Natural Variation in the Microcystin Synthetase Operon ...transcription step. No PCR products were detected by agarose electrophoresis (data not shown). Sequencing and phylogenetic

JOURNAL OF BACTERIOLOGY, May 2003, p. 2774–2785 Vol. 185, No. 90021-9193/03/$08.00�0 DOI: 10.1128/JB.185.9.2774–2785.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Natural Variation in the Microcystin Synthetase Operon mcyABC andImpact on Microcystin Production in Microcystis Strains

Bjørg Mikalsen,1 Gudrun Boison,2 Olav M. Skulberg,3 Jutta Fastner,4 William Davies,1Tove M. Gabrielsen,1 Knut Rudi,5 and Kjetill S. Jakobsen1*

Department of Biology, University of Oslo, 0315 Oslo,1 NIVA, Norwegian Institute for Water Research, 0411 Oslo,3 andMATFORSK, Norwegian Food Research Institute, 1430 Ås,5 Norway, and Institute of Botany, University of

Cologne, D-50923 Cologne,2 and Technical University of Berlin, 10587 Berlin,4 Germany

Received 1 July 2002/Accepted 30 December 2002

Toxic Microcystis strains often produce several isoforms of the cyclic hepatotoxin microcystin, and more than65 isoforms are known. This has been attributed to relaxed substrate specificity of the adenylation domain. Ourresults show that in addition to this, variability is also caused by genetic variation in the microcystin synthetasegenes. Genetic characterization of a region of the adenylation domain in module mcyB1 resulted in identifi-cation of two groups of genetic variants in closely related Microcystis strains. Sequence analyses suggested thatthe genetic variation is due to recombination events between mcyB1 and the corresponding domains in mcyC.Each variant could be correlated to a particular microcystin isoform profile, as identified by matrix-assistedlaser desorption ionization–time of flight mass spectrometry. Among the Microcystis species studied, we found11 strains containing different variants of the mcyABC gene cluster and 7 strains lacking the genes. Further-more, there is no concordance between the phylogenies generated with mcyB1, 16S ribosomal DNA, and DNAfingerprinting. Collectively, these results suggest that recombination between imperfect repeats, gene loss, andhorizontal gene transfer can explain the distribution and variation within the mcyABC operon.

Cyanobacteria are phototrophic organisms that often formwater blooms in eutrophic or estuarine waters. These waterblooms undergo fluctuations and may exhibit toxic states. Onecommon genus in such water blooms, Microcystis, producesthe hepatotoxin microcystin (6). There are approximately 65known isoforms of microcystin, representing a family of cyclicheptapeptides having the common structure cyclo(D-Ala–L-X–D-MeAsp–L-Z–Adda–D-Glu–Mdha), where L-X and L-Z arevariable L amino acids, Adda is 3-amino-9-methoxy-2,6,8,-tri-methyl-10-phenyl-4,6-decandienoic acid, D-MeAsp is 3-methyl-aspartic acid, and Mdha is N-methyl-dehydroalanine (Fig. 1).

Microcystin is produced nonribosomally by the microcystinsynthetase enzyme complex via a thio-template mechanism (2).Nonribosomal peptide synthetase genes consist of modulesthat are built up of domains, and each module activates oneamino acid, which is incorporated into the growing peptidechain in the order in which the modules are arranged. Mostmodules contain adenylation, thiolation, and condensationdomains, and the adenylation domain is responsible for rec-ognition of the specific amino acid. After the amino acid isactivated to its acyladenylate, the aminoacyl adenylate is trans-ferred to the 4�-phosphopantetheine carrier within the thiola-tion domain. Peptide bond formation between two activatedamino acids is mediated by the condensation domain (for re-views see references 24 and 30). There are 10 conserved motifs,designated A1 to A10, within the adenylation domain. The 10amino acid residues lining the substrate-binding pocket arealso located within this region and are believed to be respon-

sible for the substrate specificity. Nine of these amino acids arelocated between core motifs A4 and A5.

Microcystins mediate their toxicity through inhibition of theeukaryotic serine/threonine protein phosphatase 1 and 2A ac-tivities (13, 14, 20, 28). There have been several reports ofdeath of livestock and humans due to massive hepatic hemor-rhage (5, 6, 21). Immunoassays, phosphatase inhibition assays,and other analytical techniques (e.g., matrix-assisted laser de-sorption ionization–time of flight [MALDI-TOF] mass spec-trometry) have been developed for microcystin assays and tox-icity measurement (1, 12, 32).

Several attempts have been made to link toxin production toother molecular markers. Previous studies include, among oth-ers, studies of random amplified polymorphic DNA (RAPD)(33, 35), repetitive DNA elements (3, 39), and 16S rRNAgenes (34, 37, 43, 44, 50). None of these analyses revealed anysimple correlation with toxicity, although some recent studieshave demonstrated that it may be feasible to generate geneticprobes that are indicators of toxicity (4, 50). Although therelationship between toxicity and phylogeny within the genusMicrocystis has not been resolved, phylogenetic analyses withdifferent markers have suggested a monophyletic origin of Mi-crocystis (50).

Despite intensive research, the biological function(s) of mi-crocystins has not been determined yet. Putative roles for mi-crocystin include feeding deterrence of zooplankton grazers,siderophoric scavenging of and binding to trace metals (such asiron), and involvement in quorum sensing (9, 10, 51). Notably,synthetase gene knockout by insertional mutagenesis revealedno apparent effects on laboratory cultures (11). This and otherinconclusive investigations of microcystin function may suggestthat the role of this family of secondary metabolites can beaddressed most efficiently by studies of the various microcystin

* Corresponding author. Mailing address: Division of MolecularBiology, Department of Biology, University of Oslo, P.O. Box 1031Blindern, 0315 Oslo, Norway. Phone: 47 22 85 46 02. Fax: 47 22 85 4605. E-mail: [email protected].

