A novel short L-arginine responsive protein-coding gene (laoB) antiparallel overlapping to a CadC-like transcriptional regulator in Escherichia coli O157:H7 Sakai originated by overprinting

Abstract

BackgroundDue to the DNA triplet code, it is possible that the sequences of two or more protein-coding genes overlap to a large degree. However, such non-trivial overlaps are usually excluded by genome annotation pipelines and, thus, only a few overlapping gene pairs have been described in bacteria. In contrast, transcriptome and translatome sequencing reveals many signals originated from the antisense strand of annotated genes, of which we analyzed an example gene pair in more detail.ResultsA small open reading frame of Escherichia coli O157:H7 strain Sakai (EHEC), designated laoB (L-arginine responsive overlapping gene), is embedded in reading frame −2 in the antisense strand of ECs5115, encoding a CadC-like transcriptional regulator. This overlapping gene shows evidence of transcription and translation in Luria-Bertani (LB) and brain-heart infusion (BHI) medium based on RNA sequencing (RNAseq) and ribosomal-footprint sequencing (RIBOseq). The transcriptional start site is 289 base pairs (bp) upstream of the start codon and transcription termination is 155 bp downstream of the stop codon. Overexpression of LaoB fused to an enhanced green fluorescent protein (EGFP) reporter was possible. The sequence upstream of the transcriptional start site displayed strong promoter activity under different conditions, whereas promoter activity was significantly decreased in the presence of L-arginine. A strand-specific translationally arrested mutant of laoB provided a significant growth advantage in competitive growth experiments in the presence of L-arginine compared to the wild type, which returned to wild type level after complementation of laoB in trans. A phylostratigraphic analysis indicated that the novel gene is restricted to the Escherichia/Shigella clade and might have originated recently by overprinting leading to the expression of part of the antisense strand of ECs5115.ConclusionsHere, we present evidence of a novel small protein-coding gene laoB encoded in the antisense frame −2 of the annotated gene ECs5115. Clearly, laoB is evolutionarily young and it originated in the Escherichia/Shigella clade by overprinting, a process which may cause the de novo evolution of bacterial genes like laoB.Electronic supplementary materialThe online version of this article (10.1186/s12862-018-1134-0) contains supplementary material, which is available to authorized users.

Are Antisense Proteins in Prokaryotes Functional?

“…A strand-specific genomic knock-out mutant was shown to provide a growth advantage specific to media supplemented with arginine. Further, the differential phenotype was replicated with the addition of inducible plasmid constructs bearing ΔlaoB and WT laoB, showing that the phenotype is removed through complementation (Hücker et al, 2018b). How to mechanistically interpret such a growth advantage following gene knockout is unclear, but the condition-specific clear phenotype implies a functional role.…”

Improving Bacterial Ribosome Profiling Data Quality

“…However, the necessary amount of reads is highly dependent on the characteristics of the experiment. If the particular interest lies in using ribosome profiling experiments to detect weakly expressed genes [28,30,56,57], a certain read number matching a gene is needed to confidently confirm expression (e.g. RPKM above a certain threshold).…”

“…Nevertheless, overall this supports the result that 20 million reads are sufficient to detect most of the annotated genes in RIBO-seq experiments. Extensive further work on this question is however required, with recent improvements in gene prediction from RIBO-seq data [58][59][60] and many studies on previously unrecognised small proteins to take into consideration [16,30,57,61,62].…”

“…Phenotypes for recently detected genes overlapping an annotated one (overlapping genes, OLGs) in enterohemorrhagic E. coli (EHEC) have been found in different environmental conditions, e.g. salt stress [56], anaerobiosis [28] or arginine supplementation [57] implying that they are present in a relatively low amount when cultivated in standard LB media. Increasing read depth and sampling in other media conditions might improve the ability to predict such low abundance proteins.…”

A novel pH-regulated, unusual 603 bp overlapping protein coding gene pop is encoded antisense to ompA in Escherichia coli O157:H7 (EHEC)

“…The human pathogenic bacterium Escherichia coli O157:H7 (EHEC) and its genome are well 64 characterized, especially with respect to virulence and the associated diseases like 65 enterocolitis, diarrhea and hemolytic uremic syndrome (Betz et al, 2016, Lim et al, 2010 Stevens and Frankel, 2014). However, the coding capacity of EHEC's genome is likely to be 67 significantly underestimated, both regarding short intergenic genes (Hücker et al, 2017, 68 Neuhaus et al, 2016) as well as non-trivially overlapping genes (Fellner et al, 2014, Fellner 69 et al, 2015, Hücker et al, 2018a, Hücker et al, 2018b, Vanderhaeghen et al, 2018. 70…”