“Pseudo-pseudogenes” in bacterial genomes: Proteogenomics reveals a wide but low protein expression of pseudogenes in Salmonella enterica

Abstract

Pseudogenes (genes disrupted by frameshift or in-frame stop codons) are ubiquitously present in the bacterial genome and considered as nonfunctional fossil. Here, we used RNA-seq and mass-spectrometry technologies to measure the transcriptomes and proteomes of Salmonella enterica serovars Paratyphi A and Typhi. All pseudogenes’ mRNA sequences remained disrupted, and were present at comparable levels to their intact homologs. At the protein level, however, 101 out of 161 pseudogenes suggested successful translation, with their low expression regardless of growth conditions, genetic background and pseudogenization causes. The majority of frameshifting detected was compensatory for -1 frameshift mutations. Readthrough of in-frame stop codons primarily involved UAG; and cytosine was the most frequent base adjacent to the codon. Using a fluorescence reporter system, fifteen pseudogenes were confirmed to express successfully in vivo in Escherichia coli. Expression of the intact copy of the fifteen pseudogenes in S. Typhi affected bacterial pathogenesis as revealed in human macrophage and epithelial cell infection models. The above findings suggest the need to revisit the nonstandard translation mechanism as well as the biological role of pseudogenes in the bacterial genome.

Complementary Ribo-seq approaches map the translatome and provide a small protein census in the foodborne pathogenCampylobacter jejuni

“…TTS profiling could reveal additional examples that are challenging to infer from the genome, such as those generated by ribosomal frameshifting. Recent MS-based proteomics analysis in Salmonella has led to the hypothesis that some pseudogenes, a hallmark of host-adapted strains, maintain full-length transcription and can likewise also show partial full-length translation, possibly via re-coding (via frameshifting or codon re-definition) mechanisms at or near the introduced nonsense mutation, to expand coding potential to provide opportunities in a more generalist niche (Feng et al, 2022). TTS profiling could, in principle, be applied to identify examples of such loci, or even - under relevant conditions - measure the ratio of full-length vs. prematurely terminated translation.…”

Loss to gain: pseudogenes in microorganisms, focusing on eubacteria, and their biological significance

“…They revealed that 101 out of 161 pseudogenes could be successfully translated in S. Paratyphi A and S . Typhi (Feng et al 2022 ). Transcribed pseudogenes have also been observed in Shigella flexneri (Cervantes-Rivera et al 2020 ; Chanin et al 2019 ).…”

Pseudogenes in plasmid genomes reveal past transitions in plasmid mobility

“…The comparison of pseudogenes with their functional homologs showed that most pseudogenes are truncated (480,349, 71%). Recent studies suggest that early stop codons in many truncated pseudogenes correspond to nonsense mutations (24), or transient alleles that may be reversed under purifying selection (25). Comparing the evolutionary rates of truncated and non-truncated pseudogenes we find that truncated pseudogenes are characterized by higher dN and dS, as well as higher dN/dS compared to non-truncated pseudogenes (Supplementary Figure S3), as is expected for DNA evolution following a relaxation of selection pressure (i.e., gene non-functionalization).…”