Genome-Wide Fitness Analyses of the Foodborne Pathogen Campylobacter jejuni in In Vitro and In Vivo Models
Genome-Wide Fitness Analyses of the Foodborne Pathogen Campylobacter jejuni in In Vitro and In Vivo Models
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食源性病原体空肠弯曲菌体外和体内模型的全基因组适应性分析
DOI：
10.1101/085720
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发表时间：
2016
期刊：
影响因子：
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通讯作者：
De Vries S
中科院分区：
文献类型：
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作者：
De Vries S
结果鉴定出适合度所需的基因。为了研究空肠梭菌生长和存活的遗传基础，对3株具有良好特征的空肠梭菌[M1cam 13、14、NCTC 11168（以下简称11168）10和81-176 15]采用Tn诱变法随机灭活基因。通过Tn插入位点测序（Tn-seq 16）对Tn突变文库进行了表征（表S1），为文库中每个Tn突变体的相对丰度提供了一个衡量标准。生长和生存所需的基因，以下称为“适应度”基因，不能耐受Tn插入，或者这些基因中的Tn突变在文库中严重不足。为了鉴定适合基因，在m1中分析了23,334个独特的染色体Tn插入，在11168中分析了15,008个，在81-176中分析了17,827个（表S1），就可能失活的基因数量而言，达到了接近饱和（图1a）。除了染色体上的Tn插入，在81-176个质粒pVir 17和pVir 18中分别有2009个和1919个独特的插入（表S1）。没有观察到明显的Tn插入偏差（图S1），每个Tn库主要产生唯一的Tn插入（图S2）。基因适合度评分（观察到的与预期序列读数之间的Log2倍变化）密度图遵循双峰分布，“左侧”群体代表体外生长和存活所需的基因（图1b）。M1cam共需要445个基因，81-176需要413个基因，11168需要499个基因（表S2）。有趣的是，菌株81-176的ppet质粒中的cpp13似乎有助于适应。空肠C. 81-176存在一种失去ppet的变体（D. Hendrixson, personal communication），这意味着cpp13不是必需的，但可能有助于健康，或者它可能是一种未表征的毒素-抗毒素系统的抗毒素。出乎意料的是，在M1cam的第352位和81-176的第1115位dna中发现了1323 bp的Tn插入。因此，dna没有通过严格的适应度基因标准。在罕见的二次位点突变20的情况下，或者由于merodiploids 21的存在，dna在基因的3 '端被破坏是可以容忍的。
ResultsIdentification of genes required for fitness. To assess the genetic basis of C. jejuni growth and survival, genes were randomly inactivated using Tn mutagenesis in three well-characterized C. jejuni strains [M1cam 13, 14, NCTC 11168 (hereafter referred to as 11168) 10 and 81–176 15]. Tn mutant libraries were characterised by Tn insertion site sequencing (Tn-seq 16)(Table S1), providing a measure for the relative abundance of each Tn mutant in the library. Genes that are required for growth and survival, hereafter referred to as “fitness” genes, cannot tolerate Tn insertions, or Tn mutants in these genes are severely underrepresented in the libraries. To identify fitness genes, 23,334 unique chromosomal Tn insertions were analysed in M1cam, 15,008 in 11168, and 17,827 in 81–176 (Table S1), reaching near-saturation in terms of the number of genes that could be inactivated (Fig. 1a). In addition to chromosomal Tn insertions, 2,009 and 1,919 unique insertions were in the 81–176 plasmids pVir 17 and pTet 18, respectively (Table S1). No apparent Tn insertion bias was observed (Fig. S1) and each Tn library predominately yielded unique Tn insertions (Fig. S2).Gene fitness score (Log2 fold-change between the observed vs expected sequence reads 19) density plots followed a bimodal distribution with the “left” population representing genes required for in vitro growth and survival (Fig. 1b). In total, 445 genes were required for fitness in M1cam, 413 genes in 81–176 and 499 in 11168 (Table S2). Interestingly, cpp13 in the pTet plasmid in strain 81–176 appeared to contribute to fitness. A variant of C. jejuni 81–176 that has lost pTet exists (D. Hendrixson, personal communication), which implies that cpp13 is not obligate essential but may contribute to fitness, or it could be antitoxin of an uncharacterised toxin-antitoxin system. Unexpectedly, Tn insertions were observed in dnaA (1,323 bp) at bp position 352 in M1cam and at position 1,115 in 81–176. Consequently, dnaA did not pass the stringent fitness gene criteria. Disruption of dnaA may be tolerated at the 3′ end of the gene, in the rare event of a secondary site mutation 20, or due to the existence of merodiploids 21.