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Oscillatoriales
Nostocales
100
806040200
STRR LTRR
Phormidium sp.Planktothrix rubescens
Planktothrix agardhii
Limnothrix redekeir = 0.97
Phormidium sp.
Planktothrix pseudoagardhii
100
806040200 STRR LTRRAnabaena spiroidesAnabaena planctonica
Anabaenopsis circularisAnabaena flos-aquaeAnabaena sp.
Aphanizomenon ovalisporum
r = 0.87
Anabaena circinalis gen I
Cylindrospermopsis raciborskii
Aphanizomenon gracile gen I
Aphanizomenon gracile gen IIAnabaena circinalis gen IIAphanizomenon issatschenkoi
Aphanizomenon flos-aquae
Valério et al., 2009. Microbiology 155: 642–656
STRR / LTRR FINGERPRINTING
PATTERNS
Primer Reference
STRR1F 5’- CCCCARTCCCCART -3’ Mazel et al. 1990
STRR3F 5’- CAACAGTCAACAGT -3’ Mazel et al. 1990
LTRR5’-
GTTTTAACTAACAAAAATCCCTATCAGGGATTGAAAG-3’
Masepohl et al. 1996
Identification potential
Use of repeated sequences specific from the Cyanobacteria genome to reach the identification of most isolates
01
Valério et al., 2009. Microbiology 155: 642–656
M13 / ERIC FINGERPRINTING
PATTERNS
Primer Reference
M13 5’-GAGGGTGGCGGTTCT-3’ Meyer et al. 1993
ERICERICIR (5’-ATGTAAGCTCCTGGGGATTCAC-3’)ERIC2 (5’-AAGTAAGTGACTGGGGTGAGCG-3’) Versalovic et al. 1991
Differentiation potential
Use of RADP (Random Amplification of Polymorphic DNA) primers to differentiate cyanobacteria strains
Detection of a toxic strainresident in Montargil reservoir
MontargilMontargilMontargil
100
806040200
M13 ERICLMECYA 142LMECYA 144LMECYA 91BLMECYA 127LMECYA 128LMECYA 13LMECYA 97LMECYA 92CLMECYA 110LMECYA 92ALMECYA 93LMECYA 92BLMECYA 103LMECYA 113LMECYA 3LMECYA 7LMECYA 160LMECYA 1
23-07-0123-07-0113-11-9927-11-0027-11-0011-07-9628-11-9914-11-9911-03-0013-11-9913-11-9913-11-9918-11-9928-10-0010-05-9627-06-96ND10-06-96
r = 0.94
Isolado Data de isolamento
NT
NT
NT
T
75%
1234
5
6
78
910
Genomic relationship
02
Valério et al., 2009. Microbiology 155: 642–656
M13 / ERIC FINGERPRINTING
PATTERNS
100
806040200
M13 ERICLMECYA 142LMECYA 144LMECYA 91BLMECYA 127LMECYA 128LMECYA 13LMECYA 97LMECYA 92CLMECYA 110LMECYA 92ALMECYA 93LMECYA 92BLMECYA 103LMECYA 113LMECYA 3LMECYA 7LMECYA 160LMECYA 1
23-07-0123-07-0113-11-9927-11-0027-11-0011-07-9628-11-9914-11-9911-03-0013-11-9913-11-9913-11-9918-11-9928-10-0010-05-9627-06-96ND10-06-96
= 0.94
Isolado Data de isolamento
NT
NT
NT
T
75%
1234
5
6
78
910
Genomic relationship
Temporal relationship
02
de novo genomic types
2001
2000
1999
2
1998
1997
1996
1 3 4 5 6 7 8 9 10
T NT NT NT
16S / rpoC1 PHYLOGENIES
03
rRNA 16S rpoC1
0.1
Anabaena macrospora AJ293115Anabaena spiroides AJ293116Anabaena planctonica LMECYA 177 EU078522Anabaena planctonica AB093488Anabaena solitaria AY701552Anabaena spiroides AJ293118Anabaena spiroides LMECYA 161 EU078524Anabaena circinalis LMECYA 17 EU078518Anabaena circinalis LMECYA 126 EU078521Anabaena circinalis AF247586
Aphanizomenon flos-aquae LMECYA 125 EU078542Aphanizomenon flos-aquae LMECYA 77A EU078538Aphanizomenon flos-aquae AJ630442Aphanizomenon flos-aquae AJ293126Aphanizomenon flos-aquae LMECYA 88 EU078540
