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In Cyanobacteria International Journal of Environmental Research and Public Health Article Molecular Probes to Evaluate the Synthesis and Production Potential of an Odorous Compound (2-methylisoborneol) in Cyanobacteria Keonhee Kim 1, Youngdae Yoon 1,2, Hyukjin Cho 3 and Soon-Jin Hwang 1,2,* 1 Human and Eco-Care Center, Department of Environmental Health Science, Konkuk University, Seoul 05029, Korea; [email protected] (K.K.); [email protected] (Y.Y.) 2 Department of Environmental Health Science, Konkuk University, Seoul 05029, Korea 3 Hangang River Regional Division, Department of Water Resources Management, K-Water, Gwacheon 13841, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-2-450-3748 Received: 30 January 2020; Accepted: 14 March 2020; Published: 16 March 2020 Abstract: The volatile metabolite, 2-Methylisoborneol (2-MIB) produced by cyanobacterial species, causes odor and taste problems in freshwater systems. However, simple identification of cyanobacteria that produce such off-flavors may be insufficient to establish the causal agent of off-flavor-related problems as the production-related genes are often strain-specific. Here, we designed a set of primers for detecting and quantifying 2-MIB-synthesizing cyanobacteria based on mibC gene sequences (encoding 2-MIB synthesis-catalyzing monoterpene cyclase) from various Oscillatoriales and Synechococcales cyanobacterial strains deposited in GenBank. Cyanobacterial cells and environmental DNA and RNA were collected from both the water column and sediment of a eutrophic stream (the Gong-ji Stream, Chuncheon, South Korea), which has a high 2-MIB concentration. Primer sets mibC196 and mibC300 showed universality to mibC in the Synechococcales and Oscillatoriales strains; the mibC132 primer showed high specificity for Pseudanabaena and Planktothricoides mibC. Our mibC primers showed excellent amplification efficiency (100–102%) and high correlation among related variables (2-MIB concentration with water RNA r = 689, p < 0.01; sediment DNA r = 0.794, p < 0.01; and water DNA r = 0.644, p < 0.05; cyanobacteria cell density with water RNA and DNA r = 0.995, p < 0.01). These primers offer an efficient tool for identifying cyanobacterial strains possessing mibC genes (and thus 2-MIB-producing potential) and for evaluating mibC gene expression as an early warning of massive cyanobacterial occurrence. Keywords: 2-Methylisoborneol (2-MIB); cyanobacteria; molecular probes; mibC; real-time PCR 1. Introduction Naturally occurring 2-methylisoborneol (2-MIB) and geosmin produced by cyanobacteria and actinomyces are regarded as a worldwide problem in freshwater systems because of their muddy or earthy odorous properties [1,2]. Contamination of drinking water resources is of particular concern, motivating intensive studies to identify new methods for efficiently detecting and removing these compounds [1,3,4]. Cyanobacteria are now recognized as the major producer of the unpleasant odorous 2-MIB; thus, the odor is more severe during cyanobacterial blooms. Prior studies indicated that blooms of cyanobacteria coincided with the occurrence of odor substances in various freshwater systems [5,6], including Huajiang Lake in Mongol, where the maximum concentration of 2-MIB reached 102 ng/L during cyanobacterial blooms [7]. The major cyanobacterial genus in the North Han River in Korea has shifted from Anabaena to Pseudanabaena since 2013, accompanied by an increase in the occurrence and Int. J. Environ. Res. Public Health 2020, 17, 1933; doi:10.3390/ijerph17061933 www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2020, 17, 1933 2 of 13 concentration of 2-MIB with the maximum concentration of 74.5 ng/L recorded in 2014 [8,9]. However, a direct causal relationship between the blooms of cyanobacteria as a whole and the generation of odor substances is unclear because the cyanobacterial community in freshwater systems is composed of a wide variety of species related to odor production, making it difficult to specify which species are responsible for the production of the odor-causing compounds [10,11]. There has been increasing research in the development of methods related to 2-MIB detection in recent decades as this odorous compound has become increasingly problematic for drinking water resources [12]. Various research groups have proposed diverse approaches for detecting odorous compounds. In particular, because even rarely occurring species of cyanobacteria in certain freshwater systems can produce odorous substances, it is necessary to apply highly sensitive and specific methods. Molecular biological approaches such as polymerase chain reaction (PCR), are very relevant in this regard [13,14]. The biochemical mechanisms and genes involved in 2-MIB synthesis in various cyanobacteria