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Widespread stop-codon recoding in bacteriophages may regulate translation of lytic genes

Nature Microbiology volume 7, pages 918–927 (2022)Cite this article

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Abstract

Bacteriophages (phages) are obligate parasites that use host bacterial translation machinery to produce viral proteins. However, some phages have alternative genetic codes with reassigned stop codons that are predicted to be incompatible with bacterial translation systems. We analysed 9,422 phage genomes and found that stop-codon recoding has evolved in diverse clades of phages that infect bacteria present in both human and animal gut microbiota. Recoded stop codons are particularly over-represented in phage structural and lysis genes. We propose that recoded stop codons might function to prevent premature production of late-stage proteins. Stop-codon recoding has evolved several times in closely related lineages, which suggests that adaptive recoding can occur over very short evolutionary timescales.

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Fig. 1: Identification of recoded phage in human and animal microbiomes.
Fig. 2: Phylogeny of recoded phages and suppressor tRNA usage.
Fig. 3: Evolutionary relationships among phages according to genetic code.
Fig. 4: Preferential recoding of lysis-related genes in recoded phages.
Fig. 5: Recoded prophages integrated into bacterial genomes.
Fig. 6: A model for recoding in the phage life cycle.

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Data availability

Accessions for MIUVIG-compliant genomes27 and associated reads for alternatively coded phages and relatives are provided in Supplementary Table 3. Genomes and predicted proteins for alternatively coded phages and relatives, the terminase phylogenetic tree file, closely related Agate and crAss-like genomes, and untrimmed lysogenic contigs are available through Zenodo (10.5281/zenodo.6410225). The UniRef100 database is available through ftp.uniprot.org/pub/databases/uniprot/uniref/uniref100. Source data are provided with this paper.

Code availability

Python script used to analyse coding density and predict genetic code is available on GitHub: https://github.com/borgesadair1/AC_phage_analysis/releases/tag/v1.0.0.

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Acknowledgements

We thank Y. Song, J. Cate, K. Seed, G. Chadwick, L.-X. Chen, J. West-Roberts and S. Diamond for helpful discussions; K. K. L. Law and J. Hoff for technical support. This work was supported by a Miller Basic Research Fellowship to A.L.B; an NSF Graduate Research Fellowship to B.A-S (No. DGE 1752814); and an NIH award RAI092531A, a Chan Zuckerberg Biohub award and Innovative Genome Institute funding to J.F.B.

Author information

Authors and Affiliations

  1. Innovative Genomics Institute, University of California, Berkeley, CA, USA

    Adair L. Borges, Yue Clare Lou, Rohan Sachdeva, Basem Al-Shayeb & Jillian F. Banfield

  2. Environmental Science, Policy and Management, University of California, Berkeley, CA, USA

    Adair L. Borges & Jillian F. Banfield

  3. Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA

    Yue Clare Lou, Basem Al-Shayeb & Alexander L. Jaffe

  4. Earth and Planetary Science, University of California, Berkeley, CA, USA

    Rohan Sachdeva, Petar I. Penev, Shufei Lei & Jillian F. Banfield

  5. Department of Structural and Molecular Biology, Division of Biosciences, University College London, London, UK

    Joanne M. Santini

  6. Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Jillian F. Banfield

  7. The University of Melbourne, Parkville, Victoria, Australia

    Jillian F. Banfield

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Contributions

A.L.B and J.F.B. developed the project, led analyses and wrote the manuscript with input from all authors. A.L.B, J.F.B, Y.C.L, R.S. and S.L. compiled the phage dataset. B.A-S assembled public metagenome data and provided support for phage genome analyses. Phage genomes were manually curated by J.F.B. P.I.P contributed to phage tRNA analyses. A.L.J. and Y.C.L contributed to design of statistical analyses. J.M.S. contributed DNA samples from animal and arsenic-exposed human gut microbiomes.

Corresponding author

Correspondence to Jillian F. Banfield.

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Competing interests

J.F.B. is a founder of Metagenomi. The other authors declare no competing interests.

Peer review

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Nature Microbiology thanks Sebastian Pechmann and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Identification of recoded phages in the Giant Tortoise microbiome.

a. Dereplicated complete or near complete (>=90%) phage genomes from the Giant Tortoise gut microbiome. Phages are plotted by size and coding density (CD) in standard code (Code11) b. Replotting of phage genomes from panel A, but with coding density of the alternatively coded phage calculated with the predicted genetic code instead of standard code. In all plots, phages that have recoded the TGA stop codon are indicated in green, and phages that have recoded the TAG stop codon are indicated in orange.

Source data

Extended Data Fig. 2 True coding density of standard and alternatively coded phages.

a-f. Replotting of phage genomes from Fig. 1 in the main text, but this time the coding density of alternatively coded phage was calculated with their predicted genetic code, not standard code. In all plots, symbol color represents genetic code (TGA recoding = green, TAG recoding = orange, standard code = grey).