Aphanizomenon flos-aquae AY196084Aphanizomenon flos-aquae LMECYA 10 EU078537
Aphanizomenon gracile AJ630445Aphanizomenon gracile LMECYA 9 EU078530Aphanizomenon gracile AJ293125Aphanizomenon gracile LMECYA 64 EU078532
Aphanizomenon gracile LMECYA 40 AY354194Aphanizomenon gracile LMECYA 148 EU078533Anabaena cylindrica AJ293119
Anabaena circinalis LMECYA 213 EU078526Anabaena circinalis 90 AJ133156Aphanizomenon issatschenkoi AJ630446Aphanizomenon issatschenkoi LMECYA 31 AY196088
Cylindrospermopsis raciborskii AF516739Cylindrospermopsis raciborskii LMECYA 134 AY699989Anabaena flos-aquae AF317628Anabaena flos-aquae LMECYA 165 EU078523
Anabaena sp. LMECYA 182 EU078545Anabaenopsis circularis LMECYA 205 EU078528Anabaenopsis circularis LMECYA 206 EU078529Anabaenopsis sp. AY038033
Nodularia spumigena DQ185241Nostoc linckia AB074503
Tolypothrix sp. AB093486Fischerela muscicola AB074505Nostochopsis sp. AJ544081Chlorogloeopsis fritschii AB093489
Stigonema ocellatum AJ544082Chroococcidiopsis sp. AB074506
Scytonema sp. AB093483Pseudanabaena sp. AF091108Planktothrix agardhii AB045958Planktothrix agardhii LMECYA 153E EU078515Planktothrix rubescens AB045950Planktothrix rubescens LMECYA 203 DQ497635
Planktothrix pseudagardhii LMECYA 162 EU078517Planktothrix pseudagardhii AB045922
Spirulina platensis AY672713Prochloron sp. X63141
Microcystis aeruginosa LMECYA 81 EU078493Microcystis aeruginosa LMECYA 110 EU078499Microcystis aeruginosa LMECYA 2 EU078483Microcystis aeruginosa LMECYA 8 EU078486Microcystis aeruginosa LMECYA 136 EU078500Microcystis ichthyoblabe AB035550Microcystis aeruginosa LMECYA 170 EU078505Microcystis aeruginosa Z28699Microcystis aeruginosa LMECYA 159 EU078504Microcystis aeruginosa LMECYA 188 EU078506Microcystis aeruginosa LMECYA 91B EU078497Microcystis novacekii AB012337Microcystis aeruginosa LMECYA 151 EU078502Microcystis wesenbergii AB012334Microcystis viridis AB035552Microcystis aeruginosa LMECYA 157 EU078503Microcystis aeruginosa LMECYA 82 EU078494Microcystis aeruginosa LMECYA 142 EU078501
Synechocystis sp. LMECYA 68 EU078508Synechocystis sp. AB041937
Gloeocapsa sp. AB039000Dermocarpella incrassata AJ344559
Xenococcus sp. AB074510Pleurocapsa sp. AB074511Myxosarcina sp. AJ344562
Stanieria cyanosphaera AB039008Limnothrix redekei LMECYA 145 EU078512Limnothrix redekei AJ505943
Prochlorothix hollandica AF132792Phormidium sp. LMECYA 79 EU078509Phormidium sp. LMECYA 173 EU078510
LeptolyngbyaAB039012Phormidium sp. LMECYA 214 EU078511Phormidium sp. AB058219
Synechococcus nidulans LMECYA 156 EU078507Prochlorococcus marinus NC007577
Synechococcus sp. AY151250Cyanobium sp. AY183115
Gloeobacter violaceus NC005125E. coli NC000913
0.801.001.001.00
1.00
1.00
1.00
1.001.00
0.97
0.97
0.89
1.00
1.00
0.99
1.00
1.001.00
1.00
0.67
1.00
1.00
1.00
0.99
1.00
1.001.00
0.75
0.93
0.98
1.00
0.66
1.00
1.001.00
1.00
1.00
0.89
1.00
0.74
1.000.961.00
0.91
0.96
1.00
