Appearance of Planktothrix rubescens Bloom with [D-Asp3, Mdha7]MC-RR in Gravel Pit Pond of a Shallow Lake-Dominated Area

Toxins 2013, 5, 2434-2455; doi:10.3390/ oxins5122434 oxins ISSN 2072-6651 www.mdpi.com/jou nal/ oxins A icle Appea ance o Plank o h ix ubescens Bloom wi h [D-Asp3, Mdha7]MC–RR in G a el Pi Pond o a Shallow Lake-Domina ed A ea Gábo Vasas 1,*, Oszká Fa kas 1, Gábo Bo ics 2, Tamás Fel öldi 3, Gábo S amkó 1,4, Gyula Ba a 5, Is án Bácsi 6 and Sándo Gonda 1 1 Depa men o Bo any, Uni e si y o Deb ecen, Egye em é 1, Deb ecen H-4032, Hunga y; E-Mails: [email p o ec ed] (O.F.); [email p o ec ed] (G.S.); [email p o ec ed] (S.G.) 2 MTA Cen e o Ecological Resea ch, Depa men o Tisza Resea ch, 18/c. Bem squa e, Deb ecen H-4026, Hunga y; E-Mail: bo[email p o ec ed] 3 Depa men o Mic obiology, Eö ös Lo ánd Uni e si y, Pázmány Pé e sé ány 1/C, Budapes H-1117, Hunga y; E-Mail: [email p o ec ed] 4 MTA-ELTE-MTM Ecology Resea ch G oup, Pázmány Pé e sé ány 1/C., H1117 Budapes , Hunga y 5 Depa men o O ganic chemis y, Uni e si y o Deb ecen, Egye em é 1., Deb ecen H-4032, Hunga y; E-Mail: [email p o ec ed] 6 Depa men o Hyd obiology, Uni e si y o Deb ecen, Egye em é 1, Deb ecen H-4032, Hunga y; E-Mail: is [email protected] * Au ho o whom co espondence should be add essed; E-Mail: asas.ga[email p o ec ed]; Tel.: +36-52-512-900/62632; Fax: +36-52-512-943. Recei ed: 10 Sep embe 2013; in e ised o m: 3 Decembe 2013 / Accep ed: 4 Decembe 2013 / Published: 12 Decembe 2013 Abs ac : Blooms o oxic cyanobac e ia a e well-known phenomena in many egions o he wo ld. Mic ocys in (MC), he mos equen cyanobac e ial oxin, is p oduced by en i ely di e en cyanobac e ia, including unicellula , mul icellula ilamen ous, he e ocy ic, and non-he e ocy ic bloom- o ming species. Plank o h ix is one o he mos impo an MC-p oducing gene a in empe a e lakes. The eddish colo o cyanobac e ial blooms iewed in a g a el pi pond wi h he appea ance o a dense 3 cm hick laye (bio olume: 28.4 mm3 L−1) was an unexpec ed obse a ion in he shallow lake-domina ed allu ial egion o he Ca pa hian Basin. [D-Asp3, Mdha7]MC–RR was iden i ied om he blooms sample by MALDI-TOF and NMR. Concen a ions o [D-Asp3, Mdha7]MC–RR OPEN ACCESS Toxins 2013, 5 2435 we e measu ed by capilla y elec opho esis o compa e he mic ocys in con en o he ield samples and he isola ed, labo a o y-main ained P. ubescens s ain. In analyzing he MC gene clus e o he isola ed P. ubescens s ain, a dele ion in he space egion be ween mcyE and mcyG and an inse ion we e loca ed in he space egion be ween mcyT and mcyD. The inse ion elemen s we e sequenced and pa ly iden i ied. Al hough some in asi e opical cyanobac e ial species ha e been gi en a g ea deal o a en ion in many ecen s udies, ou esul s d aw a en ion o he sp ead o he alpine o ganism P. ubescens as a MC-p oducing, bloom- o ming species. Keywo ds: Plank o h ix; wa e bloom; mic ocys ins; MALDI-TOF; cyanobac e ia 1. In oduc ion Blooms o pho oau o ophic o ganisms, like algae and cyanobac e ia, a e well-known phenomena ha ha e been ound in many ypes o esh and ma ine wa e s o e he pas ew decades [1,2]. Nea o he spec acula discolo a ion o he habi a s, se e al unpleasan accompanying incidences we e de ec ed wi h heal h and economic consequences, such as human and animal poisonings, ish-kills, and decline in