Comparison of microcystin synthetase genes.
Although cyanobacteria are essential microorganisms on earth, some cyanobacteria produce toxins known as cyanotoxins, threatening humans and animals’ health. Hence, it is imperative to rapidly and accurately identify those toxic cyanobacteria. Unfortunately, traditional microscopic methods have limitations for accurate identification due to the lack of discernable morphological difference between toxic and non-toxic strains within the same cyanobacterial species or genus. In contrast, their genetic profiles are inherently conserved; therefore, nucleic acid-based assays can be more reliable for precise identification. Furthermore, molecular assays can provide high throughput and significantly reduce the turnaround time of test results. Such advantages make those assays a preferred method for rapid detection and early warning of potential toxicity. Toxigenic cyanobacterial species have synthetase genes (DNAs) for toxin production, which can be excellent marker genes. Numerous molecular assays targeting cyanotoxin synthetase genes have been developed for the identification of toxigenic cyanobacteria at various taxonomic levels. Polymerase chain reaction (PCR)-based assays are the most prevailing. Among different versions of PCR assays, the real-time quantitative PCR can be utilized to quantify the genes of interest in samples, fulfilling the purpose of both taxonomic recognition and biomass estimation. Reverse transcription (RT)-PCR assays can be used to detect transcripts (i.e., mRNAs) from toxin synthetase genes, probably enhancing the predictive value of PCR detection for toxin production from observed cyanobacterial species. Nevertheless, the utility of toxin synthetase gene- or its transcript-based PCR assays for routine cyanotoxin monitoring needs to be further evaluated on a large scale.
- toxin synthetase genes
- molecular techniques
- polymerase chain reaction
Cyanobacteria are essential microorganisms on earth as they produce oxygen and account for a large part of primary aquatic productivity. Simultaneously, some freshwater cyanobacteria can produce various toxins, named cyanotoxins, some of which are potently poisonous to humans and animals. A well-known cyanotoxicosis in humans was reported from Brazil in association with medical malpractice in 1996. In this incident, 126 patients in a hemodialysis unit were affected, and 60 of them died due to using microcystin-contaminated water from a local reservoir. A cyanobacterial bloom was found in that reservoir concurrently . Besides, there have been reports concerning human cyanotoxin poisoning by drinking water or via injury after contacting recreational water . Apart from humans, numerous animal poisoning cases have also taken place because they can reach the unprocessed natural water directly so that the risk of being poisoned becomes higher. These cases involve livestock, pets, and wildlife [3, 4, 5, 6, 7, 8, 9, 10].
Cyanobacterial blooms occurred more frequently in recent years, which may have been attributed to the aggravating eutrophication in freshwater and global warming. As such, cyanotoxin poisoning incidents have also been increasingly reported. Nowadays, freshwater cyanobacterial blooms have broader geographical and temporal impacts on local water bodies that act as vital municipal or agricultural water supplies. With the possibility of cyanotoxin contamination, humans and animals residing in surrounding areas continue to be threatened. Therefore, testing for toxic cyanobacteria or cyanotoxins is imperative for detection and preventive measures.
Although cyanobacteria can be observed under a microscope, their toxigenicity cannot be determined by microscopy because the toxigenic cyanobacteria do not have unique morphological characteristics. Some laboratories have adopted a testing strategy that combines microscopic observation and cyanotoxin detection to indicate the existence of toxigenic cyanobacteria in samples. Although this strategy may seem reasonable and pragmatic, it needs collaboration between chemical analysts and microalgal biologists to reach an agreement on the conclusion. Furthermore, it neglects the complex phenomena of the same toxin production by different species or genera, leading to an incorrect judgment of the truly culpable toxin producers.
Cyanotoxin testing has been in place. Yet, available tests have shortcomings. For example, commercial enzyme-linked immunosorbent assays (ELISAs) have been widely employed in water testing for cyanotoxins. However, it still has issues, such as low sensitivity  or inaccuracy. Erroneous detection is due to the cross-reactivity of isomorphic substances with targets. False-positive results can occur in a worst-case scenario . The high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC–MS) are the most accurate analytical methods and have been often employed in cyanotoxin testing [11, 13, 14, 15, 16]. But they require exquisite instruments and complicated operations, making them not as affordable as ELISA-based testing. Aside from these limitations, chemical testing can only tell the presence and/or quantity of cyanotoxins without identifying the toxin producer(s). However, it is crucial to recognize the existence of toxigenic cyanobacteria in water bodies for monitoring and early warning of cyanotoxin poisoning incidents.
