High Throughput Method for Analysis of Repeat Number for 28 Phase Variable Loci of Campylobacter jejuni Strain NCTC11168.
High Throughput Method for Analysis of Repeat Number for 28 Phase Variable Loci of Campylobacter jejuni Strain NCTC11168.
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DOI：
10.1371/journal.pone.0159634
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Bayliss CD
中科院分区：
文献类型：
--
作者：
Lango-Scholey L;Aidley J;Woodacre A;Jones MA;Bayliss CD
简单序列重复序列中的突变是包括空肠弯曲菌在内的几种细菌中相位变化的主要机制。位于阅读框内的片段重复数的变化可以在基因表达中在ON和OFF状态之间产生高频率的可逆开关。C. jejuni菌株NCTC 11168含有29个具有7个或更多重复的polyG/polyC片段的位点。该方案概述了一种方法--28个基因座-CJ 11168 PV分析测定--用于快速确定大量C.空肠菌株NCTC 11168。该方法将一系列多重PCR测定与片段分析测定和片段长度、重复数和表达状态的自动提取相结合。这种高通量的多重测定具有检测群体内和群体之间随时间推移的相位变化状态的变化以及探索相位变化对适应不同选择压力的影响的实用性。应用该方法分析了90 ℃下28个polyG/polyC片段。空肠集落检测到，当肠段从G8延长到G11时，滑移产物增加2.5倍，但相似长度的肠段之间没有差异，表明侧翼序列不影响滑移率。将这一观察到的滑移与先前测量的C. jejuni表明DNA样品的PCR扩增将高估相位变化频率20-35倍。28-基因座-CJ 11168 PV分析测定的重要输出是无法通过其他方法确定的组合表达状态。该方法也适用于其它C.空肠菌株和各种细菌物种。
Mutations in simple sequence repeat tracts are a major mechanism of phase variation in several bacterial species including Campylobacter jejuni. Changes in repeat number of tracts located within the reading frame can produce a high frequency of reversible switches in gene expression between ON and OFF states. The genome of C. jejuni strain NCTC11168 contains 29 loci with polyG/polyC tracts of seven or more repeats. This protocol outlines a method—the 28-locus-CJ11168 PV-analysis assay—for rapidly determining ON/OFF states of 28 of these phase-variable loci in a large number of individual colonies from C. jejuni strain NCTC11168. The method combines a series of multiplex PCR assays with a fragment analysis assay and automated extraction of fragment length, repeat number and expression state. This high throughput, multiplex assay has utility for detecting shifts in phase variation states within and between populations over time and for exploring the effects of phase variation on adaptation to differing selective pressures. Application of this method to analysis of the 28 polyG/polyC tracts in 90 C. jejuni colonies detected a 2.5-fold increase in slippage products as tracts lengthened from G8 to G11 but no difference between tracts of similar length indicating that flanking sequence does not influence slippage rates. Comparison of this observed slippage to previously measured mutation rates for G8 and G11 tracts in C. jejuni indicates that PCR amplification of a DNA sample will over-estimate phase variation frequencies by 20-35-fold. An important output of the 28-locus-CJ11168 PV-analysis assay is combinatorial expression states that cannot be determined by other methods. This method can be adapted to analysis of phase variation in other C. jejuni strains and in a diverse range of bacterial species.