The molecular genetics of cyanobacterial toxicity as a basis for monitoring water quality and public health risk
Introduction
The cyanobacteria or ‘blue-green algae’ as they are commonly termed, comprise a diverse group of oxygenic photosynthetic bacteria that possess the ability to synthesise chlorophyll-a and the phycobilin proteins, phycocyanin (PC) and phycoerythrin. Certain aquatic, bloom-forming species of cyanobacteria are also capable of producing biologically active secondary metabolites (cyanotoxins), which are highly toxic to many eukaryotic organisms. The release of cyanotoxins into recreational and drinking water supplies has obvious and far-reaching implications for public health and the environment [1]. Hence, considerable research efforts have been directed towards the detection, control and removal of cyanobacteria and their toxins from our waterways.
The cyanotoxins span four different classes according to their pathology: the neurotoxins, hepatotoxins, cytotoxins and irritant toxins (lipopolysaccharides). While all cyanotoxins can produce adverse symptoms in humans, the hepatotoxins (microcystin and nodularin) and neurotoxins (anatoxin, saxitoxin and cylindrospermopsin) are of greatest concern to public health.
Early diagnostic methods for toxic cyanobacteria were complicated by the fact that physiological traits and morphological characteristics (cell size and shape) do not correlate well with toxicity. In fact, toxin profiles vary widely across and within the five orders of cyanobacteria [2]. Before the development of analytical and molecular detection methods for these organisms, researchers relied primarily on animal bioassays to assess the toxicity of blooms [3]. However, low sensitivity, ethical issues and high associated costs have driven the search for alternative testing methods.
While sensitive physicochemical detection methods, such as high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) spectrometry have been established for most cyanotoxins [3, 4], they frequently necessitate the use of laborious sample preparation protocols, as well as expensive machinery and purified toxin standards that are often difficult to obtain.
Various immunological and biochemical detection methods have proven useful for detecting cyanotoxins in the laboratory and in environmental samples. For example, sensitive ELISA and colorimetric (protein phosphatase inhibition) assays are available for the detection of the hepatotoxins, microcystin and nodularin [5]. However, molecular detection methods, particularly those based on the polymerase chain reaction (PCR), are proving to be increasingly popular in this field. In addition to being simple, rapid and cost-effective, current molecular detection methods for toxic cyanobacteria are extremely sensitive, specific and amenable to high-throughput analysis.
This review encompasses the development and implementation of current molecular methods for monitoring toxic cyanobacteria in the environment. In addition to presenting early research efforts based on cyanobacterial phylogeny, we describe the discovery and exploitation of cyanotoxin biosynthesis genes as molecular diagnostic targets. Particular emphasis is given to technologies based on the microcystin (mcy) and nodularin (nda) gene clusters where most work has been focused.
Section snippets
The development of molecular tests
We now know that toxin production in cyanobacteria is dependent on the functional expression of specific sets of biosynthesis genes encoded within large operons (see below). However, before the discovery of these cyanotoxin gene clusters, a variety of alternative molecular tests were employed in an attempt to identify and classify toxic cyanobacteria.
Genetic polymorphisms within the 16S rRNA gene and internal transcribed spacer (ITS) were explored as a possible means of detecting and
Characterisation of cyanotoxin gene clusters
Microcystin and nodularin are synthesised non-ribosomally via the thiotemplate function of large multifunctional enzyme complexes containing both non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) modules [13]. The gene clusters encoding these biosynthetic enzymes, mcy (microcystin) and nda (nodularin), have recently been sequenced and partially characterised in several cyanobacterial species, including Microcystis, Anabaena, Planktothrix and Nodularia [13, 14, 15, 16] (
Conventional PCR tests
The initial discovery of the mcy (microcystin synthetase) gene cluster in M. aeruginosa was achieved via a degenerate PCR targeting the NRPS gene, mcyB [20, 21]. The oligonucleotide primers designed for the task, MTF2 and MTR, were based on the highly conserved amino acid acyladenylation domains within the NRPSs of other bacteria and fungi. Following the amplification, sequencing and alignment of mcyB from M. aeruginosa PCC7608 and HUB524, unique, non-conserved regions within the NRPS module
Quantitative real-time PCR
Various quantitative PCR tests have been adapted from the conventional PCR studies mentioned throughout this review (Table 2). At least four different genes from the mcy/nda gene clusters have been targeted for real-time PCR [31, 36, 37, 38, 39•], while at least three putative cylindrospermopsin biosynthesis genes have been investigated using this method [40]. These quantitative PCR assays are usually very sensitive, with reliable detection limits of only a few cells per reaction.
Quantitative
Microarray (DNA chip) technology
Oligonucleotide microarrays are proving to be increasingly popular diagnostic tools for analysing complex clinical and environmental samples. While only recently applied to the study of cyanobacterial diversity (Table 3), microarray technology is beginning to show great promise for the high-throughput analysis of bloom samples.
Initial microarry-based investigations for cyanobacteria focused on 16S rRNA polymorphisms. For example, Rudi et al. [41] designed a small cyanobacterial-specific DNA
Conclusions
The application of molecular biological, and DNA amplification technology in particular, for the detection of toxic cyanobacteria has provided significant advances in the area of water quality management. The ability to identify minor microbial constituents within complex natural populations, based on specific nucleic acid sequences, allows the definitive identification of potentially toxic species. The speed, economy and sensitivity of the methods described in this review make them ideal for
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
The authors are financially supported by the Australian Research Council, the Sydney Catchment Authority and Diagnostic Technology P/L.
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