Lehrstuhl für Mikrobielle Ökologie, Wissenschaftszentrum Weihenstephan, Technische Universität München, Weihenstephaner Berg 3, 85354, Freising, Germany.
ZIEL - Institute for Food & Health, Technische Universität München, Freising, Germany.
Sci Rep. 2018 Dec 14;8(1):17875. doi: 10.1038/s41598-018-35756-y.
Only a few overlapping gene pairs are known in the best-analyzed bacterial model organism Escherichia coli. Automatic annotation programs usually annotate only one out of six reading frames at a locus, allowing only small overlaps between protein-coding sequences. However, both RNAseq and RIBOseq show signals corresponding to non-trivially overlapping reading frames in antisense to annotated genes, which may constitute protein-coding genes. The transcription and translation of the novel 264 nt gene asa, which overlaps in antisense to a putative TEGT (Testis-Enhanced Gene Transfer) transporter gene is detected in pathogenic E. coli, but not in two apathogenic E. coli strains. The gene in E. coli O157:H7 (EHEC) was further analyzed. An overexpression phenotype was identified in two stress conditions, i.e. excess in salt or arginine. For this, EHEC overexpressing asa was grown competitively against EHEC with a translationally arrested asa mutant gene. RT-qPCR revealed conditional expression dependent on growth phase, sodium chloride, and arginine. Two potential promoters were computationally identified and experimentally verified by reporter gene expression and determination of the transcription start site. The protein Asa was verified by Western blot. Close homologues of asa have not been found in protein databases, but bioinformatic analyses showed that it may be membrane associated, having a largely disordered structure.
在研究得最好的细菌模式生物大肠杆菌中，只有少数重叠基因对是已知的。自动注释程序通常在一个基因座上注释六个阅读框中的一个，只允许蛋白质编码序列之间有很小的重叠。然而，RNAseq 和 RIBOseq 都在注释基因的反义位置检测到与非平凡重叠阅读框相对应的信号，这些信号可能构成蛋白质编码基因。新型 264nt 基因 asa 在致病性大肠杆菌中转录和翻译，与假定的 TEGT（睾丸增强基因转移）转运蛋白基因反义重叠，但在两种非致病性大肠杆菌菌株中没有检测到。对大肠杆菌 O157:H7（EHEC）中的基因进行了进一步分析。在两种应激条件下，即盐或精氨酸过量时，鉴定出了过表达表型。为此，EHEC 过表达 asa 与翻译受阻的 asa 突变基因的 EHEC 进行竞争生长。RT-qPCR 显示依赖于生长阶段、氯化钠和精氨酸的条件表达。通过报告基因表达和转录起始位点的确定，计算鉴定了两个潜在的启动子并进行了实验验证。通过 Western blot 验证了 Asa 蛋白。在蛋白质数据库中没有发现 asa 的密切同源物，但生物信息学分析表明它可能与膜相关，具有很大的无序结构。