1.00
0.99
1.000.61
1.00
1.00
0.96
0.990.92
1.00
1.00
1.00
1.00
1.00
1.00
1.00
Nostocales
Oscillatoriales
Chroococcales
Oscillatoriales
Chroococcales
Microcystis aeruginosa LMECYA 2 EU078483
Nostocales
0.1
Anabaena macrospora AJ293115Anabaena spiroides AJ293116Anabaena planctonica LMECYA 177 EU078522Anabaena planctonica AB093488Anabaena solitaria AY701552Anabaena spiroides AJ293118Anabaena spiroides LMECYA 161 EU078524Anabaena circinalis LMECYA 17 EU078518Anabaena circinalis LMECYA 126 EU078521Anabaena circinalis AF247586
Aphanizomenon flos-aquae LMECYA 125 EU078542Aphanizomenon flos-aquae LMECYA 77A EU078538Aphanizomenon flos-aquae AJ630442Aphanizomenon flos-aquae AJ293126Aphanizomenon flos-aquae LMECYA 88 EU078540
Aphanizomenon flos-aquae AY196084Aphanizomenon flos-aquae LMECYA 10 EU078537
Aphanizomenon gracile AJ630445Aphanizomenon gracile LMECYA 9 EU078530Aphanizomenon gracile AJ293125Aphanizomenon gracile LMECYA 64 EU078532
Aphanizomenon gracile LMECYA 40 AY354194Aphanizomenon gracile LMECYA 148 EU078533Anabaena cylindrica AJ293119
Anabaena circinalis LMECYA 213 EU078526Anabaena circinalis 90 AJ133156Aphanizomenon issatschenkoi AJ630446Aphanizomenon issatschenkoi LMECYA 31 AY196088
Cylindrospermopsis raciborskii AF516739Cylindrospermopsis raciborskii LMECYA 134 AY699989Anabaena flos-aquae AF317628Anabaena flos-aquae LMECYA 165 EU078523
Anabaena sp. LMECYA 182 EU078545Anabaenopsis circularis LMECYA 205 EU078528Anabaenopsis circularis LMECYA 206 EU078529Anabaenopsis sp. AY038033
Nodularia spumigena DQ185241Nostoc linckia AB074503
Tolypothrix sp. AB093486Fischerela muscicola AB074505Nostochopsis sp. AJ544081Chlorogloeopsis fritschii AB093489
Stigonema ocellatum AJ544082Chroococcidiopsis sp. AB074506
Scytonema sp. AB093483Pseudanabaena sp. AF091108Planktothrix agardhii AB045958Planktothrix agardhii LMECYA 153E EU078515Planktothrix rubescens AB045950Planktothrix rubescens LMECYA 203 DQ497635
Planktothrix pseudagardhii LMECYA 162 EU078517Planktothrix pseudagardhii AB045922
Spirulina platensis AY672713Prochloron sp. X63141
Microcystis aeruginosa LMECYA 81 EU078493Microcystis aeruginosa LMECYA 110 EU078499Microcystis aeruginosa LMECYA 2 EU078483Microcystis aeruginosa LMECYA 8 EU078486Microcystis aeruginosa LMECYA 136 EU078500Microcystis ichthyoblabe AB035550Microcystis aeruginosa LMECYA 170 EU078505Microcystis aeruginosa Z28699Microcystis aeruginosa LMECYA 159 EU078504Microcystis aeruginosa LMECYA 188 EU078506Microcystis aeruginosa LMECYA 91B EU078497Microcystis novacekii AB012337Microcystis aeruginosa LMECYA 151 EU078502Microcystis wesenbergii AB012334Microcystis viridis AB035552Microcystis aeruginosa LMECYA 157 EU078503Microcystis aeruginosa LMECYA 82 EU078494Microcystis aeruginosa LMECYA 142 EU078501
Synechocystis sp. LMECYA 68 EU078508Synechocystis sp. AB041937
Gloeocapsa sp. AB039000Dermocarpella incrassata AJ344559
Xenococcus sp. AB074510Pleurocapsa sp. AB074511Myxosarcina sp. AJ344562
