Transcriptomic study of E. coli 042 with a variant of aggR gene with a FRT sequence inserted in its 3´UTR by RNA-seq

A common genomic feature of most EAEC strains is the presence of a virulence plasmid termed pAA. Plasmid-encoded virulence determinants are, among others, a transcriptional activator termed AggR, a member of the AraC-XylS family of transcription factors. We have previously determined the direct correlation between (p)ppGpp, expression of AggR and biofilm development in strain EAEC 042 (https://doi.org/10.3389/fmicb.2018.00717). In this work we characterize a novel variant of the aggR gene. We modified its 3´UTR by insertion of a FRT sequence, which have generated a series of different phenotypes. We used RNA-seq to compare the transcriptome of the wt strain and its aggR3UTRFRT variant grown at 37ºC in LB medium. E. coli 042 and derivative aggR3UTRFRT mRNA profiles were generated by Illumina RNA-seq. RNA extraction, DNase treatment, evaluation of RNA quality and cDNA libraries for Illumina sequencing were performed by Vertis Biotechnologie AG, Freising-Weihenstephan, Germany. Bacterial cells were grown until O.D.600 of 2.0 in LB medium at 37ºC with 200rpm of agitation. Three replicates were grown from each strain, 1 ml of each replicate was collected and bacterial pellets were maintained at -80ºC. Bacterial pellets were sent to Vertis Biotechnologie AG, Freising-Weihenstephan, Germany in dry ice. E. coli 042 wt was used as genome reference (NC_017626 for the chromosome and NC_017627 for the pAA plasmid) for analysis.

大多数肠聚集性大肠杆菌（Enteroaggregative Escherichia coli, EAEC）菌株的共同基因组特征是携带一种名为pAA的毒性质粒。质粒编码的毒力决定簇包括隶属于AraC-XylS家族转录因子的转录激活因子AggR。我们此前已在EAEC 042菌株中明确了(p)ppGpp、AggR表达与生物膜形成之间的直接关联（https://doi.org/10.3389/fmicb.2018.00717）。本研究对aggR基因的一种新型变体进行了表征。我们通过插入FRT序列修饰了其3'非翻译区（3´UTR），由此产生了一系列不同的表型。我们采用RNA测序（RNA-seq）技术，对比了野生型菌株与其aggR3UTRFRT变体在37℃ LB培养基中生长时的转录组。大肠杆菌042及其衍生的aggR3UTRFRT菌株的mRNA表达谱通过Illumina RNA-seq生成。RNA提取、DNase处理、RNA质量评估以及用于Illumina测序的cDNA文库构建工作均由德国弗赖辛-魏恩施泰滕的Vertis Biotechnologie AG完成。细菌在37℃、200rpm振荡条件下于LB培养基中培养至光密度O.D.600为2.0。每个菌株设置三个生物学重复，每个重复收集1mL菌液，菌体沉淀保存于-80℃。随后将菌体沉淀置于干冰中寄送至德国弗赖辛-魏恩施泰滕的Vertis Biotechnologie AG。本分析以大肠杆菌042野生型菌株作为基因组参考（染色体序列登录号为NC_017626，pAA质粒序列登录号为NC_017627）。