quali y o d inking wa e [3]. Many cyanobac e ial and algal s ains can p oduce se e al oxic me aboli es wi h di e se chemis y and bioac i i y which may cause hese p oblems [4,5]. While he ha m ul algal blooms (HAB) a e mainly domina ed by euka yo ic algal species (Dinophyceae, Bacilla iophyceae) in ma ine wa e s, cyanobac e ia occu much mo e equen ly in eshwa e s and cause hese phenomena [6,7]. Mic ocys in (MC) as he mos equen cyanobac e ial oxin is p oduced by en i ely di e en cyanobac e ia, including unicellula , mul icellula ilamen ous, he e ocy ic, and non-he e ocy ic bloom- o ming species. MCs a e syn hesized ia non- ibosomal pep ide syn he ases (NRPS) and polyke ide syn hases (PKS) assembled in o la ge mul i unc ional p o eins encoded by he mcy gene clus e [8]. The gene al chemical s uc u e o MC is cyclo (D-Ala1,X2,D-MeAsp3,Z4,Adda5, D-Glu6,Mdha7), whe e D-MeAsp is he non-p o einogenic amino acid D-e y h o-iso-aspa ic acid (me hyl aspa a e), Mdha is N-me hyl-dehyd oalanine and Adda is an amino acid wi h a C10-chain: (2S,3S,8S,9S)-3-amino-9-me hoxy-2,6,8- ime hyl-10-phenyldeca-4,6-dienoic acid. X and Z ep esen a iable L-amino acids in posi ions 2 and 4, espec i ely [5]. Recen ly, p og ess has been made in he elucida ion o he gene ic basis o MC syn hesis o all h ee main MC p oduce s occu ing in eshwa e , i.e., Anabaena, Mic ocys is and Plank o h ix. Th ee gene clus e s esponsible o he biosyn hesis o MCs, con aining 9 o 10 genes (depending on he genus) and spanning 55 kb, ha e been sequenced. The co esponding genes o Mic ocys is ae uginosa K-139 and PCC 7806, Plank o h ix aga dhii CYA 126, and Anabaena sp. s ain 90 ha e been comple ely sequenced [9–11]. Plank o h ix is one o he mos impo an MC-p oducing gene a in empe a e lakes [12]. O he MC-p oducing geno ypes wi hin his genus, he ed-pigmen ed phycoe y h in (PE)- ich geno ypes a e assigned o Plank o h ix ubescens, while he g een-pigmen ed phycocyanin (PC)- ich geno ypes a e equen ly assigned o Plank o h ix aga dhii [13]. Gene ally, Plank o h ix ubescens is ound in deep, Toxins 2013, 5 2436 s a i ied and oligo- o meso ophic wa e s in which me alimne ic laye s can be buil up. Plank o h ix aga dhii has a b oade dis ibu ion and inhabi shallow, polymic ic wa e bodies in he meso ophic o hype ophic nu ien ange [1]. P. ubescens was epo ed in he ollowing Eu opean subalpine lakes: Zu ich (Swi ze land), Ga da (I aly), Mondsee (Aus ia), Nan ua (F ance) and Bou ge (F ance) [14–18]. Va ious chemical, physical, and biological pa ame e s a e known o con ibu e o he de elopmen al and spa ial dis ibu ion o cyanobac e ial popula ions [1], bu he de e minism o cyanobac e ial blooms and hei impac a he lake scale a e no clea ly unde s ood. Plank o h ix spp. di e in hei cellula MC con en s as well as he p oduc ion o MC a ian s [12,19]. Di e en MC s uc u al a ian s we e cha ac e ized o Plank o h ix s ains isola ed om lakes in he Alps: he me hyl-dehyd o-alanine esidue (Mdha) geno ype, which was ound o syn hesize s uc u al a ian s con aining only Mdha in posi ion 7; he bu y ic acid (Dhb) geno ype, which was ound o con ain Dhb ins ead o Mdha in he same posi ion; and he homo y osine (H y) geno ype, which was ound o con ain H y and Leu in posi ion 2 bu ne e A g. The H y a ian has always been ound o co-occu wi h Dhb in posi ion 7 o he