Stanieria cyanosphaera AB039008Limnothrix redekei LMECYA 145 EU078512Limnothrix redekei AJ505943
Prochlorothix hollandica AF132792Phormidium sp. LMECYA 79 EU078509Phormidium sp. LMECYA 173 EU078510
LeptolyngbyaAB039012Phormidium sp. LMECYA 214 EU078511Phormidium sp. AB058219
Synechococcus nidulans LMECYA 156 EU078507Prochlorococcus marinus NC007577
Synechococcus sp. AY151250Cyanobium sp. AY183115
Gloeobacter violaceus NC005125E. coli NC000913
0.801.001.001.00
1.00
1.00
1.00
1.001.00
0.97
0.97
0.89
1.00
1.00
0.99
1.00
1.001.00
1.00
0.67
1.00
1.00
1.00
0.99
1.00
1.001.00
0.75
0.93
0.98
1.00
0.66
1.00
1.001.00
1.00
1.00
0.89
1.00
0.74
1.000.961.00
0.91
0.96
1.00
1.00
0.99
1.000.61
1.00
1.00
0.96
0.990.92
1.00
1.00
1.00
1.00
1.00
1.00
1.00
Nostocales
Oscillatoriales
Chroococcales
Oscillatoriales
Chroococcales
Microcystis aeruginosa LMECYA 2 EU078483
Nostocales
0.1
Anabaena circinalis LMECYA 17 EU078447Anabaena circinalis LMECYA 126 EU078460Anabaena circinalis AF159373Anabaena circinalis gen IA AF199424Anabaena spiroides LMECYA 161 EU078470Anabaena spiroides AF199427Anabaena solitaria AF199426
Anabaena planctonica AB096729Anabaena planctonica LMECYA 177 EU078474
Aphanizomenon gracile LMECYA 40 EU078449Aphanizomenon gracile LMECYA 64 EU078450Aphanizomenon gracile LMECYA 148 EU078465
Anabaena circinalis LMECYA 213 EU078480Anabaena circinalis AY424996
Aphanizomenon gracile LMECYA 9 EU078445Aphanizomenon issatschenkoi LMECYA 31 EU078448Aphanizomenon flos-aquae LMECYA 77A EU078452
Anabaena cylindrica AB074793Aphanizomenon flos-aquae LMECYA 125EU078459Aphanizomenon flos-aquae LMECYA 10 EU078446Aphanizomenon flos-aquae LMECYA 88 EU078456Anabaena flos-aquae LMECYA 165 EU078471Anabaena flos-aquae AF199429Aphanizomenon gracile AF199431
Anabaena sp. LMECYA 182 EU078475Cylindrospermopsis raciborskii LMECYA 134 EU078461Cylindrospermopsis raciborskii AF159371
Anabaenopsis circularis LMECYA 205 EU078478Anabaenopsis circularis LMECYA 206 EU078479
Chlorogloeopsis fritschii AB096735Fischerella muscicola AB074804
Scytonema sp. AB096731Chroococcidiopsis sp. AB074809
Tolypothrix sp. AB096730Nodularia spumigena AY424999
Anabaena variabilis AB074795Nostoc linckia AB074792Planktothrix agardhii LMECYA 153E EU078467Planktothrix agardhii AB074790Planktothrix agardhii AY425002Planktothrix rubescens LMECYA 203 EU078477
Spirulina platensis AB074788Microcystis aeruginosa LMECYA 188 EU078476Microcystis viridis AY425001Microcystis aeruginosa LMECYA 91B EU078457Microcystis aeruginosa LMECYA 2 EU078443Microcystis aeruginosa LMECYA 8 EU078444Microcystis aeruginosa LMECYA 157 EU078468Microcystis aeruginosa LMECYA 110 EU078458Microcystis aeruginosa LMECYA 170 EU078472Microcystis aeruginosa AY425000Microcystis aeruginosa LMECYA 142 EU078463Microcystis aeruginosa LMECYA 82 EU078455Microcystis aeruginosa LMECYA 159 EU078469Microcystis aeruginosa LMECYA 81 EU078454Microcystis wesenbergii AB074794Microcystis aeruginosa LMECYA 136 EU078462Microcystis aeruginosa LMECYA 151 EU078466
