Role of RfaH in Yersinia enterocolitica O:3. Yersinia enterocolitica

To assess the effects of rfaH mutation on gene expression, RNA sequencing was carried out using the RNA extracted from rfaH mutant and wild type bacteria cultivated at both 22˚C and 37˚C. Additionally, to differentiate between genes affected directly by the rfaH mutation and those affected by the O-antigen negative phenotype, the Y. enterocolitica O:3 strain YeO3-R1 missing the O-antigen was included in the RNA-sequencing study. At RT-grown bacteria altogether 77 (45 up- and 32 down-regulated) differentially expressed genes were identified. At 37ºC, on the other hand, 44 genes were differentially expressed; 14 genes up- and 30 genes down-regulated. The RNA-sequencing data of YeO3-R1 was available only for bacteria grown at 22˚C and the comparison revealed that 22 of the 102 genes were similarly differentially expressed both in rfaH and YeO3-R1 mutants. To detect genes regulated directly or indirectly by Hfq, deep RNA-seq was performed with RNA isolated from Y. enterocolitica wild-type strain 6471/76 and the YeO3-hfq::Km strain. Total RNA was isolated from two biological replicas of bacteria grown in LB to logarithmic phase (OD600 = 0.6) at two different temperatures, RT and 37°C. knocking out of Hfq affected the transcription of 346 genes at RT and 541 genes at 37°C, i.e., ca. 8 and 12.5 % of the Y. enterocolitica genes, respectively. At RT, of the 346 genes 216 genes were down- and 132 up-regulated, and at 37°C, of the 541 genes, 96 were down- and 445 up-regulated. A total of 95 genes were differentially expressed both at RT and 37°C; 27 showed down- and 59 up-regulated. For 9 genes opposite changes took place depending on the growth temperature. Overall design: Bacterial RNA profiles of wild type, rfaH- and hfq- strains were generated using deep sequencing, in duplicates, under two growth temperatures (22°C and 37°C). The RNA-sequencing data of YeO3-R1 was generated only from bacteria grown at 22˚C.

为评估rfaH突变对细菌基因表达的影响，本研究采用分别在22℃与37℃下培养的rfaH突变株与野生型细菌中提取的总RNA，开展了RNA测序（RNA sequencing）实验。为区分受rfaH突变直接调控的基因与受O-抗原（O-antigen）阴性表型间接影响的基因，本研究将缺失O-抗原的小肠结肠炎耶尔森氏菌（Y. enterocolitica）O:3菌株YeO3-R1纳入本次RNA测序研究。于室温（Room Temperature，RT）培养的细菌样本中，共鉴定出77个差异表达基因（differentially expressed genes），其中45个基因表达上调、32个基因表达下调；而在37℃培养的样本中，共鉴定出44个差异表达基因，其中14个上调、30个下调。YeO3-R1的RNA测序数据仅来自22℃培养的细菌，对比分析显示，102个基因中共有22个在rfaH突变株与YeO3-R1突变株中呈现相似的差异表达趋势。为检测受Hfq直接或间接调控的基因，本研究采用从小肠结肠炎耶尔森氏菌野生型菌株6471/76以及YeO3-hfq::Km突变株中分离的总RNA，开展了深度RNA测序（deep RNA-seq）实验。实验收集了两种培养温度（RT与37℃）下、于LB培养基中培养至对数生长期（OD600=0.6）的细菌样本，每份样本设置两份生物学重复，从中提取总RNA。Hfq基因敲除分别在RT与37℃下影响了346个和541个基因的转录，分别约占小肠结肠炎耶尔森氏菌总基因数的8%与12.5%。其中，RT条件下的346个差异表达基因中，216个下调、132个上调；37℃条件下的541个差异表达基因中，96个下调、445个上调。共有95个基因在RT与37℃两种培养条件下均呈现差异表达，其中27个下调、59个上调；另有9个基因的表达变化趋势随培养温度不同而发生反转。整体实验设计：分别在22℃与37℃两种培养温度下，通过深度测序（设置两次生物学重复）获取野生型、rfaH突变株与hfq突变株的细菌RNA表达谱；YeO3-R1的RNA测序数据仅来源于22℃培养的细菌。