Cyanotoxin Screening in BACA Culture Collection: Identification of New Cylindrospermopsin Producing Cyanobacteria

Microcystins (MCs), Saxitoxins (STXs), and Cylindrospermopsins (CYNs) are some of the more well-known cyanotoxins. Taking into consideration the impacts of cyanotoxins, many studies have focused on the identification of unknown cyanotoxin(s)-producing strains. This study aimed to screen strains from the Azorean Bank of Algae and Cyanobacteria (BACA) for MCs, STX, and CYN production. A total of 157 strains were searched for mcy, sxt, and cyr producing genes by PCR, toxin identification by ESI-LC-MS/MS, and cyanotoxin-producing strains morphological identification and confirmation by 16S rRNA phylogenetic analysis. Cyanotoxin-producing genes were amplified in 13 strains and four were confirmed as toxin producers by ESI-LC-MS/MS. As expected Aphanizomenon gracile BACA0041 was confirmed as an STX producer, with amplification of genes sxtA, sxtG, sxtH, and sxtI, and Microcystis aeruginosa BACA0148 as an MC-LR producer, with amplification of genes mcyC, mcyD, mcyE, and mcyG. Two nostocalean strains, BACA0025 and BACA0031, were positive for both cyrB and cyrC genes and ESI-LC-MS/MS confirmed CYN production. Although these strains morphologically resemble Sphaerospermopsis, the 16S rRNA phylogenetic analysis reveals that they probably belong to a new genus.

Microcystins are hepatotoxins produced by non-ribosomal pathways, and the most common and more well-studied cyanotoxins, with over 240 variants (e.g., MC-LR, MC-LY, MC-RR). Mostly produced by Microcystis aeruginosa [11], MCs are, however, currently known to be produced by over 40 species [12]. The gene cluster responsible for the biosynthesis of MCs (mcy) contains 10 encoding genes organized in two operons (mcyA-C Table 1. Detection of genes involved in microcystin, cylindrospermopsin, and saxitoxin production, and ESI-LC-MS/MS analysis on BACA strains. Only strains with positive detection of either the genes or the toxins are shown.

Strains
Habitat STX producing genes were detected in six strains, however, the only strain with amplification of all the searched STX-encoding genes, Aphanizomenon gracile BACA0041, was the only one confirmed as saxitoxin producer by ESI-LC-MS/MS (Table 1).
CYN producing genes were detected in five strains, although only BACA0025 and BACA0031 had amplification of both searched cyrB and cyrC genes. These two strains were also the only ones with CYN detection by ESI-LC-MS/MS. The remaining three strains with positive results for the detection of CYN encoding genes only amplified the cyrB gene (Table 1).

Phylogenetic Characterization
Strains BACA0025 and BACA0031 had similarities with sequences deposited in the GenBank NCBI between 96 and 97%, which shows the distinctiveness of these strains, while strains BACA0041 and BACA0148 had similarities greater than 98% with Aphanizomenon gracile and Microcystis aeruginosa strains, respectively (Table S3). In the 16S rRNA phylogenetic tree strains BACA0041 and BACA0148 are positioned in clades with strains close to their initial identification, while strains BACA0025 and BACA0031 had high phylogenetic distances from the closest morphological identified strains.