Synechocystis sp. LMECYA 68 EU078451Synechocystis sp. BA000022
Dermocarpa sp. U52341Xenococcus sp. AB074803
Pleurocapsa sp. AB074806Stanieria cyanosphaera AB074800
Prochloron sp. Z11161Phormidium sp. LMECYA 79 EU078453Phormidium sp. LMECYA 173 EU078473
Leptolyngbya sp. AB074808Synechococcus sp. AF448112
Phormidium sp. AB074798Cyanobium sp. AJ621014
Prochlorococcus marinus NC005071Prochlorothrix hollandica Z11154
Synechococcus elongatus NC006576Pseudanabaena sp. AB074799
Limnothrix redekei LMECYA 145 EU078464Gloeobacter violaceus NC005125Gloeobacter violaceus U52340
E. coli NC000913
0.99
0.51
0.990.99
0.63
1.00
0.66
1.00
1.00
1.000.97
1.00
0.761.00
1.00
1.00
1.001.00
1.00
0.64
1.00
0.550.72
0.94
1.00
0.501.00
1.00
0.94
1.00
1.000.99
0.981.00
1.00
0.66
1.00
0.92
1.000.99
0.98
0.51
0.84
1.00
0.99
0.99
0.85
1.00
1.00
Nostocales
Nostocales
Oscillatoriales
Chroococcales
Oscillatoriales
OscillatorialesChroococcales
Nostocales
0.1
Anabaena circinalis LMECYA 17 EU078447Anabaena circinalis LMECYA 126 EU078460Anabaena circinalis AF159373Anabaena circinalis gen IA AF199424Anabaena spiroides LMECYA 161 EU078470Anabaena spiroides AF199427Anabaena solitaria AF199426
Anabaena planctonica AB096729Anabaena planctonica LMECYA 177 EU078474
Aphanizomenon gracile LMECYA 40 EU078449Aphanizomenon gracile LMECYA 64 EU078450Aphanizomenon gracile LMECYA 148 EU078465
Anabaena circinalis LMECYA 213 EU078480Anabaena circinalis AY424996
Aphanizomenon gracile LMECYA 9 EU078445Aphanizomenon issatschenkoi LMECYA 31 EU078448Aphanizomenon flos-aquae LMECYA 77A EU078452
Anabaena cylindrica AB074793Aphanizomenon flos-aquae LMECYA 125EU078459Aphanizomenon flos-aquae LMECYA 10 EU078446Aphanizomenon flos-aquae LMECYA 88 EU078456Anabaena flos-aquae LMECYA 165 EU078471Anabaena flos-aquae AF199429Aphanizomenon gracile AF199431
Anabaena sp. LMECYA 182 EU078475Cylindrospermopsis raciborskii LMECYA 134 EU078461Cylindrospermopsis raciborskii AF159371
Anabaenopsis circularis LMECYA 205 EU078478Anabaenopsis circularis LMECYA 206 EU078479
Chlorogloeopsis fritschii AB096735Fischerella muscicola AB074804
Scytonema sp. AB096731Chroococcidiopsis sp. AB074809
Tolypothrix sp. AB096730Nodularia spumigena AY424999
Anabaena variabilis AB074795Nostoc linckia AB074792Planktothrix agardhii LMECYA 153E EU078467Planktothrix agardhii AB074790Planktothrix agardhii AY425002Planktothrix rubescens LMECYA 203 EU078477
Spirulina platensis AB074788Microcystis aeruginosa LMECYA 188 EU078476Microcystis viridis AY425001Microcystis aeruginosa LMECYA 91B EU078457Microcystis aeruginosa LMECYA 2 EU078443Microcystis aeruginosa LMECYA 8 EU078444Microcystis aeruginosa LMECYA 157 EU078468Microcystis aeruginosa LMECYA 110 EU078458Microcystis aeruginosa LMECYA 170 EU078472Microcystis aeruginosa AY425000Microcystis aeruginosa LMECYA 142 EU078463Microcystis aeruginosa LMECYA 82 EU078455Microcystis aeruginosa LMECYA 159 EU078469Microcystis aeruginosa LMECYA 81 EU078454Microcystis wesenbergii AB074794Microcystis aeruginosa LMECYA 136 EU078462Microcystis aeruginosa LMECYA 151 EU078466
