Multiple Alternate Transcripts Direct the Biosynthesis of Microcystin, a Cyanobacterial

The mcyABCDEFGHIJ gene cluster of Microcystis aeruginosa encodes the mixed polyketide synthase/nonribosomal peptide synthetase (microcystin synthetase) which is responsible for biosynthesis of the potent liver toxin microcystin. The sequence and orientation of the mcy genes have previously been reported, but no transcriptional analysis had been performed prior to this study. The mcyABCDEFGHIJ genes are transcribed as two polycistronic operons, mcyABC and mcyDEFGHIJ, from a central bidirectional promoter between mcyA and mcyD. Two transcription start sites were detected for both mcyA and mcyD when cells were exposed to light intensities of 68 and 16 μmol of photons m(−2) s(−1). The start sites, located 206 and 254 bp upstream of the translational start for mcyD under high and low light conditions, respectively, indicate long untranslated leader regions. Putative transcription start sites were also identified for mcyE, mcyF, mcyG, mcyH, mcyI, and mcyJ but not for mcyB and mcyC. A combination of reverse transcription-PCR and rapid amplification of cDNA ends was employed throughout this work, which may have been one of the first transcriptional analyses of a large nonribosomal polyketide gene cluster.

铜绿微囊藻（Microcystis aeruginosa）的mcyABCDEFGHIJ基因簇，编码兼具聚酮合酶与非核糖体肽合成酶活性的复合酶（polyketide synthase/nonribosomal peptide synthetase，又称微囊藻毒素合成酶microcystin synthetase），该酶负责强效肝毒素微囊藻毒素的生物合成。此前已有研究报道了mcy基因的序列与排布方向，但本研究开展前尚未针对该基因簇进行转录水平分析。mcyABCDEFGHIJ基因以两个多顺反子操纵子（polycistronic operon）的形式进行转录，即mcyABC与mcyDEFGHIJ，转录起始于mcyA与mcyD之间的核心双向启动子（bidirectional promoter）。当细胞暴露于68与16 μmol光子·m⁻²·s⁻¹的光照强度下时，研究人员在mcyA与mcyD中均检测到两个转录起始位点（transcription start site）。这两个转录起始位点分别位于强光与弱光条件下mcyD翻译起始位点上游206 bp与254 bp的位置，表明该区域存在较长的非翻译前导区域（untranslated leader region）。研究还在mcyE、mcyF、mcyG、mcyH、mcyI与mcyJ中鉴定到了推定的转录起始位点，但未在mcyB与mcyC中发现此类位点。本研究全程采用了逆转录PCR（reverse transcription-PCR，简称RT-PCR）与cDNA末端快速扩增技术（rapid amplification of cDNA ends，简称RACE）相结合的实验方案，该研究可能是首批针对大型非核糖体聚酮基因簇开展转录水平分析的工作之一。