The Novel Anaerobiosis-Responsive Overlapping Gene ano Is Overlapping Antisense to the Annotated Gene ECs2385 of Escherichia coli O157:H7 Sakai

Abstract

Current notion presumes that only one protein is encoded at a given bacterial genetic locus. However, transcription and translation of an overlapping open reading frame (ORF) of 186 bp length were discovered by RNAseq and RIBOseq experiments. This ORF is almost completely embedded in the annotated L,D-transpeptidase gene ECs2385 of Escherichia coli O157:H7 Sakai in the antisense reading frame -3. The ORF is transcribed as part of a bicistronic mRNA, which includes the annotated upstream gene ECs2384, encoding a murein lipoprotein. The transcriptional start site of the operon resides 38 bp upstream of the ECs2384 start codon and is driven by a predicted σ70 promoter, which is constitutively active under different growth conditions. The bicistronic operon contains a ρ-independent terminator just upstream of the novel gene, significantly decreasing its transcription. The novel gene can be stably expressed as an EGFP-fusion protein and a translationally arrested mutant of ano, unable to produce the protein, shows a growth advantage in competitive growth experiments compared to the wild type under anaerobiosis. Therefore, the novel antisense overlapping gene is named ano (anaerobiosis responsive overlapping gene). A phylostratigraphic analysis indicates that ano originated very recently de novo by overprinting after the Escherichia/Shigella clade separated from other enterobacteria. Therefore, ano is one of the very rare cases of overlapping genes known in the genus Escherichia.

Are Antisense Proteins in Prokaryotes Functional?

“…Similarly to laoB, a knock-out mutant showed a conditionspecific phenotype. In this case it occurs in anaerobic conditions, and could be partially complemented with a plasmid construct (Hücker et al, 2018a). The putative protein-coding gene aatS was found in the pathogenic E. coli strain ETEC H10407 fully embedded in antisense to the ATP transporter ATB binding protein AatC (Haycocks and Grainger, 2016).…”

“…While the evolutionary analysis of antisense proteins in prokaryotes awaits further investigation of strong overlapping gene candidates, those discovered so far are typically relatively young (e.g. (Fellner et al, 2014, Fellner et al, 2015, Hücker et al, 2018a. This may be seen as a point against their functionality, particularly for candidates limited to just one species.…”

Improving Bacterial Ribosome Profiling Data Quality

“…Interestingly, RNase I is claimed to not work in bacteria and especially E. coli, as it is bound by the 30S ribosome subunit and therefore inhibited [6,25]. Nevertheless, ribosome profiling experiments have been conducted successfully using only RNase I [26,27], or in combination with other enzymes [28] producing footprints of a size of ~23 nt. Due to the assumption of RNAse I unsuitability, the enzyme micrococcal nuclease (MNase) is normally used for prokaryotic experiments instead, despite this enzyme having some sequence specificity [29], resulting in greater variability in ribosome footprint lengths.…”

“…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).…”

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…”