Molecular Basis and Phylogenetic Implications of Deoxycylindrospermopsin Biosynthesis in the Cyanobacterium Raphidiopsis curvata

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>New insights into the distribution and biochemistry of the cyanotoxin cylindrospermopsin (CYN) have been provided by the recent determination of its biosynthesis gene cluster (<jats:italic>cyr</jats:italic>) in several cyanobacterial species.<jats:named-content content-type="genus-species">Raphidiopsis curvata</jats:named-content>CHAB1150 isolated from China was analyzed for CYN analogues. Only 7-deoxy-CYN was detected in the cell extracts. The<jats:italic>cyr</jats:italic>gene cluster of<jats:named-content content-type="genus-species">R. curvata</jats:named-content>CHAB1150 was sequenced, and the<jats:italic>cyr</jats:italic>genes of this strain were found to have extremely high similarities (96% to 100%) to those from other nostocalean species. These species include<jats:named-content content-type="genus-species">Cylindrospermopsis raciborskii</jats:named-content>AWT205,<jats:named-content content-type="genus-species">Aphanizomenon</jats:named-content>sp. strain 10E6, and<jats:named-content content-type="genus-species">Aphanizomenon ovalisporum</jats:named-content>ILC-146. Insertion mutation was identified within the<jats:italic>cyrI</jats:italic>gene, and transcripts of<jats:italic>cyrI</jats:italic>and another functional gene<jats:italic>cyrJ</jats:italic>were detected in<jats:named-content content-type="genus-species">R. curvata</jats:named-content>CHAB1150. General congruence between the phylogenetic trees based on both<jats:italic>cyr</jats:italic>and 16S<jats:italic>rrn</jats:italic>was displayed. Neutral evolution was found on the whole sequences of the<jats:italic>cyr</jats:italic>genes, and 0 to 89 negative selected codons were detected in each gene. Therefore, the function of CyrI is to catalyze the oxygenation of 7-deoxy-CYN in CYN biosynthesis. The transcripts of the mutated<jats:italic>cyrI</jats:italic>gene may result from polycistronic transcription. The high conservation of the<jats:italic>cyr</jats:italic>genes may be ascribed to purifying selection and horizontal gene transfer.</jats:p>

Sporadic Distribution and Distinctive Variations of Cylindrospermopsin Genes in Cyanobacterial Strains and Environmental Samples from Chinese Freshwater Bodies

“…First, the cyr genes and flanking sequences were amplified and sequenced according to the PCR methods described earlier (38). Second, genome sequencing was performed using a Hiseq 2000 (Illumina, USA) according to the manufacturer's instructions.…”

“…Cyanobacterial cells from 2 ml of culture at the exponential phase were harvested by centrifugation. RNA extraction, DNase digestion and cDNA synthesis were performed as described previously (38). The DNase-digested RNA extracts and cDNA were used as the templates for transcription detection.…”

“…Maximum-likelihood (ML) algorithm was used to carry out phylogenetic analysis by PHYML v3.0 (56) and PAUP v4.0b10 with 1,000 bootstrap replicates. Bayesian phylogenetic inference was performed using MrBayes v3.1.2 (57), and the parameters were set as described earlier (38). Neighbor-joining (NJ) trees were constructed by MEGA v4 (58) using Kimura two-parameter model with 1,000 bootstrap replicates.…”

“…The cyr gene cluster that encodes amidinotransferase, peptide synthetase (PS), polyketide synthase (PKS), and tailoring enzymes involved in CYN production has been described in C. raciborskii (37), R. curvata (38), Aphanizomenon sp. (39), and Oscillatoria sp.…”

“…CyrI has been proven to catalyze hydroxylation at the C-7 of 7-deoxy-CYN (42), and CyrK has been proposed to be a potential transporter. Although cyr genes are highly conserved, the rearrangements of the cyr gene cluster and the insertion mutation of the cyrI gene have been reported (38). The cyrN and cyrO genes are found only in the end of the cyr gene cluster of C. raciborskii and are suggested to be excluded from the core set of cyr genes (30,38).…”