It is known that cyanotoxin synthesis is catalyzed by a string of relevant enzymes encoded by toxin synthetase genes [17, 18, 19, 20, 21, 22, 23]. Lack of essential genes for forming a toxin backbone or disruption of the enzymatic cascade toward toxin production results in the failure of toxin synthesis. Therefore, the detection of toxin synthetase genes in samples by a molecular test can disclose the presence or absence of toxigenic cyanobacteria. In this chapter, we review the application of molecular techniques, particularly PCR-based assays, for detecting toxigenic cyanobacteria in freshwater.
2. General genomic organization of toxigenic cyanobacteria
Like other bacteria, cyanobacteria often have one circular chromosome and a few plasmids that consist of the whole genome. The cyanobacterial chromosome is a few megabases in size and contains most of the genes, while plasmids play a role in transferring DNA elements. Compared to the eukaryotic microalgae, the cyanobacterial genome is highly compressed but still contains all genes essential for aquatic and photosynthetic life. Some species even have genes that can facilitate competitive superiority in the environment. For example, gas vesicle genes in
Cyanotoxin synthetase genes often cluster together in the genome and constitute one or more operons that are transcribed in identical or opposite directions [19, 21, 22, 23, 26]. The reason for such an arrangement is likely that the transcription can be well regulated so that all pertaining genes are transcribed simultaneously. This process may ensure that all necessary enzymes/proteins are present for subsequent toxin synthesis. The whole-genome sequencing of toxic cyanobacteria to date has demonstrated only a single copy of the toxin gene cluster in the cyanobacterial genome [27, 28, 29]. The toxin synthetase genes have conserved sequences encoding conserved domains/motifs in the corresponding proteins with specific functions during toxin syntheses, such as polyketide synthesis, adenylation, and methylation. The genes are always clustered closely with whose proteins conduct successive functions in a cascade reaction. It should be reiterated that the synthetase genes are indispensable for toxin production, making them the ideal targets for molecular detection.
Cyanotoxins are traditionally named after the first identified toxin-producing genus, as in the case of microcystin (
3. Cyanotoxins and toxin biosynthesis
Microcystin is the most common cyanotoxin implicated in human and animal poisoning incidents [36, 37, 38]. It is a hepatotoxin and thus can cause severe impairment in the liver when ingested by the casualties. The toxin is known to be produced by several genera of cyanobacteria, such as
Microcystin is a cyclic heptapeptide that inhibits the eukaryotic protein phosphatase type 1 and 2A in humans and animals by forming an irreversible covalent bond to a cysteine in the catalytic domain of these enzymes. It consists of the following amino acids: D-alanine, X, D-MeAsp (D-erythro-ß-methyl-aspartic acid), Z, Adda ((2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid), D-glutamic acid, and Mdha (N-methyldehydroalanine). X and Z represent variable L amino acids. It has reportedly over 80 variants, mostly differing in amino acids at the positions X and Z .