Discussion
The ability to produce cyanotoxins depends on the simultaneous existence of several genes involved in their biosynthesis pathways [42]. As seen in previous studies, the sxt cluster has suffered several modifications and its gene composition and organization varies between taxa [6,[43][44][45]. Although the amplification of the sxtA gene and the absence of toxin production in Aphanizomenon strains was seen in other studies [10,25,46,47], in the studied strains only strains with sxtA amplification were able to produce STX (Table  1). Cirés et al. [47] states that the sxtA gene does not allow the distinction between STXproducing and non-producing Anabaena/Aphanizomenon strains, nonetheless the presence of sxtA in non-producing strains could have been due to gene loss/inactivation within the sxt cluster [46]. The presence of sxtG or sxtH amplifications in several non-STX producing genera (Table 1; Table S2; Figure S3) in this study are interesting new results that require further investigation to fully understand sxt gene distribution among cyanobacteria taxa. Nonetheless, to our knowledge, the presence of sxtG, and/or sxtH genes in Kamptonema, Leptodesmis, Anathece minutissima, or Leptolyngbya strains, have not been reported before.
Aphanizomenon gracile BACA0041 was confirmed as an STX producer with amplification of the sxtA, sxtG, sxtH, and sxtI genes. The ESI-LC-MS/MS spectra of A. gracile BACA0041 matched the fragmented pattern of STX standard spectra ( Figure S4), with identification of the precursor ion 300 m/z and product ions 282 m/z, 265 m/z, 241 m/z, 240.25 m/z, 204 m/z, and 186 m/z [10]. Phylogenetic analysis confirms Aphanizomenon gracile BACA0041 identification's, this strain is positioned in a well-supported clade of several STX-producing A. gracile strains (Figure 2), as A. gracile UAM531 [47] and A. gracile PMC 638.10 [10]. In our study, we report another freshwater STX-producing A. gracile strain, the first STX producer identified in the Azores islands.
Strains BACA0025 and BACA0031 are quite similar, these were both initially identi-