Synechocystis sp. LMECYA 68 EU078451Synechocystis sp. BA000022
Dermocarpa sp. U52341Xenococcus sp. AB074803
Pleurocapsa sp. AB074806Stanieria cyanosphaera AB074800
Prochloron sp. Z11161Phormidium sp. LMECYA 79 EU078453Phormidium sp. LMECYA 173 EU078473
Leptolyngbya sp. AB074808Synechococcus sp. AF448112
Phormidium sp. AB074798Cyanobium sp. AJ621014
Prochlorococcus marinus NC005071Prochlorothrix hollandica Z11154
Synechococcus elongatus NC006576Pseudanabaena sp. AB074799
Limnothrix redekei LMECYA 145 EU078464Gloeobacter violaceus NC005125Gloeobacter violaceus U52340
E. coli NC000913
0.99
0.51
0.990.99
0.63
1.00
0.66
1.00
1.00
1.000.97
1.00
0.761.00
1.00
1.00
1.001.00
1.00
0.64
1.00
0.550.72
0.94
1.00
0.501.00
1.00
0.94
1.00
1.000.99
0.981.00
1.00
0.66
1.00
0.92
1.000.99
0.98
0.51
0.84
1.00
0.99
0.99
0.85
1.00
1.00
0.1
Anabaena circinalis LMECYA 17 EU078447Anabaena circinalis LMECYA 126 EU078460Anabaena circinalis AF159373Anabaena circinalis gen IA AF199424Anabaena spiroides LMECYA 161 EU078470Anabaena spiroides AF199427Anabaena solitaria AF199426
Anabaena planctonica AB096729Anabaena planctonica LMECYA 177 EU078474
Aphanizomenon gracile LMECYA 40 EU078449Aphanizomenon gracile LMECYA 64 EU078450Aphanizomenon gracile LMECYA 148 EU078465
Anabaena circinalis LMECYA 213 EU078480Anabaena circinalis AY424996
Aphanizomenon gracile LMECYA 9 EU078445Aphanizomenon issatschenkoi LMECYA 31 EU078448Aphanizomenon flos-aquae LMECYA 77A EU078452
Anabaena cylindrica AB074793Aphanizomenon flos-aquae LMECYA 125EU078459Aphanizomenon flos-aquae LMECYA 10 EU078446Aphanizomenon flos-aquae LMECYA 88 EU078456Anabaena flos-aquae LMECYA 165 EU078471Anabaena flos-aquae AF199429Aphanizomenon gracile AF199431
Anabaena sp. LMECYA 182 EU078475Cylindrospermopsis raciborskii LMECYA 134 EU078461Cylindrospermopsis raciborskii AF159371
Anabaenopsis circularis LMECYA 205 EU078478Anabaenopsis circularis LMECYA 206 EU078479
Chlorogloeopsis fritschii AB096735Fischerella muscicola AB074804
Scytonema sp. AB096731Chroococcidiopsis sp. AB074809
Tolypothrix sp. AB096730Nodularia spumigena AY424999
Anabaena variabilis AB074795Nostoc linckia AB074792Planktothrix agardhii LMECYA 153E EU078467Planktothrix agardhii AB074790Planktothrix agardhii AY425002Planktothrix rubescens LMECYA 203 EU078477
Spirulina platensis AB074788Microcystis aeruginosa LMECYA 188 EU078476Microcystis viridis AY425001Microcystis aeruginosa LMECYA 91B EU078457Microcystis aeruginosa LMECYA 2 EU078443Microcystis aeruginosa LMECYA 8 EU078444Microcystis aeruginosa LMECYA 157 EU078468Microcystis aeruginosa LMECYA 110 EU078458Microcystis aeruginosa LMECYA 170 EU078472Microcystis aeruginosa AY425000Microcystis aeruginosa LMECYA 142 EU078463Microcystis aeruginosa LMECYA 82 EU078455Microcystis aeruginosa LMECYA 159 EU078469Microcystis aeruginosa LMECYA 81 EU078454Microcystis wesenbergii AB074794Microcystis aeruginosa LMECYA 136 EU078462Microcystis aeruginosa LMECYA 151 EU078466