genera, including Pseudanabaena, Planktothricoides, Lyngbya, and Oscillatoria (Limnothrix), have been revealed; therefore, cyanobacterial strains capable of synthesizing 2-MIB can now be detected by PCR analysis using sequence-specific primers for the mibC gene encoding a monoterpene cyclase, which is the main enzyme involved in 2-MIB synthesis [12,15–18]. Using a PCR approach, the odor compound-producing potential in an Australian eutrophic reservoir was found to be positively correlated with the concentrations of geosmin compounds but not to the cell density of cyanobacteria known to produce the odor compounds [19]. To facilitate more selective and specific detection of cyanobacteria possessing the mibC gene, in this study, we constructed diverse sets of primers targeting mibC among species of Synechococcales and Oscillatoriales and investigated the 2-MIB production capability in 14 different cyanobacterial strains using these primer sets. To improve the specificity and efficiency of the primers, various annealing temperatures were tested in PCR amplification, and detectability of 2-MIB was assessed through application of the target gene to field samples. 2. Materials and Methods 2.1. Preparation of Cyanobacterial Strains and Field Samples The cyanobacterial strains used in this study were Pseudanabaena galeata (NIES-512), Anabaena smithii (NIES-824), and Anabaena cylindrica (NIES-19) obtained from the National Institute of Environment Science (NIES, Japan), and Anabaena variabilis (AG10064) obtained from the Korea Collection for Type Cultures (KTCC). The other strains, including Microcystis aeruginosa, Dolichospermum circinale straight type (ST), D. circinale coiled type (CT), Planktothricoides raciborskii strain NO, P. raciborskii strain PO, Pseudanabaena mucicola, Oscillatoria redekei (Limnothrix redekei), Lyngbya murrayi, Phormidium ambiguum, and Leptolyngbya boryana, were isolated from the North Han River and identified based on DNA sequence in previous studies [8,20,21]. All cyanobacterial strains were inoculated in BG-11 medium [22] and cultured in a shaking incubator (VS-3125Qi, Vison, Korea) at 25 ◦C and a light intensity of 60 µmolE m 2 s 1 (light:dark = 14:10 h). The cyanobacterial strains were grown for over three months · − · − and the medium was replaced every month. During medium changes, the dead cells that settled at the bottom of the flask and paled into yellow-green color were removed along with the medium. Field samples were obtained from a site with extensive production of 2-MIB by cyanobacteria that coincided with a cyanobacterial bloom between 2015 and February 2016 [8]. The cyanobacterial community in both the water column and sediment was collected downstream of the Gong-ji Stream (Supplementary Figure S1) (N 37◦52033.4”, E 127◦42023.1”) using a Vandorn sampler (Horizontal water sampler, iStech, Korea) and grab sampler (Peterson grab, QT Technology, Korea), respectively, monthly from February 2015 to February 2016. The sampling site constitutes the region where the Gong-ji Stream merges with the Uiam reservoir, a large reservoir located in the mainstream of the North Han River. The surface water sample and the sediment sample were transferred to a 1 L clear PET bottle and 100 mL dark bottle, respectively. The field samples were stored at 4 ◦C in the dark before Int. J. Environ. Res. Public Health 2020, 17, 1933 3 of 13 pretreatment. Sample pretreatment was performed within 12 h. The water sample was filtered using a polycarbonate filter paper (ø25 mm, pore: 1 µm, Whatman, USA) and placed in a 2-mL microtube before DNA extraction. The sediment sample (0.8 g) was transferred to a silica bead tube. Both the polycarbonate filter-filtered water sample and the sediment sample within the silica bead tube were stored at 80 C until analysis. − ◦ 2.2. Genomic DNA and RNA Extraction For genomic DNA extraction, cyanobacterial strains (P. galeata, P. mucicola, O. redekei [L. redekei], and P. raciborskii NO) attached to the bottom of flasks were collected using a scraper, whereas the other strains (M. aeruginosa, D. circinale ST, D. circinale CT, A. variabilis, A. smithii, A. cylindrica, L. murrayi, P. ambiguum, L. boryana, and P. raciborskii strain PO) were harvested by filtration of 10 mL of culture using a polycarbonate filter (ø25 mm, pore: 1 µm, Whatman, USA). The genomic DNA of the algal strain was extracted using a DNA extraction kit for soil (Macherey-Nagal, Germany) according to the manufacturer’s instructions, after cell lysis via the physical bead beating method [23]. Both water column and sediment samples were extracted DNA using the same method as for algal strain DNA extraction. The environmental RNA collected from the water column of the Gong-ji Stream was extracted with Trizol using an RNA
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