 012G
Hope Valley, SA
21
+









The use of G-PCR2 and melting curve analysis has the additional advantage of differentiating among geosmin-producing species. In this PCR protocol, the positive Geitlerinema, Phormidium, and Anabaena species produce different characteristic melting profiles (FIGS. 6-9). For example, Anabaena circinalis gives rise to a single peak with a melting temperature (Tm) of about 84.5° C., while Anabaena laxa has a more complex melting pattern with two Tms; one at about 84.5° C. and another at about 82.3° C. A similarly complex melting pattern is seen for all Phormidium sp, with Tms at about 84.5 and about 87.5° C. and also for Geitlerinema that has Tms at about 84.5 and about 86.5° C. The use of melting curve analysis for species identification and genotyping is becoming increasingly popular, especially with the availability of Tm prediction software such as POLAND and MELTSIM (22). Furthermore, the observation of differential Tms from a single primer pair has been used elsewhere to characterise several Naegleria isolates in a diagnostic setting (23). The combination of real-time PCR for detection of geosmin synthase genes and Tm analysis may be particularly useful.
In conclusion, the production of geosmin in cyanobacteria has been demonstrated to be due to the presence of a single gene encoding the geosmin synthase enzyme. In addition, a diagnostic geosmin synthase PCR protocol has been developed that can be a valuable tool for use by water utilities in the detection of organisms responsible for geosmin production in any given water body. The flexibility and portability of real-time PCR equipment has previously been used to track toxic cyanobacteria in the field (24) and, therefore, it is foreseeable that this technology may be used for mobile monitoring of geosmin-producing cyanobacteria and assistance with current dosing protocols by pinpointing specific problem areas to increase the efficacy of treatment. The quantification of the abundance of geosmin synthase genes in a water body may become a valuable input into predictive modelling of water storages when coupled with chemical, physical, and physiochemical values, and the methods described herein may be used to predict the occurrence of taste and odor episodes before they become an operational problem.
The geosmin synthase gene in cyanobacteria may be employed to explore key regulatory mechanisms controlling geosmin production as a function of life-cycle and environmental conditions is now also possible, which may provide insight into strategies to better control the production of geosmin in water storages and eliminate the need to use environmentally controversial control methods such as copper sulfate dosing. Control and mitigation of taste and odor episodes is a frustratingly common event for water utilities and it is foreseeable that the tools described herein may be used as an adjunct to current monitoring programs to help better engineer a timely response to potential water management.

    1. Burlingame, G. A.; Dann, R. M.; Brock, G. L., A case study of geosmin in Philadelphia's water. Journal AWWA 1986, (Management and operations), 56-61.
    2. Carmichael, W. W.; Li, R., Cyanobacteria toxins in the Salton Sea. Saline Systems 2006, 2, 5.
    3. Izaguirre, G.; Taylor, W. D., Geosmin and 2-methlyisoborneol production in a major aqueduct system. Wat Sci Tech 1995, 31, 41-48.
    4. van Apeldoorn, M. E.; van Egmond, H. P.; Speijers, G. J.; Bakker, G. J., Toxins of cyanobacteria. Mol Nutr Food Res 2007, 51, 7-60.
    5. Cook, D.; Newcombe, G.; Sztajnbok, P., The application of powdered activated carbon for MIB and geosmin removal: predicting PAC doses in four raw waters. Water Res 2001, 35, 1325-1333.
    6. Ho, L.; Hoefel, D.; Bock, F.; Saint, C. P.; Newcombe, G., Biodegradation rates of 2-methylisoborneol (MIB) and geosmin through sand filters and in bioreactors. Chemosphere 2007, 66, 2210-2218.
    7. Bentley, R.; Meganathan, R., Geosmin and methylisoborneol biosynthesis in streptomycetes. Evidence for an isoprenoid pathway and its absence in non-differentiating isolates. FEBS Lett. 1981, 125, 220-222.
    8. Dickschat, J. S.; Bode, H. B.; Mahmud, T.; Muller, R.; Schulz, S., A novel type of geosmin biosynthesis in myxobacteria. J. Org. Chem. 2005, 70, 5174-5182.
    9. Dickschat, J. S.; Wenzel, S. C.; Bode, H. B.; Muller, R.; Schulz, S., Biosynthesis of volatiles by the myxobacterium Myxococcus xanthus. Chembiochem 2004, 5, 778-787.
    10. Schulz, S.; Fuhlendorff, J.; Reichenbach, H., Identification and synthesis of volatiles released by the myxobacterium Chondromyces crocatus. Tetrahedron 2004, 60, 3863-3872.
    11. Gerber, N, N, Lechavalier, H. A., Geosmin, an earthy smelling substance isolated from actinomyces. Appl microbiol 1956, 13(6), 935-938.
    12. Gust, B.; Challis, G. L.; Fowler, K.; Kieser, T.; Chater, K. F., PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc. Natl. Acad. Sci. USA 2003, 100, 1541-1546.
    13. Cane, D. E.; Watt, R. M., Expression and mechanistic analysis of a germacradienol synthase from Streptomyces coelicolor implicated in geosmin biosynthesis. Proc. Natl. Acad. Sci. USA 2003, 100, 1547-1551.
    14. Jiang, J.; Cane, D. E., Geosmin biosynthesis. Mechanism of the fragmentation-rearrangement in the conversion of germacradienol to geosmin. J Am Chem Soc 2008, 130, 428-429.
    15. Jiang, J.; He, X.; Cane, D. E., Geosmin Biosynthesis. Streptomyces coelicolor Germacradienol/Germacrene D Synthase Converts Farnesyl Diphosphate to Geosmin. J. Am. Chem. Soc. 2006, 128, 8128-8129.
    16. Jiang, J.; He, X.; Cane, D. E., Biosynthesis of the earthy odorant geosmin by a bifunctional Streptomyces coelicolor enzyme. Nat Chem Biol 2007, 3, 711-715.
    17. Nawrath, T.; Dickschat, J. S.; Muller, R.; Jiang, J.; Cane, D. E.; Schulz, S., Identification of (8S,9S,10S)-8,10-Dimethyl-1-octalin, a Key Intermediate in the Biosynthesis of Geosmin in Bacteria. J. Am. Chem. Soc. 2008, 130, 430-431.
    18. Cane, D. E.; He, X.; Kobayashi, S.; Omura, S.; Ikeda, H., Geosmin biosynthesis in Streptomyces avermitilis. Molecular cloning, expression, and mechanistic study of the germacradienol/geosmin synthase. J. Antibiot. (Tokyo) 2006, 59, 471-479.
    19. Christianson, D. W., Structural biology and chemistry of the terpenoid cyclases. Chem. Rev. 2006, 106, 3412-3442.
    20. Komatsu, M., Tsuda, M., Omura, S., Oikawa, H., Ikeda, H., Identification and functional analysis of genes controlling biosynthesis of 2-methylisoborneol. Proc. Natl. Acad. Sci. USA 2008, 105, 7422-7427.
    21. Ludwig, F.; Medger, A.; Bornick, H.; Opitz, M.; Lang, K.; Gottfert, M.; Roske, I., Identification and expression analyses of putative sesquiterpene synthase genes in Phormidium sp. and prevalence of geoA-like genes in a drinking water reservoir. Appl Environ Microbiol 2007, 73, 6988-6993.
    22. Rasmussen, J. P.; Saint, C. P.; Monis, P. T., Use of DNA melting simulation software for in silico diagnostic assay design: targeting regions with complex melting curves and confirmation by real-time PCR using intercalating dyes. BMC Bioinformatics 2007, 8, 107.
    23. Robinson, B. S.; Monis, P. T.; Dobson, P. J., Rapid, sensitive, and discriminating identification of Naegleria spp. by real-time PCR and melting-curve analysis. Appl Environ Microbio12006, 72, 5857-5863.
    24. Rasmussen, J. P.; Giglio, S.; Monis, P. T.; Campbell, R. J.; Saint, C. P., Development and field testing of a real-time PCR assay for cylindrospermopsin-producing cyanobacteria. J Appl Microbiol 2008, 104, 1503-15.