molecule [20,21]. Nume ous pape s ha e al eady in es iga ed he impac o a ious bio ic and abio ic en i onmen al ac o s on MC p oduc ion by a ious cyanobac e ial s ains. These s udies demons a ed ha MC p oduc ion can be in luenced by empe a u e, ligh , nu ien s such as ni ogen and phospho us, pH, i on, xenobio ics, and p eda o s [7,22]. Despi e inconsis en esul s, he p oduc ion o MCs by he cells seems o be linked o hei g ow h a e, which is i sel a ec ed by en i onmen al condi ions. On he o he hand, se e al s udies on a ia ions in he p opo ions o MC-p oducing cells demons a ed he po en ial in luence o nu ien concen a ions, ligh and empe a u e, sugges ing ha he e is a nega i e co ela ion be ween he p opo ions o MC-p oducing cells and he abundance o cyanobac e ial cells [23]. Du ing he las decade, gene ic me hods ha e signi ican ly con ibu ed o ou unde s anding o he dis ibu ion o genes ha a e in ol ed in he p oduc ion o MCs in cyanobac e ia causing cyanobac e ial HABs. The occu ence o inac i e mcy geno ypes (i.e. geno ypes possessing he mcy genes bu lacking MC p oduc ion) o Plank o h ix spp. and Mic ocys is spp. in na u e migh be unde s ood as suppo o he mcy gene loss hypo hesis. Mo eo e , inac i a ion o he mcy gene clus e by ansposable elemen s o poin mu a ions migh be seen as an in e media e s ep in eo ganiza ion o he mcy gene clus e owa ds cell ypes wi h modi ied MC syn hesis [24,25]. In his s udy we epo he p esence o P. ubescens bloom in a wind-shel e ed, s ably s a i ied shallow lake. Based on he unusual inding, we claim ha P. ubescens can occu and build oxic blooms in wa e s which unc ionally mimic he deep alpine lakes. The mo phome ic ea u es o he pond and he ele an physical and chemical a iables we e s udied in o de o unde s and he appea ance o his alpine cyanobac e ial species in he shallow lake-domina ed allu ial egion o he Ca pa hian Basin. In addi ion o he mo phological and molecula iden i ica ion o he species, we in ended o s udy he oxici y o he species and o analyze he oxin p o ile by MALDI–TOF and NMR analyses. The mcy gene clus e o he isola ed s ain o he unusual bloom causing P. ubescens was also in es iga ed and compa ed o he sequenced mcy gene clus e o s ain CYA126/8. Toxins 2013, 5 2437 2. Resul s 2.1. Physicochemical Pa ame e s o he S udy Si e Analyses o wa e samples e ealed high conduc i i y and alkaline cha ac e o he pond whe e he wa e bloom occu ed (Figu e 1). Physicochemical pa ame e s in he pond du ing algal blooms a e summa ized in Table 1. Due o he pond’s small size and leewa d loca ion, his ype o s anding wa e s a e s a i ied in he ege a ion pe iod wi h a 3 m me alimnion dep h [26]. Concen a ion o nu ien s (Table 1) e e o meso-eu ophic cha ac e and, a his ange, nu ien limi a ion does no de elop [27]. Table 1. Mo phome ic ea u es o he lake and he ele an physical and chemical a iables. Va iables alue uni Lake a ea 5.2 (Ha) Mean dep h 3.2 (m) Max. dep h 7 (m) Lake olume 1.6 × 105 (m3) Secchi anspa ency 1.2 (m) pH 8.34 Speci ic elec ical conduc i i y 820 (µS cm−1) COD ( sMn ) 15.8 (mg L−1) TOC 22.0 (mg L−1) DOC 15.8 (mg L−1) Ino ganic Ni ogen (IN) 1953 (µg L−1) Soluble Reac i e Phospho us (SRP) 3 (µg L−1) To al Ni ogen (TN) 3125 (µg L−1) To al Phospho us (TP) 370 (µg L−1) Figu e 1. (a) Loca ion o Kocka pond in Hunga y, indica ed by a illed ci