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producers and nonproducers in their natural habitats com-bined with the use of clearly defined mutants or genetic vari-ants. Another key issue that needs to be addressed from afunctional perspective is the evolutionary history of microcys-tin synthetase and the mechanisms that cause both the distri-bution and variation observed for microcystin biosynthesis.

The recent cloning and sequencing of the complete (andalmost complete) microcystin synthetase gene cluster (mcyABCand mcyDEFGHIJ) from two different strains, PCC 7806 (49)and K-139 (36), have provided a new tool for studying micro-cystin variation, evolution, and function. We performed a studyof sequence divergence and organization of selected regions ofthe mcyABC genes of closely related Microcystis strains previ-ously characterized at the ribosomal DNA (rDNA) level (44),with particular emphasis on mcyB. Different synthetase genesequences were correlated with synthetase mRNA transcrip-tion, as well as with the structures of the cyclic peptides pro-duced by the strains harboring a functional gene cluster. Fur-thermore, comparative phylogenetic analyses with the mcyABCregion and other molecular markers were carried out.

MATERIALS AND METHODS

Bacterial strains and culture conditions. The strains used are listed in Table1. Unialgal cultures were grown at the Norwegian Institute for Water Research(NIVA) as previously described (45); the only exceptions to this were strainsPCC 7806 and HUB 5-2-4, which were kindly provided by H. Utkilen (NationalInstitute of Public Health, Oslo, Norway).

DNA isolation and PCR amplification. DNA from Microcystis cultures wereisolated by a method designed for plant DNA (27), with the following modifi-cations. The DNA preparations were treated with RNase (50 �g per ml of

supernatant) after chloroform-isoamyl alcohol (24:1) extraction, and the firstDNA precipitation was performed with two-thirds volume of isopropanol insteadof cetyltrimethylammonium bromide precipitation buffer. Later, this method wasfurther modified by using 2� sodium dodecyl sulfate lysis buffer (100 mMTris-HCl, 50 mM EDTA, 100 mM NaCl, 2% sodium dodecyl sulfate, 0.2%mercaptoethanol) instead of cetyltrimethylammonium bromide lysis buffer andtreating the preparations with 10 �l of proteinase K (10 mg/ml) prior to phenol-chloroform (25:24) and chloroform-isoamyl alcohol (24:1) extraction.

PCRs were carried out in 25-�l mixtures containing 10 mM Tris-HCl (pH 8.8),1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, each deoxynucleoside triphos-phate (dNTP) at a concentration of 200 �M, 0.5 U of DyNAzyme II DNApolymerase (Finnzymes OY, Espoo, Finland), 5 pmol of each primer, and 1 ngof template. The primers used are listed in Table 2. The PCR programs consistedof an initial denaturation step of 94°C for 4 min, followed by 30 cycles of 95°C for30 s, the appropriate annealing temperature (Table 2) for 30 s, and 72°C for 1min, ending with an additional extension step of 72°C for 7 min. PCR productswere verified on 1.5% agarose gels stained with ethidium bromide.

RNA isolation and reverse transcriptase PCR (RT-PCR). Microcystis cultures(100 ml) from NIVA, grown as previously described, were centrifuged, and eachpellet was suspended in 800 �l of TE buffer (10 mM Tris-HCl [pH 8], 1 mMEDTA [pH 8]). Total RNA was isolated from 100 �l of the cell suspension withaddition of 1 ml of TRIzol reagent (Gibco BRL, Life Technologies, Rockville,Md.). All other steps were performed according to the manufacturer’s recom-mendations. The RNA pellet was dissolved in Milli-Q water to a concentrationof approximately 1 �g/�l, and 2.5 �l was treated with DNase I (Gibco BRL, LifeTechnologies) according to the manufacturer’s protocol. First-strand cDNA syn-thesis was performed by using 500 ng of DNase-treated total RNA, SuperScriptII (Gibco BRL, Life Technologies), the gene-specific primers 135-F and 676-R(Table 2), and RNase OUT recombinant RNase inhibitor (Gibco BRL, LifeTechnologies) according to the manufacturer’s protocol. PCR amplificationswere carried out by performing standard reactions (see above) and using 2.5 �lof the first-strand reaction mixture with primers 135-F and 676-R.

Control reactions were carried out by using the same reaction conditions andprimers with DNase-treated RNA which had not been subjected to the reverse

FIG. 1. General structure of microcystin. The general structure of microcystin is cyclo(D-Ala–L-X–D-MeAsp–L-Z–Adda–D-Glu–Mdha-), whereX and Z are variable L amino acids, Adda is 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid, D-MeAsp is D-erythro-�-methyl-aspartic acid, and Mdha is N-methyl-dehydroalanine. In the most common variant (MC-LR) X is leucine (L) and Z is arginine (R).

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transcription step. No PCR products were detected by agarose electrophoresis(data not shown).

Sequencing and phylogenetic analysis of 16S rDNA and the mcyABC region.DNA amplified with primers 135-F and 676-R, primers 2156-F and 3111-R, andprimers CC and CD (Table 2) was sequenced manually by the cyclic dideoxychain termination method by using a Thermo Sequenase radiolabeled terminatorcycle sequencing kit (U.S. Biochemical Corp., Cleveland, Ohio) according to theprotocol supplied by the manufacturer.

The nucleotide sequences were aligned with the ClustalX multiple-alignmentsoftware (48), and then the alignments were manually edited with MacClade(29). A phylogenetic tree was constructed by the minimum-evolution method byusing the Phylogenetic Analysis Using Parsimony package (47). The Kimuratwo-parameter model (23) was used to compute the distance matrix.

