Vol. 69: 135–143, 2013 AQUATIC MICROBIAL ECOLOGY Published online May 28 doi: 10.3354/ame01628 Aquat Microb Ecol A cultivation-independent approach for the genetic and cyanotoxin characterization of colonial cyanobacteria Yannick Lara1, Alexandre Lambion1, Diana Menzel2, Geoffrey A. Codd2, Annick Wilmotte1,* 1Center for Protein Engineering, University of Liège, 4000 Liège, Belgium 2Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee DD1 4HN, UK ABSTRACT: To bypass the constraint of cyanobacterial strain isolation and cultivation, a combina- tion of whole genome amplification (WGA) and enzyme-linked immunoassay (ELISA) for micro- cystin toxins (MCs) was tested on individual colonies of Microcystis and Woronichinia, taken directly from aquatic environments. Genomic DNA of boiled cells was amplified by multiple strand displacement amplification (MDA), followed by several specific PCR reactions to character- ize the genotype of each colony. Sequences of 3 different housekeeping genes (ftsZ, gltX, and recA), of 3 MC biosynthesis genes (mcyA, mcyB, and mcyE), and the Internal Transcribed Spacer (ITS) were analyzed for 11 colonies of Microcystis. MCs were detected and quantified by ELISA in 7 of the 11 Microcystis colonies tested, in agreement with the detection of mcy genes. Sequence types (ST) based on the concatenated sequences of housekeeping genes from cyanobacterial colonies from Belgian water bodies appeared to be endemic when compared to those of strains described in the literature. One colony appeared to belong to a yet undiscovered lineage. A simi- lar protocol was used for 6 colonies of the genus Woronichinia, a taxon that is very difficult to cul- tivate in the laboratory. The 16S rRNA sequences of 2 colonies were obtained and were quasi identical to that of W. naegeliana 0LE35S01. For one Woronichinia colony, the mcyE PCR gave a non-specific PCR product. The corresponding amino acid sequence was 50% identical to a Micro- cystis ketoacyl carrier protein transferase. This approach for the simultaneous detection and quan- tification of MCs with mcy genotyping, at single colony level, offers potential for the ecotoxicolog- ical characterization of environmental populations of cyanobacteria without the need for strain isolation and culture. KEY WORDS: Microcystis · Woronichinia · Microcystins · ELISA · MDA · Colony-forming cyanobacteria Resale or republication not permitted without written consent of the publisher INTRODUCTION genera (e.g. Sivonen & Jones 1999, Metcalf & Codd 2012). Microcystis represents a good model organism Microcystis is a unicellular, colony-forming cyano- for the study of MC production and is among the bacterium present as blooms in nutrient-enriched, most commonly encountered MC-producing genera standing freshwaters. It can produce microcystins worldwide (Sivonen 2008). At the genetic level, the (MCs), cyclic heptapeptides that are potent hepato- mcy gene cluster involved in MC biosynthesis con- toxins and tumor promoters. Over 90 structural MC sists of a combination of non-ribosomal peptide syn- variants have been characterized thus far, from a thase (NRPS) and polyketide synthetase (PKS) genes. wide range of planktonic and benthic cyanobacterial A recent multi locus sequence typing, carried out *Corresponding author. Email: [email protected] © Inter-Research 2013 · www.int-res.com 136 Aquat Microb Ecol 69: 135–143, 2013 mainly on strains from Asian water bodies, resulted al. 2010) and the apratoxin pathway of Lyngbya in at least 8 different cryptic or panmictic lineages bouillonii (Grindberg et al. 2011). (Tanabe et al. 2009, Tanabe & Watanabe 2011). Micromanipulation of Microcystis colonies directly So far, resolution of the comparative toxicological isolated from the environment has already been per- and corresponding genotypic characteristics within formed to characterize their MC production by environmental populations of cyanobacteria has matrix-assisted laser desorption/ionisation time of been limited by the technical need for strain isolation flight mass spectrometry (MALDI-TOF MS) analysis and cultivation. While this is possible with Microcys- and genotype by 2 to 3 PCR analyses (e.g. Janse et al. tis, loss of the colony-forming habit almost invariably 2004, Via-Ordorika et al. 2004). occurs (Visser et al. 2005). Moreover, a Microcystis The aims of the present investigation were (1) to bloom population can include several genotypes that improve the genotyping of environmental colonies of may produce MCs or not, and evolve in space and Microcystis by increasing the amount of DNA mate- time (Kardinaal & Visser 2005, Kardinaal et al. 2007). rial for multiple PCRs, to enable both genetic charac- A further limitation of the strain isolation approach terization