Chen Qin, Christiansen Guntram, Deng Li, Kurmayer Rainer
College of Natural Resources and Environment, Northwest A & F University, Taicheng Road 3, 712100, Yangling, Shaanxi Province, P. R. China.
Research Institute for Limnology, University of Innsbruck, Mondseestrasse 9, 5310, Mondsee, Austria.
BMC Microbiol. 2016 Feb 25;16:23. doi: 10.1186/s12866-016-0639-1.
Bloom-forming cyanobacteria cause toxic algae outbreaks in lakes and reservoirs. We aimed to explore and quantify mutation events occurring within the large mcy gene cluster (55 kbp) encoding microcystin (MC) biosynthesis that inactivate MC net production. For this purpose we developed a workflow to detect mutations in situ occurring anywhere within the large mcy gene cluster as amplified from one single filament of the red-pigmented cyanobacterium Planktothrix rubescens. From five lakes of the Alps eight hundred Planktothrix filaments were isolated and each individual filament was analyzed for mutations affecting the mcy genes.
Mutations inactivating MC synthesis were either through an insertion element ISPlr1 or the partial deletion of mcy genes. Neutral mutations not affecting MC biosynthesis occurred within two intergenic spacer regions, either through the insertion of a Holliday-junction resolvase RusA or ISPlr1. Altogether, the insertions affected a few mcy genes only and their location was correlated with regions similar to repetitive extragenic palindromic DNA sequences (REPs). Taking all of the filaments together, the mutations leading to the inactivation of MC synthesis were more rare (0.5-6.9%), when compared with the neutral mutations (7.5-20.6%). On a spatial-temporal scale the ratio of MC synthesis-inactivating vs. neutral mutations was variable, e.g., the filament abundance carrying partial deletion of mcyD (5.2-19.4%) and/or mcyHA (0-7.3%) exceeded the abundance of neutral mutations.
It is concluded that insertion events occurring within the Planktothrix mcy gene cluster are predictable due to their correlation with REPs. The frequency of occurrence of the REPs within the mcy gene cluster of Planktothrix relates to the rather common mutation of mcy genes in Planktothrix. Spatial-temporal variable conditions may favor the emergence of partial mcy deletion mutants in Planktothrix, in particular a higher proportion of genotypes resulting in inactivation of MC synthesis might be caused by increased ISPlr1 activity.
形成水华的蓝藻会在湖泊和水库中引发有毒藻类爆发。我们旨在探索和量化在编码微囊藻毒素（MC）生物合成的大型mcy基因簇（55千碱基对）内发生的突变事件，这些突变会使MC的净产量失活。为此，我们开发了一种工作流程，用于检测从红色色素蓝藻纤细席藻的单根丝状体扩增出的大型mcy基因簇内任何位置发生的原位突变。从阿尔卑斯山的五个湖泊中分离出800根纤细席藻丝状体，并对每根丝状体进行分析，以检测影响mcy基因的突变。
使MC合成失活的突变要么是通过插入元件ISPlr1，要么是mcy基因的部分缺失。不影响MC生物合成的中性突变发生在两个基因间隔区，要么是通过插入霍利迪连接解离酶RusA，要么是ISPlr1。总体而言，插入仅影响少数几个mcy基因，其位置与类似于重复基因外回文DNA序列（REPs）的区域相关。将所有丝状体放在一起，与中性突变（7.5 - 20.6%）相比，导致MC合成失活的突变更为罕见（0.5 - 6.9%）。在时空尺度上，MC合成失活突变与中性突变的比例是可变的，例如，携带mcyD部分缺失（5.2 - 19.4%）和/或mcyHA部分缺失（0 - 7.3%）的丝状体丰度超过了中性突变的丰度。
得出的结论是，由于纤细席藻mcy基因簇内的插入事件与REPs相关，因此是可预测的。纤细席藻mcy基因簇内REPs的出现频率与纤细席藻中mcy基因相当常见的突变有关。时空可变条件可能有利于纤细席藻中mcy部分缺失突变体的出现，特别是更高比例导致MC合成失活的基因型可能是由ISPlr1活性增加引起的。