Microcystin is a non-ribosomal oligopeptide, which means unlike most of the peptides and proteins, it is not synthesized by cellular ribosomes. The enzymes responsible for its synthesis contain the non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) modules as well as tailoring functional domains. All the enzymes are the protein products encoded by the microcystin synthetase genes (
|Gene||Size (bp) 1||Encoded domain or function 2||Existence in different genera|
|8838||NRPS, C, NMT, E||yes||yes||yes|
|6318||NRPS, A, T, C||yes||yes||yes|
|3876||NRPS, C, A, TE||yes||yes||yes|
|11721||PKS, KS, AT, KR, DH, ACP, CM||yes||yes||yes|
|10464||PKS, NRPS, KS, AT, ACP, CM, AMT||yes||yes||yes|
|7896||NRPS, PKS, KS, AT, CM, DH, KR, ACP||yes||yes||yes|
Per annotation of
The cyanobacterial alkaloid anatoxin-a has been found in different genera, such as
Although anatoxin-a doesn’t look structurally complicated, its synthesis still requires a cascade of many enzymes whose genes known as anatoxin-a synthetase genes (
|Gene||Size (bp) 1||Encoded domain or function 2||Existence in different genera|
|6438||PKS, KS, AT, DH, ER, KR, ACP||yes||yes||yes|
|5619||PKS, KS, AT, DH, KR, ACP||yes||yes||yes|
|4896||PKS, KS, AT, CM, ACP||yes||yes||yes|
To start the anatoxin-a synthesis, AnaC activates and tethers the precursor proline to AnaD, which covalently combines with the proline. Then AnaB dehydrogenates the heterocyclic ring of proline to form a “C=N” double bond. AnaE introduces a carbonyl group into its connection with the heterocycle passed from AnaD. Then AnaJ catalyzes a cyclization step to form the characteristic bicyclic ring structure of anatoxin-a by connecting the heterocyclic ring with the backbone. At the same time, the growing chain is bound to the acyl carrier protein domain of AnaF. Finally, the bicyclic thioester is transferred to AnaG for chain extension by adding an acyl group, followed by the enzymatic reaction of AnaA to break the single “SCO-C” covalent bond connecting the enzyme (AnaG) and final product for the completion and releasing of anatoxin-a. Similar to its counterpart McyH in microcystin-producing cyanobacteria, AnaI transports the toxin through the cytomembrane. The rest of the Ana proteins are not commonly shared across different genera and have their own functions. AnaH is a transposase only found in
Cylindrospermopsin can be produced by various cyanobacterial genera, such as
Cylindrospermopsin is synthesized via a string of NRPS/PKS reactions conducted by up to over a dozen Cyr proteins ( Table 3 , Figure 3 ). The cylindrospermopsin synthetase genes (
|Gene||Size (bp) 1||Encoded domain or function 2||Existence in different genera|
|8754||NRPS, PKS, PCP, KS, AT, DH, MT, KR, ACP||yes||yes||yes|
|5005||PKS, KS, AT, KR, ACP||yes||yes||yes|
|5631||PKS, KS, AT, DH, KR, ACP||yes||yes||yes|
|5667||PKS, KS, AT, DH, KR, ACP||yes||yes||yes|
|4074||PKS, KS, AT, ACP||yes||yes||yes|
|1437||Uracil ring formation||yes||yes||yes|
|1431||Uracil ring formation||yes||yes||yes|
Nodularin is a cyclic pentapeptide and has the identical chemical structure as microcystin except the lack of D-alanine and the amino acid at position X. The mechanism of its toxicity is the same as microcystin’s, i.e., inhibiting the eukaryotic protein phosphatase catalytic subunit type 1 and 2A and leading to severe liver damage. Different from the three aforementioned cyanotoxins, nodularin is solely found in
|Gene||Size (bp) 1||Encoded domain or function 2|
|2607||NRPS, A, NM, PCP, C|
|1299||C, A, PCP, TE|
|2640||NRPS, PKS, A, PCP, KS, AT, CM, KR, ACP|
|3872||PKS, KS, AT, CM, DH, KR, ACP|
|3475||PKS, NRPS, KS, AT, CM, ACP, AMT, C, A, PCP|
Nodularin synthesis is conducted putatively according to the annotated functions of each Nda protein. NdaC activates the starter unit as phenylalanine or phenylacetate, and then NdaE catalyzes the transfer of a methyl group to the growing chain. NdaD is involved in two further polyketide extension steps, and NdaF facilitates the final round of polyketide extension and the biosynthesis of Adda. Next, epimerization of L-glutamic acid is catalyzed by NdaG, followed by the peptide condensation carried out by NdaA and NdaB. During the condensation, NdaH participates in the conversion of N-methyl-L-threonine (MeThr) to N-methyldehydrobutyrine (MeDhb) with a cofactor nicotinamide adenine dinucleotide (NADH). Finally, the mature peptide chain is cyclized by NdaB and released from the enzyme-substrate complex. As an ABC-transporter, NdaI is responsible for the transmembrane transportation of nodularin for extracellular excretion.