The amino acid sequences were aligned with the multisequence alignmentalgorithm PILEUP in the Wisconsin Package, version 10.0 for Unix (GeneticsComputer Group, Madison, Wis.). A Dayhoff PAM matrix was computed withthe Protdist program (Genetics Computer Group), and then a neighbor-joiningtree was created. To infer confidence in the branch points in both trees, boot-strap analyses (17) were performed. The consensus trees were constructed from500 bootstrap replicates.

Southern hybridization. Approximately 1 �g of genomic DNA was digestedwith 15 U of HindIII overnight (37°C). Restricted DNA was separated on a 1%agarose gel and then transferred to an Amersham Hybond-N membrane bySouthern dry blotting overnight. Hybridization was performed at 68°C by usingstandard procedures (18). The probe (Fig. 2) was generated from the probe-F–probe-R PCR product from strain PCC 7806 by standard MSLP (magneticsolid-phase labeling) random primed synthesis (15) by using a random primedlabeling kit (Boehringer GmbH, Mannheim, Germany).

MALDI-TOF mass spectrometry. Portions (100 ml) of unialgal Microcystiscultures were harvested by centrifugation and freeze-dried. Lyophilized sampleswere dissolved in 50% methanol and sonicated for 15 min. A mixture of 1 �l ofsample and 1 �l of matrix (saturated �-cyano-4-hydroxycinnamic acid matrixsolubilized in 50% acetonitrile–0.03% trifluoroacetic acid) was prepared directlyon the template. The samples were analyzed by using a MALDI-TOF massspectrometer (Voyager elite; PerSeptive BioSystems, Framingham, Mass.) with anitrogen laser having a 337-nm output. The ions were accelerated with a voltageof 20 kV. Measurements were obtained in the delayed extraction mode, whichallowed determination of monoisotopic mass values. A low mass gate of 500improved the measurement by filtering out the most intense matrix ions. Themass spectrometer was used in the positive-ion detection and reflector mode.Post Source decay (PSD) measurements were obtained after peptide mass de-termination with the same samples on the template. The operating voltages ofthe reflectron were reduced stepwise to record 12 spectral segments sequentially(12, 16).

RAPD and REP fingerprinting analyses. The following 7 primers (OperonTechnologies Inc., Alameda, Calif.) of the 41 primers tested were used forRAPD analyses of the strains: A04 (5�-AATCGGGCTG-3�), A18 (5�-AGGTGACCGT-3�), C08 (5�-TGGACCGGTG-3�), C11 (5�-AAAGCTGCGG-3�), C20(5�-ACTTCGCCAC-3�), D03 (5�-GTCGCCGTCA-3�), and D18 (5�-GAGAGCCAAC-3�). These primers were selected on the basis of band quality, bandnumber, and PCR performance. PCRs were carried out in 25-�l mixtures con-taining 10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl2, 50 mM KCl, 0.1% TritonX-100, each dNTP at a concentration of 100 �M, 0.75 U of Super Taq (HC)polymerase (HT Biotechnology Ltd., Cambridge, England), 6 pmol of primer,and 1 ng of template. The PCR program consisted of an initial denaturation stepof 94°C for 3 min, followed by 35 cycles of 94°C for 15 s, 40°C for 30 s, and 72°Cfor 1 min and then an additional extension step of 72°C for 5 min. Fingerprintpatterns were visualized on 1.4% agarose gels stained with ethidium bromide.

The REP (repetitive extragenic palindromic element) PCR was performedwith primers REP1P-I (5�-IIIICGICGICATCIGGC-3�) and REP2-I (5�-ICGIC

TABLE 1. Microcystis strains investigated

Straina Species Geographic originAccession no.

mcyAB (region 135–676) mcyB1 (region 2156–3111) 16S rDNA region

N-C 31 M. aeruginosa Little Rideau Lake, Canada AJ496434 AJ492552 Y12604N-C 43 M. aeruginosa Wisconsin (strain ATCC 22663) —b —b Z82784N-C 57 M. aeruginosa Lake Frøylandsvatnet, Norway AJ496435 AJ492553 Z82785N-C 118/2 Microcystis sp. Lake Gjersjøen, Norway AJ496436 AJ492554 Y12607N-C 122/2 M. viridis Lake Finjasjon, Sweden —b —b Y12612N-C 123/1 M. aeruginosa Lake Malaren, Sweden —b —b Y12605N-C 143 M. aeruginosa Lake Akersvatnet, Norway AJ496437 AJ492555 Z82786N-C 144 M. cf. flos-aquae Lake Borrevatnet, Norway —b —b Y12610N-C 161/1 M. botrys Lake Mosvatnet, Norway AJ496438 AJ492556 Y12608N-C 166 M. aeruginosa Lake Hellesjøvatnet, Norway —b —b Y12606N-C 169/7 M. viridis Lake Arresø, Denmark AJ496439 AJ492557 Y12613N-C 172/5 M. cf. wesenbergii Lake Arresø, Denmark —b —b Y12614N-C 228/1 M. aeruginosa Lake Akersvatnet, Norway AJ496440 AJ492558 Z82783N-C 264 M. botrys Lake Frøylandsvatnet, Norway AJ496441 AJ492559 Y12609N-C 279 M. cf. ichthyoblabe Lake Østensjøvatnet, Norway —b —b Y12611N-C 324/1 Microcystis sp. Lake Tøråssjøen, Norway AJ496442 AJ492560 Z82808HUB 5-2-4 M. aeruginosa Lake Pehlitzsee, Germany AJ496443 AJ492561c —d

PCC 7806 M. aeruginosa The Netherlands AF183408 AF183408 U03402K-139 M. aeruginosa Lake Kasumigaura, Japan AB019578 AB019578 —d

a N-C, NIVA-CYA, Norwegian Institute for Water Research Cyanobacterial Culture Collection; HUB, Humboldt University of Berlin; PCC, Pasteur CultureCollection.

b The strain does not possess the mcyA and mcyB genes.c See accession no. Z28338 (31). Our sequence differs at one base.d The sequence is not available.

