Spotlight on alternative frame coding: Two long overlapping genes in Pseudomonas aeruginosa are translated and under purifying selection.

The existence of overlapping genes (OLGs) with significant coding overlaps revolutionizes our understanding of genomic complexity. We report two exceptionally long (957 nt and 1536 nt), evolutionarily novel, translated antisense open reading frames (ORFs) embedded within annotated genes in the pathogenic Gram-negative bacterium Pseudomonas aeruginosa. Both OLG pairs show sequence features consistent with being genes and transcriptional signals in RNA sequencing. Translation of both OLGs was confirmed by ribosome profiling and mass spectrometry. Quantitative proteomics of samples taken during different phases of growth revealed regulation of protein abundances, implying biological functionality. Both OLGs are taxonomically restricted, and likely arose by overprinting within the genus. Evidence for purifying selection further supports functionality. The OLGs reported here, designated olg1 and olg2, are the longest yet proposed in prokaryotes and are among the best attested in terms of translation and evolutionary constraint. These results highlight a potentially large unexplored dimension of prokaryotic genomes.

A Novel pH-Regulated, Unusual 603 bp Overlapping Protein Coding Gene pop Is Encoded Antisense to ompA in Escherichia coli O157:H7 (EHEC).

Antisense transcription is well known in bacteria. However, translation of antisense RNAs is typically not considered, as the implied overlapping coding at a DNA locus is assumed to be highly improbable. Therefore, such overlapping genes are systematically excluded in prokaryotic genome annotation. Here we report an exceptional 603 bp long open reading frame completely embedded in antisense to the gene of the outer membrane protein ompA. An active σ70 promoter, transcription start site (TSS), Shine-Dalgarno motif and rho-independent terminator were experimentally validated, providing evidence that this open reading frame has all the structural features of a functional gene. Furthermore, ribosomal profiling revealed translation of the mRNA, the protein was detected in Western blots and a pH-dependent phenotype conferred by the protein was shown in competitive overexpression growth experiments of a translationally arrested mutant versus wild type. We designate this novel gene pop (pH-regulated overlapping protein-coding gene), thus adding another example to the growing list of overlapping, protein coding genes in bacteria.