Discussion
The ability to produce cyanotoxins depends on the simultaneous existence of several genes involved in their biosynthesis pathways [42]. As seen in previous studies, the sxt cluster has suffered several modifications and its gene composition and organization varies between taxa [6,[43][44][45]. Although the amplification of the sxtA gene and the absence of toxin production in Aphanizomenon strains was seen in other studies [10,25,46,47], in the studied strains only strains with sxtA amplification were able to produce STX (Table 1). Cirés et al. [47] states that the sxtA gene does not allow the distinction between STXproducing and non-producing Anabaena/Aphanizomenon strains, nonetheless the presence of sxtA in non-producing strains could have been due to gene loss/inactivation within the sxt cluster [46]. The presence of sxtG or sxtH amplifications in several non-STX producing genera (Table 1; Table S2; Figure S3) in this study are interesting new results that require further investigation to fully understand sxt gene distribution among cyanobacteria taxa. Nonetheless, to our knowledge, the presence of sxtG, and/or sxtH genes in Kamptonema, Leptodesmis, Anathece minutissima, or Leptolyngbya strains, have not been reported before.
Aphanizomenon gracile BACA0041 was confirmed as an STX producer with amplification of the sxtA, sxtG, sxtH, and sxtI genes. The ESI-LC-MS/MS spectra of A. gracile BACA0041 matched the fragmented pattern of STX standard spectra ( Figure S4), with identification of the precursor ion 300 m/z and product ions 282 m/z, 265 m/z, 241 m/z, 240.25 m/z, 204 m/z, and 186 m/z [10]. Phylogenetic analysis confirms Aphanizomenon gracile BACA0041 identification's, this strain is positioned in a well-supported clade of several STX-producing A. gracile strains (Figure 2), as A. gracile UAM531 [47] and A. gracile PMC 638.10 [10]. In our study, we report another freshwater STX-producing A. gracile strain, the first STX producer identified in the Azores islands.
Strains BACA0025 and BACA0031 are quite similar, these were both initially identified as Sphaerospermopsis sp. (isolated from similar freshwater lakes from the same island; Table S1), with similar BlastN results (Table S3). Strains BACA0025 and BACA0031 were positioned in cluster II in the phylogenetic tree ( Figure 2) close to Nostocaceae genera, however with significant phylogenetic distance to conclude that these strains might belong to a new genus. Both were confirmed as CYN producers with detection of both cyrB and cyrC genes ( Table 1). The ESI-LC-MS/MS spectra of BACA0025 and BACA0031 both matched the fragmented pattern of CYN standard spectra ( Figure S5), with identification of the precursor ion 416 m/z and product ions 336 m/z, 318 m/z, 274 m/z, and 194 m/z [48], confirming these two strains as CYN producers.
The cyrB gene was also amplified in Nostoc sp. BACA0109, and in two thermal Leptolyngbya sp. strains BACA0142 and BACA0146, however without CYN identification in the ESI-LC-MS/MS. Amplification of cyr genes without CYN production confirmation has been reported previously, as is the case of non-CYN producing Chrysosporum bergii and Chrysosporum ovalisporum strains with amplification of cyrA, cyrB, and cyrC genes [49]. Nostoc and Leptolyngbya strains are known to produce cyanotoxins, however, MCs [12,17,50] and not CYN, and as far as we know, the presence of the cyrB and cyrC genes has not been previously reported in Nostoc or Leptolyngbya strains.
Microcystins are the most common and more well-studied cyanotoxins, being MC-LR one of the most prevalent and toxic congeners [51]. Microcystis aeruginosa was the first cyanobacteria species identified as MCs producer and is the most studied species regarding MCs [11]. Our results show that the Azorean strain M. aeruginosa BACA0148 is also an MC-LR producer, with the detection of mcyC, mcyD, mcyE, and mcyG genes. Characteristic MC-LR fragmentation pattern was observed in M. aeruginosa BACA0148 ( Figure S6), with identification of precursor ion 995 m/z and product ions 977 m/z, 866 m/z, 599 m/z, and 553 m/z [52]. The mcyE gene amplification in Nostoc sp. BACA0091, despite the absence of MC-LR ions in the ESI-LC-MS/MS, or the absence of the other searched mcy genes (mcyC, mcyD, and mcyG), can be explained due to gene(s) recombination or loss [6,14,53]. As stated by Dittmann et al. [6], the mcy gene cluster has high repetitive sequences, enabling recombination events that ultimately cause changes in the final product, which can be confirmed by the growing reported number of MCs congeners [54,55].
All strains identified as toxin producers (BACA0025, BACA0031, A. gracile BACA0041, and M. aeruginosa BACA0148) were isolated from lakes in Pico and São Miguel islands (Table S1, S2). The presence of toxic strains in these lakes represents environmental and public health hazards. Contrarily, none of the strains isolated from thermal and terrestrial habitats were identified as cyanotoxin producers, although in some of them, cyanotoxinencoding genes were detected, as in the thermal Leptolyngbya strains BACA0112, BACA0123, BACA0142, BACA0144 and BACA0146, and Coleospermum sp. BACA0119.

Conclusions
Within the BACA collection, we identified and reported another MC-LR-producing M. aeruginosa strain (BACA0148) and another STX-producing A. gracile strain (BACA0041). Phylogenetic analysis revealed evidence for new cyanobacteria taxa BACA0025 and BACA0031, confirmed as CYN producers. Further studies are necessary to confirm and describe these new taxa, with morphological characterization and 16S rRNA and ITS analysis.
The identification of new cyanotoxin-producing strains, and unreported toxins, in the Azores, confirms the risk of toxicity and threat to environmental and public health; thus, an appropriate monitoring program should be implemented/updated to search MCs, STX, and CYN. Future efforts should also be made to avoid cyanobacteria blooms and consequently cyanotoxins released in high concentrations.

BACA Strains and Growth Conditions
A total of 157 strains, isolated from various environments (Table S1), were retrieved from the Azorean Bank of Algae and Cyanobacteria (BACA) created in the framework of the REBECA project (MAC/1.1a/060). For genetic analysis, 50 mL cultures were prepared without agitation, whereas for toxin extraction, the cultures were scaled up to 1 L, with filtered aeration. All strains were grown in liquid BG-11 media (with or without combined nitrogen) [56], in a climate-controlled room with a 14:10 h light: dark (170 µmol photons m −2 s −1 ) photoperiod at 25 • C [56,57]. Cyanobacterial cells were harvested by centrifugation (4000× g for 15 min), after 3-5 weeks, and lyophilized. The lyophilized cyanobacteria biomass were stored at −20 • C.

