小蛋白由于其在整个生命领域的主要生物过程中的重要功能而受到越来越多的关注。然而,它们的小尺寸和低序列保守性使它们难以识别。因此,肠细菌ryfA作为一种小的蛋白质编码基因逃避了近20年的鉴定,这并不奇怪。自2001年鉴定以来,ryfA被认为编码非编码RNA,并与大肠杆菌的生物膜形成和志贺氏菌的发病机理有关。尽管最近的核糖体分析研究表明ryfA被翻译,未检测到相应的蛋白质产物。在这项研究中,我们提供的证据表明,ryfA编码一个小的毒性内膜蛋白,TimP,其过度表达导致细胞质膜渗漏。TimP携带N端信号序列,表明其膜定位是Sec依赖性的。TimP的表达被小RNA(sRNA)TimR抑制,与timPmRNA碱基配对以抑制其翻译。与过表达相反,TimR缺失时TimP的内源性表达允许细胞生长,可能表明细菌膜中的毒性非依赖性功能。重要性下一代测序(NGS)已经实现了跨越生命所有领域的生物体的大量基因组的揭示。为了降低新基因组序列被注释时的复杂性,通常省略长度短于50个密码子的开放阅读框(ORF)。然而,最近很明显,该程序可以分离编码具有高生物学意义的小蛋白的ORF。例如,定制的小蛋白质鉴定方法表明,细菌编码许多具有重要生理功能的小蛋白质。随着预测的小ORF数量的增加,表征相应的蛋白质变得很重要。在这项研究中,我们发现了一种保守但以前被忽视的小肠杆菌蛋白。我们证明了这种蛋白质,我们称之为TimP,是一种有效的毒素,通过靶向细胞膜来抑制细菌生长。毒性由一个小的调节RNA来缓解,结合毒素mRNA抑制毒素合成。
Small proteins are gaining increased attention due to their important functions in major biological processes throughout the domains of life. However, their small size and low sequence conservation make them difficult to identify. It is therefore not surprising that enterobacterial ryfA has escaped identification as a small protein coding gene for nearly 2 decades. Since its identification in 2001, ryfA has been thought to encode a noncoding RNA and has been implicated in biofilm formation in Escherichia coli and pathogenesis in Shigella dysenteriae Although a recent ribosome profiling study suggested ryfA to be translated, the corresponding protein product was not detected. In this study, we provide evidence that ryfA encodes a small toxic inner membrane protein, TimP, overexpression of which causes cytoplasmic membrane leakage. TimP carries an N-terminal signal sequence, indicating that its membrane localization is Sec-dependent. Expression of TimP is repressed by the small RNA (sRNA) TimR, which base pairs with the timP mRNA to inhibit its translation. In contrast to overexpression, endogenous expression of TimP upon timR deletion permits cell growth, possibly indicating a toxicity-independent function in the bacterial membrane.IMPORTANCE Next-generation sequencing (NGS) has enabled the revelation of a vast number of genomes from organisms spanning all domains of life. To reduce complexity when new genome sequences are annotated, open reading frames (ORFs) shorter than 50 codons in length are generally omitted. However, it has recently become evident that this procedure sorts away ORFs encoding small proteins of high biological significance. For instance, tailored small protein identification approaches have shown that bacteria encode numerous small proteins with important physiological functions. As the number of predicted small ORFs increase, it becomes important to characterize the corresponding proteins. In this study, we discovered a conserved but previously overlooked small enterobacterial protein. We show that this protein, which we dubbed TimP, is a potent toxin that inhibits bacterial growth by targeting the cell membrane. Toxicity is relieved by a small regulatory RNA, which binds the toxin mRNA to inhibit toxin synthesis.