TABLE 2. PCR primers

Primera Sequence Annealingtemp (°C)

135-F 5�-GACTTATAGCCATCTCATCT-3� 52676-R 5�-TTGACGCTCTGTTTGTAA-3� 522156-F 5�-ATCACTTCAATCTAACGACT-3� 523111-R 5�-AGTTGCTGCTGTAAGAAA-3� 52mcyB-R 5�-CTTCTCGCAAAATAACTACG-3� 47mcyC-R 5�-TCAGGTTTAGCCACGACT-3� 47probe-F 5�-Biotin-TTACAGCAAAAGCAAGCA-3� 50probe-R 5�-CCTACACCCTTATCTCGT-3� 50CCb 5�-TGTAAAACGACGGCCAGTCCAGAC

TCCTACGGGAGGCAGC-3�70

CDb 5�-CTTGTGCGGGCCCCCGTCAATTC-3� 70

a Compare the positions in Fig. 3.b Positions are shown in reference 44.

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TTATCIGGCCTAC-3�) (52). PCRs were carried out in 25-�l mixtures contain-ing 50 mM Tris-HCl (pH 9.0), 15 mM (NH4)2SO4, 0.1% Triton X-100, 1.7 mMMgCl2, each dNTP at a concentration of 360 �M, 1 U of Dynazyme EXT DNApolymerase (Finnzymes OY), 10 pmol of each primer, and 1 ng of template. ThePCR program consisted of an initial denaturation step of 95°C for 6 min, fol-lowed by 40 cycles of 94°C for 1 min, 40°C for 1 min, and 65°C for 8 min and thenan additional extension step of 65°C for 16 min. PCR products were verified on1.5% agarose gels stained with ethidium bromide.

All REP and RAPD PCRs were carried out in parallel with DNA isolatedfrom two independently grown cultures of the same strain. Direct sequencing ofthe 16S rDNA variable region in the different cultures confirmed that all strainshad undetectable levels of contaminating bacteria, as judged by the absence ofdouble bands in the sequencing ladder. The gels were scored conservatively,meaning that only reproducible and strong bands were scored (1, present; 0,absent).

The results were analyzed together by using the Jaccard similarity coefficient,which excludes shared absence of bands. The results were then subjected tounweighted pair group method with arithmetic averages (UPGMA) clustering byusing the program NTSYS-PC (38).

RESULTS

In this paper we present the results of molecular analyses ofthe mcyABC gene cluster in 18 closely related Microcystisstrains. The strains were investigated at the DNA organization,sequence, and functional levels by using Southern hybridiza-tion, PCR, fingerprinting, DNA sequencing, mRNA RT-PCR,and microcystin structure analysis by MALDI-TOF mass spec-trometry. The data generated were used to study the relation-ship between gene variants and the microcystin isoforms pro-duced (i.e., whether the isoforms are a result of partialrelaxation of substrate specificity or are produced by geneti-cally distinct variants of the enzyme complex) and the evolu-tionary forces creating the distribution and variation of themcyABC gene cluster.

Two major restriction fragment length polymorphism vari-

ants among the mcyABC-containing Microcystis strains. South-ern hybridization was performed by using a probe derived fromthe first condensation domain of mcyB (positions 46174 to46754 in PCC 7806 [accession no. AF183408]) (Fig. 2) withstrains collected from various locations. Altogether, 16 differ-ent NIVA-CYA (N-C) strains, including 10 strains originatingfrom Norway, 2 strains from Sweden, 2 strains from Denmark,1 strain from the United States, and 1 strain from Canada(Table 1), were used. In addition, the previously characterizedstrains HUB 5-2-4 (31) from Germany and PCC 7806 (49)from The Netherlands were investigated. As shown by theresults of the Southern analysis in Fig. 3, 11 of the 18 strainstested gave a positive hybridization signal, demonstrating thepresence of the mcyB gene. The hybridizing N-C strains fellinto two classes based on their HindIII restriction patterns; oneclass included strains N-C 31, N-C 118/2, and N-C 161/1 with afragment length of 2,400 bp, which closely resembled the frag-ment length determined for strain PCC 7806 (2,390 bp), andthe other class included strains N-C 57, N-C 143, N-C 169/7,N-C 228/1, N-C 264, and N-C 324/1 with a fragment length of5,900 bp, which was similar to the fragment length determinedfor HUB 5-2-4 (5,840 bp). This polymorphism may indicatethat there are differences in the adenylation domain in the firstmodule of mcyB (mcyB1), as displayed by a PCC 7806-likevariant and a HUB 5-2-4-like variant differing in at least oneHindIII site.

To further investigate the sequence differences among thevarious strains, PCR primers were designed based on an align-ment of the PCC 7806 and HUB 5-2-4 sequences. The forwardprimer 2156-F anneals to both sequence variants, and the tworeverse primers, mcyB-R and mcyC-R, are specific for the PCC7806-like sequence and the HUB 5-2-4-like sequence, respec-

FIG. 2. Organization of the mcyABC gene cluster in strain PCC 7806. The module arrangement and HindIII restriction sites are shown. In thediagram at the bottom, the relative positions of primers (arrows) and the Southern probe are shown. Abbreviations: H, HindIII restriction sites;s, spacer (His motif); A1, adenylation core motif 1; T, thiolation core motif. For simplicity, condensation core motifs are not shown.