cle; (b) The Plank o h ix ubescens bloom in he g a el pi pond; and (c) a mic oscopic obse a ion o Plank o h ix ubescens ichomes om he pond. Toxins 2013, 5 2438 2.2. Mo phology-Based Iden i ica ion o he HAB Causing O ganism P io o he molecula analyses, he collec ed bloom samples we e in es iga ed by ligh mic oscope (Figu e 1). T ichomes we e s aigh , soli a y wi hou shea h, and pale pu ple in colo . Cells we e cylind ical, no cons ic ed a c oss-walls, and mos ly isodiame ic wi h a diame e o 6–8 (8) µm. Cells a e di ision we e conside ably sho e (3–4 µm). All he cells had nume ous ae o opes and seemed densely g anula ed. Mos o he ilamen s had widely ounded e minal cells, he wall o he dis al end o hese cells we e no hickened. Occasionally, some ilamen s a enua ed o he ends and had sligh ly conical e minal cells wi h hickened ou e cell wall. These mo phological ea u es a e iden ical wi h hose cha ac e is ic o Plank o h ix ubescens (DeCandolle ex Gomon ) [28]. 2.3. Molecula Phylogene ic Analyses Sequence analysis o egions co e ing he almos comple e 16S RNA gene and he cpcBA-IGS o s ain BGSD-500 esul ed in 1387 and 527 n , espec i ely. Based on he 16S RNA, BGSD-500 showed high pai wise simila i y alues (99.9%–100%) o he sequence g oup con aining he ype s ain P. ubescens NIVA-CYA 18 (=PCC 7821)T and was sepa a ed om he clus e ha bo ing he ype s ain o P. aga dhii, NIES 204T (Figu e 2A). The analysis pe o med wi h cpcBA-IGS sequences showed simila esul s; BGSD-500 showed 100% pai wise simila i y alues o he clus e ha con ained mos ly P. ubescens isola es (Figu e 2B). Un o una ely, no ype s ain sequences a e a ailable cu en ly in da abases co e ing his egion, only a sho e agmen wi h 217 n om P. ubescens NIVA-CYA 18 (=PCC 7821)T (GenBank Acc. No. AJ558154), which was iden ical wi h sequences om he a o emen ioned clus e and showed ≤98.2% pai wise simila i y alues wi h he membe s o he o he cpcBA-IGS clus e . Figu e 2. (A) Maximum likelihood ees showing he phylogene ic posi ion o BGSD-500 based on he 16S RNA gene and (B) he phycocyanin ope on. In he case o he 16S RNA gene, 1329 n posi ions we e in ol ed in he analysis ha was pe o med wi h he HKY + G subs i u ion model, while o he cons uc ion o he cpcBA-IGS ee, 464 n we e used and he Kimu a 2-pa ame e model was applied. Type s ains o Plank o h ix species acco ding o Suda e al. [29] a e ma ked wi h supe sc ip T. A h ospi a pla ensis PCC 7345 was used as an ou g oup in bo h phylogene ic analyses. Boo s ap alues lowe han 70 a e no shown (based on 500 eplica es). Toxins 2013, 5 2439 Figu e 2. Con . 2.4. Iden i ica ion o MC and Compa a i e Analysis o Bloom Sample and he Isola ed P. ubescens S ain Unde he pu i ica ion p ocedu e, he oxic ac ions we e de ec ed by mus a d es (Figu e 3). Figu e 3. DEAE-52 ch oma og aphy and Blue-G een Sinapis Tes o [D-Asp3, Mdha7]MC–RR om Plank o h ix ubescens. Abso bance a 239 nm (-○-); hypoco yl leng h o h ee-day-old mus a d seedlings (-●-), g adien be ween 0 and 0.2 M NaCl in 5 mM T is-HCl bu e (---). The main oxic ac ions a e DEAE cellulose ch oma og aphy we e combined and u he pu i ied by HPLC-DAD. The majo oxin was iden i ied as [D-Asp3, Mdha7]MC–RR (Figu e 4) on he basis o he ollowing s udies. Toxins 2013, 5 2440 Figu e 4. Chemical s uc u e o he iden i ied cyanobac e ial hep apep ide [ D -Asp 3 , Mdha 7 ]MC–RR. The pu i ied MC had an abso p ion maximum a 239 nm in me hanol and exhibi ed a m/z 1024.6 [MH] + by MALDI-TOF. The cons i u ion o amino acids (Ala 1 ,A g 2 ,Asp 3 ,A g 4 ,Adda 5 ,Glu 6 ,Mdha 7 ) was con i med by MALDI pos -sou ce decay. Cha ac e is ic agmen s we e: m/z 754 ([A g 4 -ADDA 5 - Glu 6 -DHB 7 -Ala 1 +H + ] o [A g 4 -ADDA 5 -Glu 6 -MDHA 7 -Ala 1 +H + ]), 714 ([H-A g 2 -Asp 3 -A g 4 -ADDA 5 ] + , lack o Me-Asp 3 ), 216 ([Glu 6 -DHB 7 +H + ] o [Glu 6 -MDHA 7 +H + ]), 155 ([MDHA 7 -Ala 1 +H + ] o [DHB 7 -Ala 1 +H + ]), among o he s. The connec i i y and con igu a ion o N-me hyldehyd oalanine could be de e mined om TOCSY and NOESY spec a. The Asp 3 esidue showed no me hyl g oup a he C(β) posi ion, bu a he wo H–C(β) esonances. This also allowed an assignmen o he 1D 1 H NMR spec um. 2D HSQC spec a we e also eco ded. In ou sample, an H–C link was iden i ied in he HSQC spec um be ween a ca bon a 38.0 ppm and 1H a 3.32 ppm, indica ing p esence o he N-me hyl g oup. Also, he =CH 2 was ound, a pai o 1 Hs a 5.56 ppm and 5.88 ppm loca ed on a 13 C 116.0 ppm. Two anabaenopep in (B, m/z: 837 and F, m/z: 851) congene s we e also iden i ied om he P. ubescens by MALDI-TOF pos -sou ce decay. The lyophilized samples we e es ed by mus a d es and he oxici y o he samples was calcula ed. The IC 50 alue o he bloom sample was 0.97, and he BGSD-500 s ain was 2.47 (Figu e 5). Figu e 5. E ec o c ude P. ubescens-domina ed bloom-sample ex ac (-●-) and he isola ed P. ubescens BGSD-500 (-■-) on he g ow h o Sinapis alba e iola ed seedlings (Blue-G een-Sinapis-Tes ). Toxins 2013, 5 2441 Compa ing he MC con en o he samples, he concen a ion o [D-Asp3, Mdha7]MC–RR we e measu ed by capilla y elec opho esis. The amoun o MC con en calcula ed o he bloom sample was 8.57 mg g−1, and 1.85 mg g−1 o he isola ed P. ubescens s ain (Figu e 6). Figu e 6. Capilla y elec opho esis o P. ubescens-domina ed bloom-sample ex ac (A) and he isola ed P. ubescens BGSD-500 (B). Peak o [D-Asp3, Mdha7]MC–RR is indica ed by black a ow. (sepa a ion condi ions: capilla y: 64.5 cm, 50 µm i.d., bu e elec oly e: 25 mM bo a e and 75 mM SDS, pH 9.3, applied ol age: +25 kV, de ec ion: UV abso p ion a 238 nm). 2.5. Analysis o he mcy Gene Clus e Dele ions we e iden i ied by sho e - han-expec ed PCR amplicons a one si e. In one case, PCR ampli ica ion cons an ly ailed o gi e amplicons wi h he co esponding p ime pai s (posi ion: 23,612–24,003 n , [10]. This dele ion was loca ed in he space egion be ween mcyE and mcyG, and should he e o e no dis u b he ansla ion p ocess. The ampli ica ion o he MC syn hesis gene clus e yielded an unusually long PCR p oduc (a ound 1.6 kb) when using p ime pai myc3 (posi ion: 925–1399 n ); his inse ion was loca ed in he space egion be ween mcyT and mcyD. Sequencing o his amplicon yielded 1509 n and 1387 n long sequences o he o wa d and e e se ead, espec i ely, which made i possible o assemble a 1606 n long “coun ing” sequence o he egion. A s anda d nucleo ide BLAST sea ch in he nucleo ide collec ion o GenBank conduc ed on 22 June 2013 o highly simila sequences (“megablas ”) showed 99% and 98% sequence iden i y on 17% and 25% o he que y leng h wi h he MC syn he ase-associa ed hioes e ase (mcyT) gene o Plank o h ix ubescens and P. aga dhii, espec i ely. When compa ed o he e e ence