Cyanobacteria saxitoxin gene cluster and detection of cyanotoxic organisms

Abstract

The present invention relates to methods for the detection of cyanobacteria, dinoflagellates, and in particular, methods for the detection of cyanotoxic organisms. Kits for the detection of cyanobacteria, dinoflagellates, and cyanotoxic organisms are provided. The invention further relates to methods of screening for compounds that modulate the activity of polynucleotides and/or polypeptides of the saxitoxin and cylindrospermopsin biosynthetic pathways.

1 . An isolated polynucleotide comprising a nucleotide sequence sharing at least 90% sequence homology with SEQ ID NO: 1 or a fragment thereof, wherein said fragment encodes a protein of a saxitoxin biosynthetic pathway. 
     
     
         2 . The polynucleotide according to  claim 1 , wherein said fragment comprises a nucleotide sequence sharing at least 90% sequence homology with a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, and SEQ ID NO: 68. 
     
     
         3 . (canceled) 
     
     
         4 . An isolated saxitoxin biosynthetic pathway polypeptide comprising an amino acid sequence sharing at least 90% sequence homology with a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 69. 
     
     
         5 . (canceled) 
     
     
         6 . (canceled) 
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . A method for detecting cyanotoxic organism, the method comprising the steps of obtaining a sample for use in the method and analyzing the sample for the presence of a saxitoxin (SXT) cluster gene present only in saxitoxin-producing organisms, wherein said analysing comprises detecting:
 (i) a polynucleotide comprising a sequence selected from the group consisting of: SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 36, and variants and fragments thereof,   (ii) a ribonucleic acid or complementary DNA encoded by a sequence according to (i),   (iii) a polypeptide comprising a sequence selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 37, and variants and fragments thereof,   and wherein said presence is indicative of cyanotoxic organisms in the sample.   
     
     
         10 . The method according to  claim 9 , wherein said cyanotoxic organisms are cyanobacteria. 
     
     
         11 . The method according to  claim 9 , wherein said analyzing comprises amplification of DNA from the sample by polymerase chain reaction and detecting the amplified sequences. 
     
     
         12 . The method according to  claim 11 , wherein said polymerase chain reaction utilises one or more primers comprising a sequence selected from the group consisting of SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, and variants and fragments thereof. 
     
     
         13 . The method according to  claim 9 , further comprising analyzing the sample for the presence of one or more of:
 (i) a polynucleotide comprising a sequence selected from the group consisting of: SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, and variants and fragments thereof,   (ii) a ribonucleic acid or complementary DNA encoded by a sequence according to (i),   (iii) a polypeptide comprising a sequence selected from the group consisting of: SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, and SEQ ID NO: 110, and variants and fragments thereof.   
     
     
         14 . The method according to  claim 13 , wherein said analyzing comprises amplification of DNA from the sample by polymerase chain reaction utilizing one or more primers comprising a sequence selected from the group consisting of SEQ ID NO: 111, SEQ ID NO: 112, and variants and fragments thereof. 
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . (canceled) 
     
     
         23 . A kit for the detection of cyanotoxic organisms, the kit comprising at least one agent for detecting the presence an SXT cluster gene present only in saxitoxin-producing organisms, wherein said agent detects:
 (i) a polynucleotide comprising a sequence selected from the group consisting of: SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 36, and variants and fragments thereof,   (ii) a ribonucleic acid or complementary DNA encoded by a sequence according to (i),   (iii) a polypeptide comprising a sequence selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 37, and variants and fragments thereof,   and wherein said presence is indicative of cyanotoxic organisms in the sample.   
     
     
         24 . The kit according to  claim 23 , wherein said at least one agent is a primer, antibody or probe. 
     
     
         25 . The kit according to  claim 24 , wherein said primer or probe comprises a sequence selected from the group consisting of SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, and variants and fragments thereof. 
     
     
         26 . The kit according to  claim 23 , further comprising at least one additional agent for detecting the presence of one or more of:
 (i) a polynucleotide comprising a sequence selected from the group consisting of: SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, and variants and fragments thereof,   (ii) a ribonucleic acid or complementary DNA encoded by a sequence according to (i),   (iii) a polypeptide comprising a sequence selected from the group consisting of: SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, and SEQ ID NO: 110, and variants and fragments thereof.   
     
     
         27 . The kit according to  claim 26 , wherein said at least one additional agent is a primer, antibody or probe. 
     
     
         28 . The kit according to  claim 27 , wherein said primer or probe comprises a sequence selected from the group consisting of SEQ ID NO: 109, SEQ ID NO: 110, and variants and fragments thereof. 
     
     
         29 . (canceled) 
     
     
         30 . (canceled) 
     
     
         31 . (canceled) 
     
     
         32 . (canceled) 
     
     
         33 . The method according to  claim 10 , wherein said cyanotoxic organisms are dinoflagellates. 
     
     
         34 . The kit according to  claim 23 , wherein said cyanotoxic organisms are cyanobacteria. 
     
     
         35 . The kit according to  claim 23 , wherein said cyanotoxic organisms are dinoflagellates.