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tively. The results confirmed the existence of the two classes ofsequence variants among the previously uncharacterizedstrains (Table 3). The hybridization-positive strain N-C 31 didnot produce a PCR product with either of the two reverseprimers. However, the reason for this was clarified by sequenc-ing (see below). Additional PCRs with primers 135-F and

676-R (Table 2) amplifying a region from the carboxy-terminalend of mcyA to the N-terminal end of mcyB (Fig. 2) showedthat only the strains that produced a positive hybridizationsignal and an mcyB PCR product gave an mcyAB band (Fig.4A). This was confirmed by using primers 2156-F and 3111-R,which amplified both mcyB1 variants (Fig. 4B). Thus, we con-

FIG. 3. Southern blot analysis of the first module in mcyB. Genomic DNA was digested with HindIII and hybridized with an mcyB probe (Fig.2) (A) and a 16S rDNA probe (B). The 16S rDNA hybridization results are shown for loading comparisons.

TABLE 3. Overview of mcyB gene structure, transcriptional status, and microcystin production in various Microcystis strains

Strain Fragmentlength (bp)a

PCR productsbGeneticvariant

RT-PCRproductb Microcystinc

135–676 2156–3111 2156-mcyB 2156-mcyC

N-C 31 2,390 � � � � mcyB1(B) � MC-LRN-C 43 NS � � � � � �N-C 57 5,840 � � � � mcyB1(C) � [Asp3, Dha7]MC-RR, [Dha7]MC-RRN-C 118/2 2,390 � � � � mcyB1(B) � [Asp3]MC-LR, MC-LRN-C 122/2 NS � � � � � �N-C 123/1 NS � � � � � �N-C 143 5,840 � � � � mcyB1(C) � �N-C 144 NS � � � � � �N-C 161/1 2,390 � � � � mcyB1(B) � MC-YR, MC-LRN-C 166 NS � � � � � �N-C 169/7 5,840 � � � � mcyB1(C) � MC-RR, MC-LRN-C 172/5 NS � � � � � �N-C 228/1 5,840 � � � � mcyB1(C) � [Dha7]MC-RR, [Dha7]MC-LRN-C 264 5,840 � � � � mcyB1(C) � [Dha7]MC-RRN-C 279 NS � � � � � �N-C 324/1 5,840 � � � � mcyB1(C) � [Asp3, Dha7]MC-RR, [Dha7]MC-RR,

[Dha7]MC-LR, MC-LRHUB 5-2-4 5,840 � � � � mcyB1(C)d ND MC-RR, MC-LRe

PCC 7806 2,390 � � � � mcyB1(B)f ND MC-LR, [Asp3]MC-LRe

K-139 ND ND ND ND ND mcyB1(B)g ND [Asp3, Dha7]MC-LR, [Dha7]MC-LRg

a HindIII fragment lengths as determined by Southern analysis. NS, no hybridization signal; ND, not determined in this study.b �, PCR product; �, no PCR product; ND, not determined in this study.c Microcystin content as determined by MALDI-TOF mass spectrometry. MC-LR, microcystin-LR; MC-YR, microcystin-YR; MC-RR, microcystin-RR; [Asp3,

Dha7]MC-RR, 3,7-didesmetylmicrocystin-RR; [Dha7]MC-RR, 7-desmetylmicrocystin-RR; [Asp3]MC-LR, 3-desmetylmicrocystin-LR; �, no microcystin detected.d Deduced from the sequence published by Meissner et al. (31) (accession no. Z28338).e Data from reference 16.f Deduced from the sequence published by Tillett et al. (49) (accession no. AF183408).g Data from reference 36.



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cluded that these 11 hybridization-positive strains contain boththe mcyA and mcyB genes.

Sequence analysis of mcyA and mcyB: high mutation rates inmcyB. In order to unambiguously confirm the identities of thestrains, we sequenced a 560-bp highly variable region of 16SrDNA using the eubacterium-specific primers CC and CD(Table 2). The results, which showed very little variationamong the N-C strains, are in full agreement with previousresults obtained by Rudi et al. (44). In this region there areonly six point mutations, including a single informative site thatgroups strains N-C 118/2, N-C 144, N-C 161/1, N-C 169/7, N-C264, and N-C 324/1 together and strains N-C 31, N-C 43, N-C57, N-C 122/2, N-C 123/1, N-C 143, N-C 166, N-C 172/5, N-C228/1, N-C 279, and PCC 7806 together.

Sequencing of the region from 135 to 676 (Fig. 2) of mcyABalso revealed few differences between the strains. Only 11point mutations were found in the 500-bp region. Notably,strain N-C 118/2 had more point mutations (7 of the 11 mu-tations) than the other strains, including a change in the mcyBstart codon from AUG to GUG. Two informative sites werefound in this region; one site grouped N-C 57, N-C 169/7, N-C264, and N-C 324/1 together, and the other informative sitegrouped N-C 57, N-C 118/2, N-C 161/1, N-C 264, N-C 324/1,and PCC 7806 together.

Sequencing of the adenylation domain in the first module ofmcyB (mcyB1) was accomplished by using PCR products gen-erated with primers 2156-F and 3111-R (Table 2). This primerpair amplifies both the PCC 7806-like and the HUB 5-2-4-likesequence variants, and positive PCR signals were obtained forthe 11 strains shown to contain the mcyB gene. In contrast tothe mcyAB region, the mcyB1 module showed considerablevariation among the strains. Both nucleotide and amino acidalignments from the region from 2156 to 3111 (Fig. 2) indeedconfirmed that the strains are divided into two main groups(the same PCC 7806-like and HUB 5-2-4-like groups describedabove) (Fig. 5).

