Abstract
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.
Highlights
IMPORTANCE Next-generation sequencing (NGS) has enabled the revelation of a vast number of genomes from organisms spanning all domains of life
During our first experiments aimed at characterizing timP function in Salmonella, we observed that its overexpression from an inducible promoter strongly inhibited bacterial growth
Spotting dilutions of bacterial cultures on plates containing the inducer resulted in a strong reduction in the number of CFU when cells harbored the timP-inducible plasmid (Fig. S2)
Summary
IMPORTANCE Next-generation sequencing (NGS) has enabled the revelation of a vast number of genomes from organisms spanning all domains of life. ® mbio.asm.org 1 cation by combining advanced computational prediction with experimental methods [1,2,3,4,5,6,7,8,9,10,11,12,13], recently reviewed for Escherichia coli in reference 14 These studies demonstrate that small protein genes are much more abundant than previously imagined. Since type I toxin translation is generally repressed during growth in common laboratory media, most research on these systems has been done with ectopic expression of the system components These studies have shown that, when overexpressed from a plasmid, type I toxins damage the cells in different ways, often by compromising the cytoplasmic membrane [31]. It has been reported that TA systems induce cell death under unfavorable conditions (e.g., postsegregational killing and abortive infection) or that controlled activation of toxins can induce a transient state of dormancy that promotes stress tolerance [28]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