The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.
EQUIVALENTS
While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

## Claims

1. A method of detecting a geosmin producing microorganism and a 2-methylisoborneol producing microorganism in a sample, comprising the steps of:
a) amplifying nucleic acids in the sample in the presence of at least one geosmin synthase nucleic acid primer and at least one 2-methylisoborneol synthase nucleic acid primer to thereby generate amplified products; and
b) detecting a geosmin synthase nucleic acid and a methylisoborneol synthase nucleic acid in the amplified products,
whereby the presence of the geosmin synthase nucleic acid and the 2-methylioborneol synthase nucleic acid in the amplified products detects the geosmin producing microorganism and the 2-methylisoborneol producing microorganism in the sample.

2. The method of claim 1, further including the step of sequencing the amplified products.

3. The method of claim 1, wherein the microbial 2-methylisoborneol synthase is detected.

4. The method of claim 1, wherein the microbial 2-methylisobomeol synthase primer nucleic acid includes a primer for amplifying at least a portion of at least one member selected from the group consisting of a S-adenosylmethionine-dependent C-methyltransferase nucleic acid (2-methylgeranyl diphosphate synthase) and a terpene synthase nucleic acid.

5. The method of claim 1, wherein the sample includes at least one member selected from the group consisting of a potable water sample, an aquaculture sample and a substrate sample.

6. The method of claim 1, wherein the source of microbial contamination includes less than about 10 ng microbial geosmin per liter and less than about 10 ng microbial 2-methylisoborneol per liter.

7. The method of claim 1, wherein the geosmin producing microorganism and the 2-methylisoborneol producing microorganism in the sample is consequent to the presence of at least one cyanobacteria species selected from the group consisting of Phormidium sp., Phormidium calcicola, Anabaena circinalis, Anabaena laxa, Geirlerinema sp., Nostoc punctiforme, Nostoc sp., Pseudoanabaena limnetica, Pseudanabaena sp., Oscillatoria sp., Lyngbya sp., Planktothrix sp., Tyconema sp., Hyella sp., Anabaena sp, and Aphanizomenon sp.

8. The method of claim 1, further including the steps of:
a) amplifying nucleic acids in the sample in the presence of at least one microbial 2-methylgeranyl diphosphate synthase nucleic acid primer to thereby generate amplified products; and
b) detecting a microbial 2-methylgeranyl diphosphate synthase nucleic acid in the amplified products,
whereby the presence of the geosmin synthase nucleic acid, the 2-methylioborneol synthase nucleic acid and the microbial 2-methylgeranyl diphosphate synthase nucleic acid in the amplified products detects the geosmin producing microorganism and the 2-methylisoborneol producing microorganism in the sample.

9. The method of claim 1, wherein the geosmin synthase primer includes at least one member selected from the group consisting of SEQ ID NOS. 1-10.

10. The method of claim 1, wherein the 2-methylisobomeol synthase primer includes at least one member selected from the group consisting of SEQ ID NOS. 5, 6, 46-68 and 69.