Phylogenetic analyses revealed recombination between mod-ules in mcyB and mcyC. The nucleotide and amino acid se-quence alignments were used to construct phylogenetic trees.The mcyC modules in PCC 7806 (PCC 7806c) and K-139 (K-139c) were included for comparison, and the correspondingsequence in grsA (25) was used as an outgroup. Both the aminoacid and the nucleotide trees clustered the strains in two main

groups (Fig. 6A and B), in agreement with the Southern anal-ysis. The PCC 7806-like sequences comprised a B-type group[with greatest affinity to the mcyB1 module of PCC 7806; des-ignated mcyB1(B)], while the other group displayed a C type ofmcyB1 module [i.e., the greatest affinity was to the mcyC re-gion; designated mcyB1(C)]. These two main clades were alsoformed when phylogenetic analyses were performed with con-served regions A3 to A8 alone (data not shown). The PCC7806-like group, comprised of strains N-C 118/2, K-139, N-C161/1, and PCC 7806, clustered together with 100% bootstrapsupport. The N-C 31 strain created a side branch with a strongaffinity to the PCC 7806-like group (Fig. 6). The amino acidsequence alignment showed that this strain has sequence affil-iation with the HUB 5-2-4-like group after core motif A8 (Fig.5).

The HUB 5-2-4-like group (the C type of mcyB1) could befurther divided into subgroups. Although the exact topology ofthe subgroups could not be unambiguously determined due todifferences in topology between the amino acid and nucleotidetrees and low bootstrap support for some branches, the anal-ysis showed that there were two strongly supported subgroups,one that included the mcyC modules from PCC 7806 andK-139 in addition to the mcyB1 module of N-C 264 and onethat included N-C 169/7 and N-C 324/1. Notably, strains N-C169/7 and N-C 324/1 showed different affinities in the nucleo-tide and amino acid trees. These two strains showed affinitiesto HUB 5-2-4, N-C 228/1, N-C 143, and N-C 57, as well as tothe true C-type subgroup (N-C 264, K-139c, and PCC 7806c)(Fig. 6A and B).

Taken together, our results revealed the existence of chi-meric mcyB1 modules that show the greatest sequence simi-larity to the downstream mcyC module. It seems likely thatthese variants arose through recombination of the repeatedsequences (A1 to A10) in the different modules of mcyB andmcyC. Analyses based on the program LARD (version 2.2;Likelihood Analysis of Recombination in DNA) (19) indicatedthat there is a recombination breakpoint at the end of motif A3in mcyB1 (data not shown).

Variation in microcystin synthetase transcription. The nextquestion which we addressed was the transcriptional function-ality of the mcyABC gene cluster in the different strains interms of mRNA synthesis. An RT-PCR analysis of total RNAfrom the 16 N-C strains was performed with primers 135-F and

FIG. 4. PCR of two regions in the mcyABC operon. Standard PCR was performed with regions mcyAB (primers 135-F and 676-R) (A) andmcyB (primers 2156-F and 3111-R) (B) (Fig. 2). The marker used was �X174/HaeIII.



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FIG. 5. Alignment of the amino acid sequences of region 2156-3111 from the 12 strains possessing the mcyB gene and the corresponding sequencesin the mcyC gene from strains PCC 7806 and K-139 (PCC 7806c and K-139c, respectively) and the grsA gene from Bacillus brevis. Red, polar, chargedamino acids; orange, polar, uncharged amino acids; light green, hydrophobic amino acids; dark green, small amino acids. The nine amino acids putativelyinvolved in substrate recognition are indicated by arrows, core sequences are underlined, and genetic variants are indicated at the end of the alignment.

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FIG. 6. Phylogenetic and UPGMA analyses. Phylogenetic analyses were performed with both nucleotide (A) and amino acid (B) alignmentsfrom the region amplified by primers 2156-F and 3111-R. The corresponding region in grsA (accession no. M29703) from B. brevis was used as anoutgroup. (A) Tree constructed by the minimum-evolution method based on a distance matrix computed by the Kimura two-parameter model.(B) Neighbor-joining tree constructed based on distance measurements estimated by the Dayhoff PAM matrix. Bootstrap values (in percentages)from 500 replicates are indicated at the branch nodes. (C) UPGMA analysis of both the RAPD and REP matrices based on Jaccard similaritiesbetween strains. The arrows point towards the positions of the same strains in the mcyB1 (nucleotide) phylogenetic tree. Abbreviations: PCC 7806c,mcyC module from PCC 7806; K-139c, mcyC module from K-139; aer, M. aeruginosa; vir, M. viridis; sp, Microcystis sp.; bot, M. botrys; fl-a, Microcystiscf. flos-aquae; icht, Microcystis cf. ichthyoblabe; wes, Microcystis cf. wesenbergii.

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676-R (Table 2). Of the nine strains shown to contain the mcyAand mcyB genes, eight (strains N-C 31, N-C 57, N-C 118/2, N-C143, N-C 161/1, N-C 169/7, N-C 228/1, and N-C 264) gave clearpositive RT-PCR results (Fig. 7). These results show that thedifferent strains containing the B or C type of the mcyB1module are capable of producing microcystin synthetase mRNA.None of the strains lacking the mcyABC gene cluster produceddetectable levels of mRNA.

The strains investigated produce various microcystin iso-forms. The final aspect studied was determination of the mi-crocystin isoforms synthesized by the various strains. All 16N-C strains were analyzed by MALDI-TOF mass spectrome-try, and the results are shown in Table 3. Only the strains foundto contain the mcyABC cluster produced detectable levels ofmicrocystin. The B-type variants of mcyB1 produced variousmicrocystin-LR isoforms, and one strain (N-C 161/1) producedmicrocystin-YR in addition to microcystin-LR. Strain N-C 31,which formed a basal branch of the mcyB1(B) strains (Fig. 6Aand B), produced only microcystin-LR. N-C 118/2 and PCC7806 both produced large amounts of [Asp3]microcystin-LRand microcystin-LR. All strains with a C-type variant of themcyB1 module synthesized microcystin-RR, and the membersof some subgroups produced microcystin-LR in addition tomicrocystin-RR. Strain N-C 264, which possesses the true Ctype (i.e., its mcyB1 module groups together with the mcyCmodules of PCC 7806 and K-139), produced only a microcys-tin-RR isoform. This was also the case for N-C 57. ThemcyB1(C) strains (N-C 169/7, N-C 228/1, and N-C 324/1) be-longing to other C-type subgroups synthesized both microcys-tin-RR and microcystin-LR isoforms. Strain N-C 324/1, whichdid not give a detectable RT-PCR signal, produced smallamounts of microcystin-LR and demethylated isoforms of mi-crocystin-RR and microcystin-LR. HUB 5-2-4, belonging tothe C-type subgroup, produced both microcystin-RR and mi-crocystin-LR, while N-C 228/1 produced demethylated iso-forms of these two microcystins. Our data show that there isvariation in the methylation of the microcystin isoforms in thedifferent strains, but since the enzymatic activity of methylationlies outside the mcyB1 module, the genetic basis for this vari-ation could not be addressed in this study. Notably, strain N-C143 did not produce detectable levels of microcystin, eventhough it contained the mcyA and mcyB genes and synthesizedthe mRNA.

