Molecular Probes to Evaluate the Synthesis and Production Potential of an Odorous Compound (2-methylisoborneol) in Cyanobacteria

• Main Authors: Keonhee Kim, Youngdae Yoon, Hyukjin Cho, Soon-Jin Hwang
• Format: Article
• Language: English
• Published: MDPI AG 2020-03-01
• Series: International Journal of Environmental Research and Public Health
• Subjects: 2-methylisoborneol (2-mib); cyanobacteria; molecular probes; <i>mibc</i>; real-time pcr
• Online Access: https://www.mdpi.com/1660-4601/17/6/1933

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 <i>mibC</i> 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 <i>mibC</i> in the Synechococcales and Oscillatoriales strains; the mibC132 primer showed high specificity for <i>Pseudanabaena</i> and <i>Planktothricoides mibC</i>. Our mibC primers showed excellent amplification efficiency (100&#8722;102%) and high correlation among related variables (2-MIB concentration with water RNA r = 689, <i>p</i> &lt; 0.01; sediment DNA r = 0.794, <i>p</i> &lt; 0.01; and water DNA r = 0.644, <i>p</i> &lt; 0.05; cyanobacteria cell density with water RNA and DNA r = 0.995, <i>p</i> &lt; 0.01). These primers offer an efficient tool for identifying cyanobacterial strains possessing <i>mibC</i> genes (and thus 2-MIB-producing potential) and for evaluating <i>mibC</i> gene expression as an early warning of massive cyanobacterial occurrence.