Toxin extraction
The lyophilized cyanobacteria, 157 samples in total, were weighed (80-100 mg) to a glass vial and extracted with a 5% methanolic solution (2-10 mL). Solutions were then submitted to ultrasounds for 1-5 min, 60 Hz, in an ice bath, and transferred to falcons to be centrifuged (5000× g, 5 min, 4 • C). Pellet was then submitted to a second extraction and left in the dark at 4 • C overnight. Supernatants were pooled together and lyophilized (extraction solvent completely freeze-dried).
Residues were finally dissolved in 200-500 µL 50% methanol LC-MS grade acidified with 0.1% Formic Acid and filtered with a nylon membrane 0.2 µm before analysis (or centrifuged at 10,000× g for 5 min). Samples were protected from light through all the processes and stored at −80 • C until analysis.
All the standards were injected individually and then as a standard mixture with a concentration interval from 10 ppb to 300 ppb. ESI-LC-MS/MS analysis was performed to confirm the presence or absence of these three cyanotoxins in the selected cyanobacteria strains.
The eluents used were methanol (A) and water (B) both acidified with formic acid at 0.1% (v/v). The gradient program started at 10% B (held for 10 min), increasing to 50% B in 5 min, turning back to initial conditions in 5 min, equilibrating more 10 min with 10% B. The injection volume was 10 µL with a flow of 0.2 mL min −1 and column kept at 30 • C.
Mass spectrometry analysis acquisition parameters were as follows: ESI source, positive ionization using collision-induced dissociation (CID). Table S4 resumes analysis parameters for each searched toxin.

DNA Extraction, PCR Amplification, and Sequencing
Total genomic DNA was extracted with the PureLinkTM Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA), as previously described by Cordeiro et al. [58]. DNA samples were stored at −20 • C.

Phylogenetic Analysis
Partial 16S sequences were amplified for the four strains with toxin identification by ESI-LC-MS/MS. All the sequences obtained in this study were compared with sequences deposited in the GenBank NCBI by BlastN tool.
The 16S rRNA gene phylogenetic relations were calculated using maximum likelihood (ML) and Bayesian inference (BI). jModelTest 2.1.10 [69] was used to select the best-fit nu-cleotide model for our database, on which the general time-reversible evolutionary model of substitution with gamma-distributed evolutionary rates and with an estimated proportion of invariable sites (GTR+G+I) was selected. ML was calculated using the IQ-Tree online version v1.6.12 [70] with 1000 ultrafast bootstrap and BI was calculated using MrBayes v3.2.7a [71], applying two separate runs with four chains each and 50,000,000 Markov chain Monte Carlo generations (sampling every 100 generations with a 0.25 burn-in). The tree was drawn with FigTree 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree, accessed on 8 February 2021) and Inkscape 1.0.1 (https://inkscape.org/pt/, accessed on 8 February 2021). Only the ML tree is presented, with bootstrap percentages (ML) and BI probabilities for branch support, since ML and BI methods resulted in similar trees. Only probabilities above 0.9 and bootstrap percentages above 50 are shown at the branch nodes of the phylogenetic distance trees. Gloeobacter violaceus PCC 8105 (AF132791) was used as the out-group.  Table S1: Strains information, Table S2: PCR amplifications of MC (mcyC, mcyD, mcyE, mcyG), STX (sxtA, sxtG, sxtH, sxtI) and CYN (cyrB, cyrC) biosynthesis-encoding genes and ESI-LC-MS/MS toxicity confirmation, Table S3: Sequence identity (%) of 16S rRNA gene fragment between BACA strains and other cyanobacterial sequences available in GenBank (NCBI), Table S4