Clustering of M. aeruginosa strains by REP and RAPD fin-gerprinting. The REP and RAPD fingerprinting matrices, cal-culated by using REP and seven different RAPD primers, wereanalyzed together by using UPGMA. This analysis showed thatthe strains formed two clusters (Fig. 6C). One of the clusters

contained all of the strains characterized as M. aeruginosa. Thisis in agreement with 16S rDNA results (44). However, theclustering shown by fingerprinting markers did not resemblethe major groups revealed by the mcyABC region (compareFig. 6A and C).

DISCUSSION

Genetic variation in the mcyB1 module generates differentmicrocystin isoforms. The different analyses performed in thisstudy showed that Microcystis strains can have different adeny-lation domains in the mcyB1 module of the microcystin syn-thetase genes. Our initial studies (Southern blotting and PCR)showed that the strains could be divided into two groups, thePCC 7806-like variants (B type) and the HUB 5-2-4-like vari-ants (C type). With this information in hand, we performeda BLAST search using the partially sequenced module ofHUB 5-2-4 deposited in the GenBank database (accession no.Z28338), which showed that the ends of the HUB 5-2-4 se-quence are most similar to the first module in mcyB (96 and95% identity to strain PCC 7806) and that the middle (A3 toA9) is most similar to the module in mcyC (91% identity toPCC 7806). In other words, the well-characterized strain HUB5-2-4 displays unmistakable features of genetic recombination.Our detailed studies at the sequence level, including phyloge-netic analyses, supported this interpretation and suggested thatthere are several phylogenetic subgroups of the adenylationdomain in mcyB1. We designated these two main variantsmcyB1(B) (the PCC 7806-like variants) and mcyB1(C) (theHUB 5-2-4-like variants). In contrast, the upstream mcyAB re-gion displayed extensive sequence homology in all the strains,in agreement with the variable region of the 16S rRNA gene(44).

The variation in the mcyB1 module does not seem to influ-ence the functionality of the genes as both variants producemRNA (Fig. 7) and microcystins (Table 3). The investigationof the microcystin synthetase transcripts revealed potentialregulation at the translational (or posttranslational) level, asexemplified by one of the strains which synthesized the mRNAbut no detectable microcystin. However, in the absence of realquantitative measures of both transcript and microcystin levels,transcriptional regulation was not addressed in this study. Thefew differences observed in the mcyAB region do not influencefunctionality either, as strain N-C 118/2, which contains thehighest relative frequency of point mutations, including achange in the start codon from AUG to GUG, synthesizes bothmRNA and normal amounts of microcystin. It should be notedthat the unaffected transcription could be explained by recent

FIG. 7. RT-PCR for region mcyAB. A standard RT-PCR was performed with the gene-specific primers 135-F and 676-R. There is a weak signalin lane N-C 169/7.



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findings indicating that mcyA and mcyD have two transcrip-tional start points (22).

The different microcystin isoforms synthesized by each strainwere determined by MALDI-TOF mass spectrometry, and agood correlation was found between the genetic variants andthe microcystin isoforms produced by the strains. Most impor-tantly, the B-type variants produce various microcystin-LRisoforms (one strain produces microcystin-YR in addition tomicrocystin-LR), and the C-type variants synthesize either mi-crocystin-RR (the true C type) or microcystin-RR in combi-nation with microcystin-LR (the other subgroups of the Ctype). The observed substrate specificity follows the predictedamino acid sequence variation that influences the substrate-binding pocket of the module (Table 4), which consists of 10amino acids. From the deduced amino acid sequences we couldidentify nine of these amino acids in the different variants(Table 4). The substrate-binding pocket of the mcyB1(C) vari-ant is very similar to the binding pocket of the mcyC gene(Table 4), containing arginine (R) in the strains used in thisstudy, thus explaining the presence of various microcystin-RRisoforms. However, some of these strains also produce micro-cystin microcystin-LR; this may be a position-dependent prop-erty or due to residue Gly236 (Table 4). This result is par-ticularly interesting in light of the finding that the C-typesubgroups possess a genetic recombinant mcyB1 module (achimeric mcyB1/C module). The mcyB1(B) variants, whichusually activate leucine (Table 3), show a high degree of sim-ilarity to other leucine-activating domains (7, 8, 46) (Table 4).

Taken together, our results demonstrate that there is astrong correlation between the microcystin isoforms producedand the genetic variants of the mcyB1 module; the geneticvariants are the result of recombinations between modules(i.e., between mcyB1 and mcyC). The presence of multiplegenetic variants of the mcyBC region is in agreement with thefindings of Kurmayer et al. (26). However, Kurmayer et al. (26)did not perform an extensive sequence and phylogenetic anal-ysis, and for that reason the distribution of specific microcystinisoforms could not be linked to the diversity of genotypes.According to our results, identification of isoform structure byanalyses such as MALDI-TOF mass spectrometry should en-able deduction of the type of genetic organization of the mi-crocystin synthetase genes.

