Thousands of small, novel genes predicted in global phage genomes

Fremin, Brayon J.; Bhatt, Ami S.; Kyrpides, Nikos C.

doi:10.1016/j.celrep.2022.110984

Cited by 31 publications

(21 citation statements)

References 78 publications

(121 reference statements)

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“…Recently, new in silico ORF prediction tools have been developed to identify small proteins in bacteria as well as phages based on genome sequence alone (Durrant and Bhatt, 2021; Fremin et al, 2022; Miravet-Verde et al, 2019). However, two examples (ranSEPS, smORFinder) did not detect most of our validated CJsORFs ( Table S1 ).…”

Section: Discussionmentioning

confidence: 99%

“…Searches for sORFs using comparative genomics across bacterial species and even their phages have revealed thousands of potential candidates (Durrant and Bhatt, 2021; Fremin et al, 2022; Miravet-Verde et al, 2019; Sberro et al, 2019). Validating these predictions, however, is challenging as small proteins can be difficult to detect by mass spectrometry (MS) approaches despite the development of new methodologies tailored for small proteins (reviewed in (Ahrens et al, 2022)).…”

Section: Introductionmentioning

confidence: 99%

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

Froschauer

Svensson

Gelhausen

et al. 2022

Preprint

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Background: Knowing the molecules encoded by bacterial pathogens and how their expression is regulated is essential to understand how they survive, colonize, and cause disease. RNA-seq technologies that map transcriptomes have revealed a wealth of new transcripts in bacterial pathogens. However, they do not provide direct evidence or coordinates for coding potential. In particular, they miss small proteins (less than or equal to 50-100 amino acids) translated from small open reading frames (sORFs). However, this still poorly annotated component of bacterial genomes shows emerging roles in bacterial physiology and virulence. Results: Here, we present an integrated approach based on complementary ribosome profiling (Ribo-seq) techniques to map the 'translatome' of Campylobacter jejuni, the most common cause of bacterial gastroenteritis. Besides conventional Ribo-seq, we employed translation initiation site (TIS) profiling to map start codons and reveal internal sORFs. We also developed a Ribo-seq approach for mapping of translation termination sites (TTS), which revealed stop codons not apparent from the reference genome in virulence-associated loci. Our translatome map confirms translation of leaderless ORFs and leader peptides, re-annotates start or stop codons of 35 genes, and reveals isoforms generated by internal start sites. It also adds 42 novel sORFs in diverse contexts to the C. jejuni annotation, such as within small RNAs, in 5 prime untranslated regions (UTRs), or internal/out-of-frame/antisense in larger ORFs. Using epitope tagging/western blot and mass spectrometry we validated expression of almost 60 annotated and novel sORFs, including cioY, which we show encodes a conserved, 34 amino acid component of the CioAB terminal oxidase. Conclusions: Overall, we provide a blueprint for integrating several Ribo-seq approaches to refine and enrich bacterial annotations.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Froschauer

Svensson

Gelhausen

et al. 2022

Preprint

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“…Interestingly, phages can 'fight back', sometimes by producing small proteins that inhibit toxins and thus allow phages to complete their infection cycle 82,83 . Homologs of these short proteins are often only found in phage genomes 84 , and our work suggests a possible de novo origin for these proteins.…”

Section: Discussionmentioning

confidence: 57%

Selection of ade novogene that can promote survival ofE. coliby modulating protein homeostasis pathways

Frumkin

Laub

2023

Preprint

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Cells sometime adapt to challenging environments by turning non-functional loci into functional genes in a process termedde novogene birth. But how proteins with random amino acid sequences integrate into existing cellular pathways to provide a benefit remains poorly understood. Here, we screened ~108random genes for their ability to rescue growth arrest ofE. colicells producing the ribonuclease toxin MazF. Approximately 2,000 random genes could promote growth by reducing transcription from the promoter drivingmazFexpression. Additionally, one gene, namedrandomantitoxin ofMazF(ramF), whose protein product was well-tolerated in cells and neutralized MazF by interacting with chaperones, leading to MazF proteolysis. We show that the specificity of RamF for MazF relative to other toxins relies on the degron-like function of MazF's first 10 amino acids. Finally, we demonstrate that random proteins can improve during evolution by identifying beneficial mutations that turned RamF into a more efficient inhibitor. Our work provides a mechanistic basis for howde novogene birth can produce new, functional proteins that are integrated into complex cellular systems and provide a benefit to cells.

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“…Moreover, PHANOTATE predicts significantly more genes that are on average smaller than other gene prediction tools ( McNair et al , 2019 ). Small phage genes are more prevalent than predicted based on existing non-phage specific annotation tools and may encode for anti-CRISPR and antimicrobial resistance proteins ( Fremin et al , 2022 ). Alternatively, Pharokka users can specify Prodigal, a gene predictor designed for prokaryotic genes, be used for CDS prediction ( Hyatt et al , 2010 ; Fig.…”

Section: Methodsmentioning

confidence: 99%

Pharokka: a fast scalable bacteriophage annotation tool

et al. 2022

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Summary In recent years, there has been an increasing interest in bacteriophages which has led to growing numbers of bacteriophage genomic sequences becoming available. Consequently, there is a need for a rapid and consistent genomic annotation tool dedicated for bacteriophages. Existing tools either are not designed specifically for bacteriophages or are web- and email- based and require significant manual curation, which makes their integration into bioinformatic pipelines challenging. Pharokka was created to provide a tool that annotates bacteriophage genomes easily, rapidly and consistently with standards compliant outputs. Moreover, Pharokka requires only two lines of code to install and use and takes under 5 minutes to run for an average 50 kb bacteriophage genome. Availability and Implementation Pharokka is implemented in Python and is available as a bioconda package using ‘conda install -c bioconda pharokka’. The source code is available on GitHub (https://github.com/gbouras13/pharokka). Pharokka has been tested on Linux-64 and MacOSX machines, and on Windows using a Linux Virtual Machine. Supplementary information All benchmarking input FASTA and output files, including the python script calc_gff_coding_density_prokka.py script, is available at https://doi.org/10.5281/zenodo.7227091.

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Thousands of small, novel genes predicted in global phage genomes

Cited by 31 publications

References 78 publications

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

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

Selection of ade novogene that can promote survival ofE. coliby modulating protein homeostasis pathways

Pharokka: a fast scalable bacteriophage annotation tool

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