4. PCR detection of toxic cyanobacteria
PCR-based assays have been most commonly utilized in molecular identification studies because the assays are able to recognize targets accurately. The assays incorporate oligonucleotide primers explicitly designed for complementary sequences of the target gene(s). Two types of PCR methods have been used: conventional gel-based PCR and real-time PCR. In general, the real-time PCR has higher sensitivity (i.e., detect a low amount of the target) than the conventional PCR. The real-time PCR also offers better specificity than the conventional PCR since it uses an additional oligonucleotide known as a probe, which is complementary to sequences between primer-binding sequences.
Furthermore, the real-time PCR allows estimating the number of the intended target in samples when performed with standards with a known copy number of the target sequences. This procedure is referred to as quantitative real-time PCR (qPCR). In addition, reverse transcription (RT)-PCR or RT-qPCR platforms have been utilized for specifically detecting transcripts (i.e., mRNAs) from the target genes of cyanobacteria. Typically, PCR can be completed within one or two hours, much shorter than the traditional analytical methods and microscopy mentioned above.
4.1 Microcystin-producing cyanobacteria
The molecular identification of microcystin-producing cyanobacteria has been conducted using nearly all
Although most publications have been concerned about toxic/toxigenic
The rest of the
With increased bioinformatic data related to
There are a few unidentified open reading frames (ORFs) flanking the
4.2 Anatoxin-a-producing cyanobacteria
4.3 Cylindrospermopsin-producing cyanobacteria
Molecular detection of cylindrospermopsin-producing cyanobacteria has been mostly reported for
Multi-generic detection of cylindrospermopsin-producing cyanobacteria was reported as well. Campo et al. found that
There are also a few ORFs flanking the
4.4 Nodularin-producing cyanobacteria
5. Other cyanotoxins and PCR detection of the toxic cyanobacteria
Apart from the four most commonly reported cyanotoxins mentioned above, there are a few other cyanotoxins, such as saxitoxin, lyngbyatoxin, guanitoxin, β-N-methylamino-L-alanin (BMAA), aplysiatoxin, and lipopolysaccharide [18, 77, 78]. Hitherto, only the gene clusters for the biosynthesis of saxitoxin and lyngbyatoxin have been characterized.
Saxitoxin belongs to the group of carbamate alkaloid toxins composed of a tetrahydropurine group and two guanidinium moieties  and can also be produced by marine phytoplankton . It can cause paralytic shellfish poisoning syndrome and afflict human health via bioaccumulation. At least 30 clustered saxitoxin synthesis genes (
Lyngbyatoxin is characterized as a potent skin irritant produced by
No literature regarding molecular detection of cyanobacteria producing the rest of the toxins mentioned above could be searched. It is most likely because there are few reports as to the molecular mechanisms of their biosynthesis. Nevertheless, it is worthwhile to briefly introduce guanitoxin, previously known as anatoxin-a(S), to emphasize its difference from anatoxin-a. Guanitoxin was recently renamed due to its structural and toxicological disparities from anatoxin-a . It is a guanidino organophosphate neurotoxin that irreversibly inhibits acetylcholinesterase’s active site, leading to excess acetylcholine, which causes severe salvation and chromodacryorrhea, so-called “bloody tears” before respiratory arrest . Up to now, it was only found in planktonic
As various cyanobacterial genera can produce the same cyanotoxin, the development of toxigenic cyanobacteria identification needs to be multi-generic detection. Furthermore, as many genes for different toxins have sequences for the same conserved domains, designing PCR methods for all the cyanobacteria producing multiple toxins would be ideal.
Although most publications have focused on the selected cyanotoxins and their producers, more attention should be paid to other cyanotoxins and producers due to their potential of posing a significant threat to animal and human health. However, many cyanotoxin-producing cyanobacteria still lack bioinformation for the synthesis-related genes (e.g., guanitoxin), and it is thereby urgent to make further exploration to enrich the gene pools and their sequences so that a much more comprehensive understanding of the molecular mechanisms and the development of nucleic acid-based identification methods can be facilitated.
