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

ABSTRACT: The existence of overlapping genes (OLGs) with significant coding overlaps revolutionises 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 medically important Gram-negative bacterium Pseudomonas aeruginosa. Both OLG pairs show sequence features consistent with being genes and transcriptional signals in RNA sequencing data. 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 highly 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Pseudomonas Aeruginosa Pao1

SUBMITTER: Christina Ludwig

LAB HEAD: Christina Ludwig

PROVIDER: PXD023992 | Pride | 2022-03-21

REPOSITORIES: Pride

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

Kreitmeier Michaela M Ardern Zachary Z Abele Miriam M Ludwig Christina C Scherer Siegfried S Neuhaus Klaus K

iScience 20220201 2

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 <i>Pseudomonas aeruginosa</i>. Both OLG pairs show sequence features consistent with being genes and transcriptional signals in RNA sequencing. Translation of both OLGs w ...[more]

PMID: 35198897

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Project description:IntroductionBacteria inhabit the in- and outside of the human body, such as skin, gut or the oral cavity where they play an innoxious, beneficial or even pathogenic role. It is well known that bacteria can secrete membrane vesicles (MVs) like eukaryotic cells with extracellular vesicles (EVs). Several studies indicate that bacterial membrane vesicles (bMVs) play a crucial role in microbiome-host interactions. However, the composition of such bMVs and their functionality under different culture conditions are still largely unknown. MethodsTo gain a better insight into bMVs, we investigated the composition and functionality of E. coli (DSM 105380) bMVs from the culture media Lysogeny broth (LB) and RPMI 1640 throughout the different phases of growth (lag-, log- and stationary-phase). bMVs from three time points (8 h, 54 h and 168 h) and two media (LB and RPMI 1640) were isolated by ultracentrifugation and analyzed using nanoparticle tracking analysis (NTA), cryogenic electron microscopy (Cryo-EM), conventional transmission electron microscopy (TEM) and mass spectrometry-based proteomics (LC-MS/MS). Furthermore, we examined pro-inflammatory cytokines IL-1β and IL-8 in the human monocyte cell line THP-1 upon bMV treatment.ResultsParticle numbers increased with inoculation periods. The bMV morphologies in Cryo-EM/TEM were similar at each time point and condition. Using proteomics, we identified 140 proteins, such as the common bMV markers OmpA and GroEL, present in bMVs isolated from both media and at all time points. Additionally, we were able to detect growth-condition-specific proteins. Treatment of THP-1 cells with bMVs of all six groups lead to significantly high IL-1β and IL-8 expressions.ConclusionsOur study showed that the choice of medium and the duration of culturing significantly influence both E.coli bMV numbers and protein composition. Our TEM/Cryo-EM results demonstrated the presence of intact E.coli bMVs. Common E. coli proteins, including OmpA, GroEL, and ribosome proteins, can consistently be identified across all six tested growth conditions. Furthermore, our functional assays imply that bMVs isolated from the six groups retain their function and result in comparable cytokine induction.

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