Synechocystis sp. LMECYA 68 EU078451Synechocystis sp. BA000022
Dermocarpa sp. U52341Xenococcus sp. AB074803
Pleurocapsa sp. AB074806Stanieria cyanosphaera AB074800
Prochloron sp. Z11161Phormidium sp. LMECYA 79 EU078453Phormidium sp. LMECYA 173 EU078473
Leptolyngbya sp. AB074808Synechococcus sp. AF448112
Phormidium sp. AB074798Cyanobium sp. AJ621014
Prochlorococcus marinus NC005071Prochlorothrix hollandica Z11154
Synechococcus elongatus NC006576Pseudanabaena sp. AB074799
Limnothrix redekei LMECYA 145 EU078464Gloeobacter violaceus NC005125Gloeobacter violaceus U52340
E. coli NC000913
0.99
0.51
0.990.99
0.63
1.00
0.66
1.00
1.00
1.000.97
1.00
0.761.00
1.00
1.00
1.001.00
1.00
0.64
1.00
0.550.72
0.94
1.00
0.501.00
1.00
0.94
1.00
1.000.99
0.981.00
1.00
0.66
1.00
0.92
1.000.99
0.98
0.51
0.84
1.00
0.99
0.99
0.85
1.00
1.00
Nostocales
Nostocales
Oscillatoriales
Chroococcales
Oscillatoriales
OscillatorialesChroococcales
Nostocales
Valério et al., 2009. Microbiology 155: 642–656
Species identification
Toxins molecular markersMCY MARKERS
04
10 20 30 40 50 55 Kb
mcy Cmcy Bmcy Amcy Dmcy Emcy Fmcy Gmcy J I H
Microcystis aeruginosa (Tillet et al. Chem. Biol., 2000)
mcy Cmcy Bmcy Amcy D mcy Emcy T mcy G mcyJmcyH
Planktothrix agardhii (Christiansen et al. J. Bacteriol., 2003)
PS PKS Other functions
mcy Cmcy Bmcy Amcy Dmcy EF mcy Gmcy JImcy H
Anabaena circinalis (Rouhiainen et al. Appl. Environ. Microbiol., 2004)
Legend:
0
300 bp
541 bp 955 bp Mikalsen et al. J. Bacteriol., 2003
Hisbergues et al. Arch. Microbiol., 2003
Non-Ribosomal Peptide Synthesis
Microcystins
Microcystins gene cluster
Toxins molecular markersMCY MARKERS
Valério et al., 2010 Environ Toxicol. 25(3):251-260
04
Validation using 124 LMECYA culture collection isolates
LMECYA 2LMECYA 3LMECYA 7LMECYA 53LMECYA 55LMECYA 56LMECYA 57LMECYA 59LMECYA 87LMECYA 92ALMECYA 92BLMECYA 92CLMECYA 93LMECYA 103LMECYA 106LMECYA 107LMECYA 108LMECYA 110LMECYA 113LMECYA 151LMECYA 159LMECYA 160LMECYA 167LMECYA 170LMECYA 179LMECYA 203
1 Kb plus
M. aeruginosa
P. rubescensUsing only one or two mcy markers:some false positives are obtained
Using the multiplex PCR for the 3 mcy markers: no false-positives
LMECYA 123LMECYA 175LMECYA 177LMECYA 31
LMECYA 40LMECYA 64LMECYA 77ALMECYA 88LMECYA 89
LMECYA 166
LMECYA 134Marau 1LMECYA 91BLMECYA 137LMECYA 144LMECYA 183LMECYA 145LMECYA 152LMECYA 153ELMECYA 153FLMECYA 127
1 Kb plus
Anabaena circinalisAnabaena planctonica
Aphanizomenon issatschenkoi
Aphanizomenon gracile
Aphanizomenon flos-aquae
Cylindrospermopsis raciborskii
Microcystis aeruginosa
Limnothrix redekei
Planktothrix agardhii
Microcystis aeruginosa
Sensitivity: 92.3% Specificity: 100%
Toxins molecular markersMCY MARKERS
Valério et al., 2010 Environ Toxicol. 25(3):251-60
04
Odivelas Reservoir: positive (3 bands)ELISA: 2.03 g/L
Monte Novo Reservoir: negative (1 band)ELISA: negative
Application to environmental samples
Sampling