and biochemical analysis (in this case, MC was shown by Fewer et al. (2009) in studies of a concentration); (2) to use this on colony-forming mixed Anabaena/Nodularia bloom in the Gulf of cyano bacteria that are, to date, difficult or impossible Finland, from which only certain genotypes may be to cultivate such as Woronichinia. We therefore pro- selected during laboratory isolation and cultivation. posed to apply the WGA technology (before perform- Uncertainty exists regarding the production of ing multiple PCRs) together with an immunochemi- MCs by a second planktonic genus of cyanobacteria: cal assay for MCs, on single colonies from natural Woronichinia. Although high concentrations of MCs populations, with portions of the same colony in each were found in blooms dominated by Woronichinia, case being used for both WGA and immunoassay. species of other established MC-producing genera We present a dataset obtained for 11 Microcystis were also present as minor members (Willame et al. and 6 Woronichinia colonies. We suggest that this 2005). Woronichinia belongs to the sub-family Gom- approach can also be used on other colony-forming phosphaerioideae in the family Merismopediaceae. or filamentous cyanobacteria and can contribute to This colony-forming genus is characterized by the the study of cyanobacterial phylogeny. binary fission of cells in 2 perpendicular planes in successive generations and the location of cells at the end of mucilaginous stalks which radiate from the MATERIALS AND METHODS center of the colonies (Komárek & Anagnostidis 1999). Woronichinia is frequently observed in Euro- The experimental procedure was divided into 2 pean and Scandinavian lakes. It can dominate phyto- main steps (for full details, see the Supplement at plankton in oligotrophic, mesotrophic and eutrophic www.int-res.com/articles/suppl/a069p135_supp. pdf). lakes and ponds (Rajaniemi-Wacklin et al. 2006). Lit- First, the environmental colonies of Microcystis and tle is known about the genus Woronichinia since only Woronichinia spp. were isolated under sterile condi- 2 strains, 0LE35S01 and 1ES42S01, have been isolated tions and washed in 100 µl sterile BG11 medium (Rajaniemi-Wacklin et al. 2006, Willame et al. 2006). droplets to dilute potential co-occurring algal and Unfortunately, these strains could not be maintained bacterial contaminants present around the cyanobac- in culture, although their 16S rRNA sequences were terial colonies. The colonies were then photo graphed deposited in GenBank. Only one strain (1ES42S01) (100× to 400×) and suspended in 5 to 15 µl BG11 was tested for the presence of the mcyE gene and the (Rippka et al. 1979). In the second step, the individual result was negative (Willame et al. 2006). colonies were disrupted by boiling at 99°C for 1 min Recently, new approaches have been developed to in a thermocycler. From the resulting suspensions, study the genetic features of uncultured single cells 0.5 µl subsamples were used as a template for MDA including microbes, involving enzymes e.g. DNA reactions. The remaining volumes of the suspension polymerase Phi29 (Woyke et al. 2010). Phi29 is a were used for MC quantification by enzyme-linked strand displacement polymerase (Kvist et al. 2007), immunoassay (ELISA) with microcystin-LR (MC-LR) which amplifies DNA by isothermal multiple strand antibodies. The MDA reaction was performed with displacement (MDA). This whole genome amplifica- the QIAGEN REPLI-g Mini Kit following the ‘Ampli- tion (WGA) technology has already helped charac- fication of genomic DNA from blood or cells’ proto- terize the metabolism of the uncultured unicellular col. ELISA was performed following Young et al. nitrogen-fixing cyanobacterium UNCY-A (Tripp et (2008). We then used multiple sets of Microcystis Lara et al.: Genetic and cyanotoxin characterization of colonial cyanobacteria 137 and/or cyano-specific PCR primers (Table S1 in the The estimated toxin content per co lony, based on Supplement) to estimate genome amplification effi- cross-sectional area measurement (Young et al. ciency. The success of the WGA is attributed to an 2008), ranged from 70.15 to 854.9 pg. Microcystis effective dispersal and homogenization of the boiled colony cross-sectional area as an indicator of colony colonies, as well as to the availability of a sufficient volume was preferred as a more reliable indicator number of cells for the MDA reaction, shown to be than mathematical models, which tend to be specific critical by Rodrigue et al. (2009). for the populations from which they are drawn (Mor- The sequences reported in the present study were rison 2005). The MC concentrations per colony area deposited in the GenBank database, with accession ranged from 0.36 to 31.37 ng mm−2. The highest