sequence o Plank o h ix aga dhii MC syn hesis gene clus e (GenBank accession n . AJ441056; [10]), he que y sequence showed 98% iden i y on 334 n leng h om he 960 h o he 1293 d posi ion, hen a e a ca. 1.2 kb gap o an unalignable pa , ollowed by ano he 98% iden ical pa on 80 n leng h om he 1299 h o he 1378 h posi ion wi h he same mcyT gene. The unalignable egion was ound o be an inse ion in o his gene o 1194 n leng h (Figu e 7). Toxins 2013, 5 2442 Figu e 7. Localiza ion o he de ec ed and pa ly iden i ied 1194 n leng h inse ion elemen in he space egion be ween mcyT and mcyD o mcy gene clus e o Plank o h ix. When sea ching o highly simila sequences in BLAST (“megablas ”), no signi ican simila i y was ound o his inse ion. The e o e, we epea ed he BLAST sea ch bu o somewha simila sequences (“blas n”). This second sea ch has ound wo somewha simila sequences in GenBank: he i s one was a hypo he ical p o ein o a Synechococcus sp. (s ain PCC 7002; GenBank accession n . CP000951) which showed 77% iden i y on 87% leng h o he inse ion egion; whe eas he second showed 74% simila i y on 75% leng h in wo pa s: he i s pa was simila o signal ansduc ion his idine kinase, while he second pa was o RNA(Ile)-lysidine syn he ase o a Synechococcus sp. (s ain PCC 6312; GenBank accession n . CP003558). No u he simila i y was ound o he inse ed elemen . When we compa ed he sequence o he inse o he whole genome o Synechococcus sp. (s ain PCC 7002; GenBank accession n . CP000951) using he LAGAN algo i hm [30] in he web-based e sion o mVISTA [31], i iden i ied a simila pa be ween posi ions 865,594 and 867,245 o he e e ence genome, which is po en ially homologous o he inse . This egion con ains 65 n a he 3'-end o he icd gene o he p oduc isoci a e dehyd ogenase, NADP-dependen ; wo hypo he ical p o eins (co esponding o locus ags SYNPCC7002_A0839 and SYNPCC7002_A0840) in he whole leng h; and 44 n a he 5'-end o he pe D gene o he p oduc cy b6/ complex subuni IV. 3. Discussion The eddish colo o cyanobac e ial blooms iewed in Figu e 1 in he Kocka pond wi h he appea ance o a dense 3 cm hick laye (bio olume: 28.4 mm3/L) was an unexpec ed obse a ion in ou egion. The iden i ica ion o Plank o h ix ubescens as he dominan bloom- o ming species was a su p ising obse a ion, because Plank o h ix ubescens has p e iously no been iden i ied in ou egion. This species is cha ac e is ic in deep-lakes loca ed in Cen al and No he n Eu ope [32], including he lakes Zu ich, Ga da, Mondsee, Gene a, Nan ua, S eins jo den and Bou ge [16,24,33–35]. Occasionally he “Bu gundy-blood phenomenon” [32] migh also occu . The appea ance o he mass on he su ace o P. ubescens in No embe is a common phenomenon because du ing he mixing pe iod, P. ubescens is sp ead wi hin he en i e wa e column bu i is Toxins 2013, 5 2449 298 K. Residual HDO signal was sa u a ed. A wo-dimensional 1H–13C HSQC expe imen yielded a1 H/13C assignmen iden ical o ha published by Me iluo o e al. ([61], da a no shown). 4.7. Capilla y Elec opho esis o Field Samples and Isola ed P. ubescens Labo a o y S ain Samples Mic ocys in a ian s in he whole ex ac s o he samples we e analyzed by micella elec okine ic ch oma og aphy de eloped by ou labo a o y [40,41] (sepa a ion condi ions: capilla y: 64.5 cm, 50 µm i.d., bu e elec oly e: 25 mM bo a e and 75 mM SDS, pH 9.3, applied ol age: +25 kV, de ec ion: UV abso p ion a 238 nm). 