Transcriptional regulation of the cylindrospermopsin biosynthesis (cyr) gene cluster in Raphidiopsis raciborskii AWT205

Cyanobacterial harmful algal blooms (HABs), producing the hepatotoxin cylindrospermopsin (CYN), are among the most frequently reported globally. Although the gene cluster encoding CYN biosynthesis (cyr) has been sequenced, many aspects of CYN regulation are unresolved. This study investigates cyr transcriptional regulation in the cyanobacterium, Raphidiopsis raciborskii AWT205, through in silico analysis, reverse transcription quantitative PCR (RT-qPCR), and DNA-affinity capture assays (DACAs). While in silico analysis identified binding sites for environmentally responsive transcription factors throughout the cyr cluster, DACAs captured the binding of AbrB (a transcription factor that regulates the cyanobacterial response to nitrogen and iron availability) to the cyrD promoter. Surprisingly, the initiating cylindrospermopsin synthetase, CyrA, was also captured by the cyrD promoter probe. This is the first experimental evidence of CYN (and cyanotoxin) autoregulation. Our study is also the most extensive investigation of cyr transcription, concurrently targeting nine cyr genes across three growth stages. We found significant heterogeneity between transcription levels of each cyr gene, which also varied across different growth stages. Surprisingly, the ratio of cyrI (hydroxylase) to cyrJ (sulfotransferase) transcripts was inversely proportional to the ratio of deoxyCYN to CYN. Taken together, the results of this study suggest that transcription of the cyr gene cluster in R. raciborskii AWT205 is driven by multiple promoters and DNA-binding proteins, that can be responsive to changing environmental conditions. However, the production of different CYN variants did not correlate to transcription alone, with additional regulatory mechanisms proposed.