With the technical advance in PCR, researchers have been able to develop multiplex PCR methods in which many cyanotoxin biosynthesis genes can be detected simultaneously. For example, Ouahid et al. devised a multiplex PCR assay to detect six
Cyanobacteria with cyanotoxin synthetase genes in their genome are clearly equipped with the ability of toxin production. However, transcription of toxin biosynthesis genes is triggered by various environmental factors [88, 89, 90]; hence, toxin production is not consistently ongoing. It means the presence of genes itself may not always translate into the appearance of toxins unless they are inter- or extra-cellularly accumulated and detectable. Furthermore, the significant positive correlation between gene copies and toxin levels is still controversial, as described in this chapter and another review . Instead, the presence of mRNA transcripts from cyanotoxin synthase genes may be more closely associated with toxin production. Consequently, cDNA detection is justifiable to indicate an ongoing toxin synthesis, which is more critical and useful for monitoring the toxin-producing cyanobacteria. For this purpose, genes located at the end of operons should be good candidates for two reasons. One, primers designed from those genes can be directly used in cDNA testing like other genes because cyanobacteria lack introns. Two, the appearance of those genes in cDNA form signifies the successful cascade transcription of the clustered genes, gearing up all pertinent proteins for toxin synthesis. For example,
Although qPCR is preferred due to its many advantages, conventional PCR should also be considered for assessing the presence or absence of toxigenic cyanobacteria in water samples, as previously reported [49, 56]. In addition, the simplicity and cheaper operation may make conventional PCRs a more cost-effective tool for molecular detection of toxigenic cyanobacteria in comparison to qPCRs.
Besides PCR-based assays, there are other molecular technologies applicable to the identification and/or characterization of toxigenic cyanobacteria. A noteworthy method is the next-generation sequencing (NGS) technology. The technology has been widely used to identify previously unrecognized agents, non-culturable microorganisms, and/or variants because of its advanced and hypothesis-free sequencing ability  and has been applied to cyanobacteria research. Although most NGS studies have been investigations of taxonomic diversities using representative cyanobacterial genetic markers such as 16S rDNA [93, 94], the potential toxigenicity of cyanobacteria can be disclosed by sequencing the pooled libraries of toxin biosynthesis associated genes. Casero et al. revealed the existence of multiple toxigenic taxa in a summer bloom in a Spanish reservoir using
Nowadays, freshwater cyanobacterial blooms are seen more frequently than ever before because of increased eutrophication of their habitats and climate changes (e.g., global warming), which are utterly favorable to the overgrowth of cyanobacteria. Even though toxic cyanobacterial species are not always the mere culprit for these ecological disasters, they are often the dominant organisms and cause more destructive consequences because they can produce potent cyanotoxins into the water. There is no doubt that the toxic freshwater cyanobacteria pose a grave threat to human and animal health, agricultural production, tourism, to name a few. Hence, advancing techniques and technologies for rapid and reliable identification and monitoring of toxic cyanobacteria is an inevitable mission for healthcare, economy, and environmental conservation. To date, molecular assays, especially PCR-based tests, have been employed in toxic cyanobacterial identification, but their utilization should be further expanded into large-scale and long-term detection tasks and routine monitoring programs for not only the acute poisoning incidents but also the chronic impacts and preventative measures.
The authors disclosed receipt of the following financial support for the publication of this chapter: the manuscript compilation was supported in part by funding from the Innovative Swine Industry Enhancement Grant Program by the Iowa Attorney General’s Office, Iowa State University (ISU) Health Research Initiative, and ISU Veterinary Diagnostic Laboratory Research Support Fund.
Conflict of interest
The authors declare no conflict of interest.
The authors are sincerely grateful to the people who have made their contributions to the pertaining studies that are helpful for writing the chapter, including, but not limited to, Dr. Steve Ensley, Dr. Hyun-Joong Kim, Dr. Christopher Filstrup, Dr. Baoqing Guo, Dr. Paula Imerman, Dr. Grace Wilkinson, Dwayne Schrunk, and Amy Curtis.