4.8. Gene ic Analysis o he mcy Gene Clus e DNA ex ac ion om s ains and ield samples was pe o med by a s anda d phenol-chlo o o m p ocedu e. PCR ampli ica ions we e pe o med in eac ion mix u es o 20 µL as published by Ku maye e al. [20] and Ch is iansen e al. [25]. In o de o sc een he comple e Plank o h ix mcy gene clus e , 28 p ime pai s co e ing he whole mcy gene clus e we e used o ampli y agmen s o 2 kb wi hou in e up ion [10]. DNA mu a ions we e de ec ed ia he di e ence in PCR p oduc sizes in aga ose gels compa ed o he co esponding PCR p oduc s ob ained om s ain CYA126/8, whose mcy gene clus e has been sequenced [10]. The PCR he mal cycling p o ocol included an ini ial dena u a ion s ep a 94 °C o 3 min, ollowed by 35 cycles o dena u a ion empe a u e o 94 °C o 30 s, annealing empe a u e o 60 °C o 30 s, and elonga ion empe a u e o 72 °C o 2 min. The p ime pai s o Ch is iansen e al. [10,25,86], ampli ying agmen s o ca. 500 bp, we e used o de ec size di e ences in he clus e . PCR-p oduc s wi h possible inse ed o dele ed elemen s we e sequenced di ec ly om he same PCR p oduc s (sequencing ollowed he same p ocedu e desc ibed in phylogene ic analyses). 5. Conclusions In his mul idisciplina y s udy we epo ed he p esence o P. ubescens bloom om a wind-shel e ed, s ably s a i ied shallow lake wi h low phospha e and high ni ogen loads, whe e he Secchi anspa ency was 1.2 m. The eddish colo o cyanobac e ial blooms was an unexpec ed obse a ion in ou egion and he causa i e o ganism was iden i ied by classic mo phological ma ke s and by 16S RNA gene and phycocyanin ope on (cpcBA-IGS) as molecula ma ke s. The esul s ob ained in he Kocka pond hus con i m ha he Plank o h ix bloom sample con ained compa ably high amoun s o MC. The MALDI-TOF and he CE-analyses demons a ed ha he P. ubescens bloom sample and he isola ed s ain (BGSD-500) p ima ily con ain one main MC congene , a deme hyla ed a ian o MC-RR. Analysis o MALDI-TOF spec a and 2D HSQC NMR spec a p o ided e idence ha he molecule is iden ical o [D-Asp3,Mdha7]MC–RR. Compa ing he concen a ion o he MC congene in he bloom sample and in he isola ed s ain i can be clea ly seen ha he bloom sample con ained i e imes mo e MC han he isola ed s ain. This di e ence may due o speci ic en i onmen al condi ions bu i is impo an o no e a dele ion in he space egion be ween mcyE and mcyG, and an inse ion we e de ec ed a one si e binding o he space egion be ween mcyT and mcyD. Al hough ou elemen has p obably no in luence on he MC Toxins 2013, 5 2450 syn hesis, conside ing he unc ion o he p oduc o he pa ly simila sequences, i is wo h discussing his possibili y. Al hough some in asi e opical cyanobac e ial species ha e ecen ly come o he o e in many s udies, in ou pape , we d aw he a en ion o he possible sp ead o an alpine ha m ul o ganism, P. ubescens. Acknowledgmen s This wo k has been suppo ed by Hunga ian Na ional Resea ch Founda ion G an s OTKA K81370, F046493 and K105459, GVOP-3.2.1.-2004-04-0110/3.0, GVOP-TST 3.3.1-05/1-2005-05-0004/3.0. The wo k/publica ion is suppo ed by he TÁMOP-4.2.2/B-10/1-2010-0024 p ojec . Tamás Fel öldi and Is án Bácsi we e suppo ed by he János Bolyai Resea ch Schola ship o he Hunga ian Academy o Sciences. 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