Odivelas
Reservoirs
Monte Novo
Toxins molecular markersCYL MARKERS
04
Non-Ribosomal Peptide Synthesis
Cylindrospermopsins
Cylindrospermopsin gene cluster
PSPKSAmidinotransferase
Legend:
Shalev-Alon et al. FEMS Microbiol. Lett., 2002
Aphanizomenon ovalisporum
* Schembri et al. Environm. Toxicol., 2001
# Fergusson & Saint Environm. Toxicol., 2003
650 bp* 600 bp*
422 bp# 597 bp#
“PKS fragment” “PS fragment”
Komarek 2013 Cyanoprokaryota 3. Teil/3rd Part: Heterocytous genera
Toxins molecular markersCYL MARKERS
Valério et al., 2005 Harmful Algae . 4(6):1044-1052
04
T T
PS gene
PKS gene
Validation using 120 LMECYA culture collection isolates
2 positive controls: Aphanizomenon ovalisporumCylindrospermopsis raciborskii
118 non-toxic
Sensitivity: 100% Specificity: 100%
05 Expression levels MCY MARKERS
Salvador et al., 2016 J Microbiol Methods. 123:4-12
Effect of light intensity on gene expression
Legend:4 µmol photons m-2 s-1
20 µmol photons m-2 s-1
30 µmol photons m-2 s-1
Planktothrix agardhii Microcystis aeruginosa
Light intensities tested had distinct influences on microcystin production and mcyA expression levels, presenting considerabledifferences between P. agardhii and M. aeruginosa.
The verified trend concerning the increase of the mcyA expression levels prior to the increase in microcystins content per cellneed further confirmation, however it could help water management authorities foresee the peaks of toxicity, which would bepreceded by a peak in mcyA transcripts.
06 Real-Time PCRCELL COUNTS
Churro et al., 2012 Arch Microbiol. 194(9):749-757
Correlation between Ct values and log cell numbers from serial dilutions of DNA extracts from P. agardhii LMECYA 153B
Correlation between optical light microscopy cellcount numbers and real-time qPCR estimative cellconcentration for a serial tenfold dilutions range between 79,102 and 8 cells / µL of P. agardhiiLMECYA 153B
Using Real-Time PCR to determine cellcounts – Planktothrix agardhii
06 Real-Time PCRCELL COUNTS
Churro et al., 2012 Arch Microbiol. 194(9):749-757
P. agardhii quantification in environmental samples
06 Real-Time PCRMONITORING
Churro et al., 2015 4ºCIC
2 year monitoring of a reservoir (2012-2014)
Real-Time quantification of:- rpoC1 specific for Planktothrix (Churro et al. 2012)
Microcystins (ug.L
-1)Gen
e co
pies
nº.m
L-1
Time2012 2014
rpoC1 gene (copies.mL-1)
06 Real-Time PCRMONITORING
Churro et al., 2015 4ºCIC
Real-Time quantification of:- mcyA for cyanobacteria (Hisbergues et al. 2003)
2 year monitoring of a reservoir (2012-2014)
Microcystins (ug.L
-1)Gen
e co
pies
nº.m
L-1
Time2012 2014
mcyA gene (copies.mL-1)
06 Real-Time PCRMONITORING
Churro et al., 2015 4ºCIC
Real-Time quantification of:- mcyA specific for Planktothrix (Briand et al. 2008)
2 year monitoring of a reservoir (2012-2014)
Microcystins (ug.L
-1)Gen
e co
pies
nº.m
L-1
mcyA gene (copies.mL-1)
Time2012 2014
Spearman correlation coeficient0.84
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