Source data

Extended Data Fig. 3 Evolution of alternative coding.

a. Global alignment of an 80 kilobase (kb) partial TGA recoded Agate genome (Cattle_ERR2019405_scaffold_1063) and a close standard code relative (pig_ID_3053_F60_scaffold_12). Homologous collinear sequences are shown with colored blocks (red and green here), where color corresponds to nucleotide alignment between the two genomes and lack of color represents lack of alignment. Genome structure for each phage is shown under the alignment graph, with DNA replication machinery represented as yellow bars and structural and lysis genes with pink bars. TGA stop codons have predominantly arisen in structural and lysis genes (individual recoded genes below in green).

Source data

Extended Data Fig. 4 Genomic maps of Jade, Sapphire, Agate and Topaz phages.

a-d. TGA recoded genomes (A) contain genes with in-frame TGA codons (green) while TAG recoded genomes (B-D) have genes with in-frame TAG codons (orange). Suppressor tRNAs (tRNA TGA or tRNA TAG, red) are predicted to suppress translation termination at TGA and TAG stop codons, respectively. Regions of the genome encoding structural and lysis genes (pink) coincide with high use of alternative code. Contrastingly, genes involved in DNA replication (yellow) are variably encoded in alternative code. Genomes with a GC skew patterns indicative of bidirectional replication and clear origins and termini (C) have unique replichores marked in alternating shades of blue. Genomes with GC skew patterns most consistent with unidirectional replication (A-B,D) have no replication-related annotation. In some cases, unique or interesting genes have been noted with text. Clade representatives: Jade = JS_HF2_S141_scaffold_159238, Sapphire= SRR1747018_scaffold_13, Agate = Cattle_ERR2019359_scaffold_1067472, Topaz = pig_ID_1851_F40_2_B1_scaffold_1589.

Source data

Extended Data Fig. 5 Genomic maps of Lak, Garnet, and Amethyst phages.

a-c. TAG recoded genomes have genes with in-frame TAG codons (orange). Suppressor tRNAs (tRNA TAG, red) are predicted to suppress translation termination at TAG stop codons. Regions of the genome encoding structural and lysis genes (pink) coincide with high use of alternative code. In Lak phage (A), genes involved in DNA replication (yellow) are mostly encoded in alternative code. Origins and termini are unmarked in these genomes as we were unable to define clear replichores for Lak (A) and Garnet and Amethyst (B-C) appear to utilize unidirectional genome replication based on GC skew patterns. In some cases, unique or interesting genes have been noted with text. Clade representatives: Lak = C1–CH_A02_001D1_final, Garnet = pig_ID_3640_F65_scaffold_1252, Amethyst = pig_EL5596_F5_scaffold_275.

Source data

Extended Data Fig. 6 Code change machinery in two TGA-recoded Jade phages.

a. An operon implicated in changing the genetic code from standard code (TGA=Stop) to code 4 (TGA=W) is directly upstream of the lysis cassette. The code change genes themselves are encoded in standard code, while some genes in the lysis cassette have in frame TGA codons (green). TrpRS = Tryptophanyl tRNA synthetase, RF1=Release Factor 1, TM-domain = Transmembrane domain.

Extended Data Fig. 7 Read mapping to Garnet and Topaz lysogens.

a. Reads were mapped against a manually curated Garnet lysogen. Read coverage for the Prevotella DNA is ~2x higher than the read coverage of the Garnet prophage, indicating that the bacterial population in this sample is incompletely lysogenized. Supporting this conclusion are paired reads that span the prophage (not shown), as well as some individual reads which show imperfect mapping to the lysogen consensus sequence (marked with asterisk), which represent the contiguous bacterial sequence. Identical sequence blocks are indicated with color. b. Reads were mapped against a manually curated Topaz lysogen. Read coverage for the integrated Topaz phage genome is ~50x higher than the neighboring Oscillospiraceae sequence. This indicates that the phage is actively replicating in this sample. Supporting this conclusion are paired reads that span the length of the prophage (not shown), as well as individual reads which show imperfect mapping to the lysogen consensus sequence at the 5’ end of the prophage (light blue) and the 3’ end of the prophage (dark blue). The reads correspond to circularized sequences. Identical sequence blocks are indicated with color.

Extended Data Fig. 8 Alignments of free and integrated phage genomes.

a. A 25 kb circular TAG-recoded Garnet phage aligned to a prophage integrated in a Prevotella genome (Prevotella genes = brown). The prophage boundaries are marked by the phage integrase (pink) and the host tRNA Met. b. A 24 kb circular TAG-recoded Topaz phage aligned to a prophage integrated into a Oscillospiraceae genome (Oscillospiraceae genes = blue). The prophage boundaries are marked by the phage integrase (pink) and the host tRNA Thr.

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2.

Reporting Summary

Peer Review File

Supplementary Tables 1–5

Table 1. Source metagenomes used in this study. Table 2. Clades of alternatively coded phages found in this study. Table 3. Alternatively coded phage genomes and relatives from this study. Table 4. tRNAs for all alternatively coded phage genomes and relatives from this study. Table 5. Table of release factor and tRNA synthetase counts in alternatively coded phage clades.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

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Borges, A.L., Lou, Y.C., Sachdeva, R. et al. Widespread stop-codon recoding in bacteriophages may regulate translation of lytic genes. Nat Microbiol 7, 918–927 (2022). https://doi.org/10.1038/s41564-022-01128-6

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