Evolution of the microcystin synthetase genes through hor-izontal gene transfer, lateral recombination, and gene loss. Ofthe 18 strains used in this study, 7 were shown not to containthe mcyA and mcyB genes (Fig. 3 and 4). Our results, togetherwith data from a recent study by Tillett et al. (50) showingconservation of the chromosomal location of the mcyABC genecluster in all Microcystis strains, as well as the same gene ar-rangement in the synthetase-negative strains, suggest that themcyA and mcyB genes (or possibly the whole operon) havebeen lost in some strains. Thus, microcystin synthesis is prob-ably an ancestral feature of the genus Microcystis.

Phylogenetic studies of prokaryotic adenylation domainshave shown that they normally cluster according to function,making it unlikely that they arose by convergent evolution (7).Obviously, the selection forces acting upon these genetic mod-ules have been significant during evolution. The amino acidsequences in the two main variants of mcyB1 are highly diver-gent from motif A3 (amino acid 20 in Fig. 5) onward. The ratioof nonsynonymous to synonymous mutations is elevated in thisregion compared to amino acids 1 to 20 (Fig. 5) and to themcyAB (135-676) region. This could be seen as evidence ofdiversifying selection, which would be the case if the mcyB1(C)variants originated from recombination between mcyB1 andmcyC modules. Accordingly, selection is not sufficient to ex-plain the distribution of the mcyABC region, as well as thephylogenetic relationships between the module variants as op-posed to other markers, such as 16S rDNA and DNA finger-printing (RAPD, REP). Thus, the peptide synthetases appearto be the result of an evolutionary mechanism involving hori-zontal transfer, since structurally related peptides occur indiverse microorganisms (24). Notably, horizontal transfer pro-cesses have recently been suggested to be important evolution-ary mechanisms involved in genomic stability and variation inseveral closely related cyanobacterial strains (44), as mani-fested in the rbcLX (43) and trnl(UAA) genes (40–42). Theevolutionary processes leading to the distribution and geneticvariation of the microcystin synthetase gene cluster, includingthe two major genetic variants of the mcyB1 module, are likelyto be caused by horizontal gene transfer, lateral recombina-tion, and gene loss, particularly as suggested by the chimericmcyB1 modules and the nonconcordance between the micro-cystin phylogenies and the fingerprinting results. In this context

TABLE 4. Putative binding pocket constituents

Varianta Substrate(s)bResidue at positionc: Putative

substrate(s)d235 236 239 279 299 301 322 330 331

PCC 7806-like Leu D A W F I G N V V LeuN-C 161/1 Leu, Tyr D A L F I G N V V LeuN-C 264 Arg D V W T I G A V D D/L-PhePCC 7806 (mcyC) Arge D V W T I G A V D D/L-Phee

K-139 (mcyC) Arge D V W T I G A V E D/L-Phee

HUB 5-2-4-like Arg, Leue D G W T I G A V E D/L-Phe, Valc

grsA Phe D A W T I A A I C D/L-Phe

a PCC 7806-like includes PCC 7806, K-139, N-C 118/2, and N-C 31; PCC 7806 (mcyC), corresponding region in module mcyC; K-139 (mcyC), corresponding regionin module mcyC; HUB 5-2-4-like includes HUB 5-2-4, N-C 169/7, N-C 324/1, N-C 228/1, N-C 143, and N-C 57.

b Substrate(s) as predicted by MALDI-TOF mass spectrometry analyses (Table 3).c Positions as described by Conti et al. (8).d Substrate(s) as predicted from the specificity-inferring code of adenylation domains (http://www.jbu.edu/chem/townsend/).e Data from references 16 (PCC 7806 and HUB 5-2-4) and 36 (K-139). Note that the predictions from the specificity-inferring code of adenylation domains in these

cases are not in agreement with the empirically determined data. This was also seen for the other C-type strains (i.e., HUB 5-2-4-like).



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it is interesting that uma4, a gene downstream of mcyC, en-codes a peptide with 45% identity to TnpA, a transposase fromAnabaena sp. strain PCC 7120 (49).

The few differences in the 16S rDNA or mcyAB regions didnot exhibit any correspondence to the variants found in themcyB gene or to properties of toxicity or nontoxicity. This is inagreement with other studies that were also unable to identifydifferences in the 16S rDNA genes that were correlated withtoxic or nontoxic strains (34, 37, 50).

Does the genetic variation and recombination of themcyABC gene cluster enhance our understanding of microcys-tin function? The biological function or functions of microcys-tin are currently unknown, although there are many theories,ranging from iron binding (51) to involvement in quorum sens-ing (10). The diversity of microcystin isoforms is unlikely tohave a restraining effect on the function of microcystin in thecyanobacterial cell. Rather, it seems more likely that the di-versity of isoforms is crucial to the various strains in theirnatural habitats. Furthermore, the genetic processes generat-ing the genetic diversity, particularly in the substrate-bindingpockets, are also expected to be of crucial biological impor-tance. Based on these results, it should now be possible todesign genetic constructs of the mcyB1 variants and introducethese into identical genetic backgrounds (strains) in order totest for phenotypic effects in complex laboratory-designed eco-systems.

ACKNOWLEDGMENTS

We thank Randi Skulberg for providing the N-C strains, KamranShalchian-Tabrizi for help with the phylogenetic analyses, and TonjeFossheim for modifications of the DNA isolation protocol. Thanks arealso due to Hans Utkilen and Arne Mikalsen for providing the PCC7806 and HUB 5-2-4 strains and for fruitful discussions.

This work was supported by grant 107622/420 from the NorwegianResearch Council to K.S.J.

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Natural Variation in the Microcystin Synthetase Operon ...transcription step. No PCR products were detected by agarose electrophoresis (data not shown). Sequencing and phylogenetic