Hughes Rebecca-Ayme, Zhang Yunlong, Zhang Ran, Williams Philip G, Lindsey Jonathan S, Miller Eric S
Department of Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina, USA.
Department of Chemistry, North Carolina State University, Raleigh, North Carolina, USA.
Appl Environ Microbiol. 2017 Sep 15;83(19). doi: 10.1128/AEM.01068-17. Print 2017 Oct 1.
The cyanobacterial culture HT-58-2 was originally described as a strain of with the ability to produce tolyporphins, which comprise a family of distinct tetrapyrrole macrocycles with reported efflux pump inhibition properties. Upon reviving the culture from what was thought to be a nonextant collection, studies of culture conditions, strain characterization, phylogeny, and genomics have been undertaken. Here, HT-58-2 was shown by 16S rRNA analysis to closely align with strains and not with isolates. Light, fluorescence, and scanning electron microscopy revealed cyanobacterium filaments that are decorated with attached bacteria and associated with free bacteria. Metagenomic surveys of HT-58-2 cultures revealed a diversity of bacteria dominated by , 97% of which are species. A dimethyl sulfoxide washing procedure was found to yield enriched cyanobacterial DNA (presumably by removing community bacteria) and sequence data sufficient for genome assembly. The finished, closed HT-58-2Cyano genome consists of 7.85 Mbp (42.6% G+C) and contains 6,581 genes. All genes for biosynthesis of tetrapyrroles (e.g., heme, chlorophyll , and phycocyanobilin) and almost all for cobalamin were identified dispersed throughout the chromosome. Among the 6,177 protein-encoding genes, coding sequences (CDSs) for all but two of the eight enzymes for conversion of glutamic acid to protoporphyrinogen IX also were found within one major gene cluster. The cluster also includes 10 putative genes (and one hypothetical gene) encoding proteins with domains for a glycosyltransferase, two cytochrome P450 enzymes, and a flavin adenine dinucleotide (FAD)-binding protein. The composition of the gene cluster suggests a possible role in tolyporphin biosynthesis. A worldwide search more than 25 years ago for cyanobacterial natural products with anticancer activity identified a culture (HT-58-2) from Micronesia that produces tolyporphins. Tolyporphins are tetrapyrroles, like chlorophylls, but have several profound structural differences that reside outside the bounds of known biosynthetic pathways. To begin probing the biosynthetic origin and biological function of tolyporphins, our research has focused on studying the cyanobacterial strain, about which almost nothing has been previously reported. We find that the HT-58-2 culture is composed of the cyanobacterium and a community of associated bacteria, complicating the question of which organisms make tolyporphins. Elucidation of the cyanobacterial genome revealed an intriguing gene cluster that contains tetrapyrrole biosynthesis genes and a collection of unknown genes, suggesting that the cluster may be responsible for tolyporphin production. Knowledge of the genome and the gene cluster sharply focuses research to identify related cyanobacterial producers of tolyporphins and delineate the tolyporphin biosynthetic pathway.
蓝藻培养物HT - 58 - 2最初被描述为一种能够产生托利卟啉的菌株，托利卟啉是一类独特的四吡咯大环化合物，据报道具有外排泵抑制特性。从被认为已不存在的保藏物中复苏该培养物后，我们开展了培养条件、菌株特征、系统发育和基因组学研究。在这里，通过16S rRNA分析表明HT - 58 - 2与[具体名称1]菌株密切相关，而与[具体名称2]分离株不相关。光学、荧光和扫描电子显微镜显示，蓝藻丝体上附着有细菌，并与游离细菌相关联。对HT - 58 - 2培养物的宏基因组调查揭示了以[具体细菌名称]为主的多种细菌，其中97%是[具体细菌名称]物种。发现一种二甲基亚砜洗涤程序能够产生富集的蓝藻DNA（可能是通过去除群落细菌）以及足以进行基因组组装的序列数据。完成的、封闭的HT - 58 - 2蓝藻基因组由785万个碱基对组成（G + C含量为42.6%），包含6581个基因。所有四吡咯生物合成基因（如血红素、叶绿素a和藻蓝胆素）以及几乎所有钴胺素生物合成基因都被鉴定出分散在整个染色体上。在6177个蛋白质编码基因中，除了将谷氨酸转化为原卟啉原IX的八种酶中的两种之外，其余所有酶的编码序列（CDS）也都在一个主要基因簇中被发现。该基因簇还包括10个推定基因（和一个假设基因），这些基因编码的蛋白质具有糖基转移酶、两种细胞色素P450酶和一个黄素腺嘌呤二核苷酸（FAD）结合蛋白的结构域。该基因簇的组成表明其在托利卟啉生物合成中可能发挥作用。25多年前在全球范围内搜索具有抗癌活性的蓝藻天然产物时，从密克罗尼西亚分离出一种产生托利卟啉的培养物（HT - 58 - 2）。托利卟啉与叶绿素一样都是四吡咯，但具有一些已知生物合成途径范围之外的深刻结构差异。为了开始探究托利卟啉的生物合成起源和生物学功能，我们的研究集中在对该蓝藻菌株的研究上，此前关于该菌株几乎没有任何报道。我们发现HT - 58 - 2培养物由蓝藻和一群相关细菌组成，这使得确定是哪种生物体产生托利卟啉的问题变得复杂。对蓝藻基因组的解析揭示了一个有趣的基因簇，其中包含四吡咯生物合成基因和一系列未知基因，这表明该基因簇可能负责托利卟啉的产生。基因组和基因簇的知识使研究能够更精准地聚焦于识别托利卟啉的相关蓝藻生产者，并描绘托利卟啉的生物合成途径。
