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Gamma-Mobile-Trio systems are mobile elements rich in bacterial defensive and offensive tools

Nature Microbiology volume 9, pages 3268–3283 (2024)Cite this article

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Abstract

The evolutionary arms race between bacteria and phages led to the emergence of bacterial immune systems whose diversity and dynamics remain poorly understood. Here we use comparative genomics to describe a widespread genetic element, defined by the presence of the Gamma-Mobile-Trio (GMT) proteins, that serves as a reservoir of offensive and defensive tools. We demonstrate, using Vibrio parahaemolyticus as a model, that GMT-containing genomic islands are active mobile elements. Furthermore, we show that GMT islands’ cargoes contain various anti-phage defence systems, antibacterial type VI secretion system (T6SS) effectors and antibiotic-resistance genes. We reveal four anti-phage defence systems encoded within GMT islands and further characterize one system, GAPS1, showing it is triggered by a phage capsid protein to induce cell dormancy. Our findings underscore the need to broaden the concept of ‘defence islands’ to include defensive and offensive tools, as both share the same mobile elements for dissemination.

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Fig. 1: GMT proteins define a class of widespread, mobile genomic islands.
Fig. 2: VPaI-6 can be horizontally shared via a conjugatable plasmid.
Fig. 3: GMT islands contain a diverse cargo of offensive and defensive tools.
Fig. 4: Four anti-phage defence systems identified within GMT islands.
Fig. 5: GAPS1 induces cell dormancy upon activation by a phage capsid protein.
Fig. 6: E. coli GAPS1 homologues protect against various coliphages.

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All data related to the manuscript are available in the main text, the supplementary material and the source data files. Source data for the figures are provided with this paper.

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Acknowledgements

We thank members of the Salomon, Qimron and Bosis laboratories for helpful discussions and suggestions; A. Endimiani (University of Bern) for gifting us the E. coli ZH142-A strain; and A. Harms (ETH Zurich) for generously sharing the BASEL phage collection. D.S. and E.B. received funding from the Israel Science Foundation (ISF grant number 1362/21). U.Q. was supported by the European Research Council – Horizon 2020 research and innovation programme, grant no. 818878. U.Q. also received funding from the Israeli Ministry of Health in the framework of the ERANET-JPI-AMR, grant no. 15370. K.K. was supported by a PhD scholarship from the Tel Aviv University Center for Combatting Pandemics.

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Authors and Affiliations

  1. Department of Clinical Microbiology and Immunology, School of Medicine, Faculty of Medical and Health Sciences, Tel Aviv University, Tel Aviv, Israel

    Tridib Mahata, Katarzyna Kanarek, Moran G. Goren, Rameshkumar Marimuthu Ragavan, Udi Qimron & Dor Salomon

  2. Department of Biotechnology Engineering, Braude College of Engineering, Karmiel, Israel

    Eran Bosis

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  2. Katarzyna Kanarek
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Contributions

T.M., E.B., U.Q. and D.S. conceived the work; T.M., K.K., E.B., U.Q. and D.S. designed experiments; E.B. performed bioinformatic analysis and D.S. assisted in data interpretation; T.M. and K.K. carried out most of the experiments; M.G.G. generated mutant phages and identified escape phages; R.M.R. assisted in performing phage plaque assays; E.B., U.Q. and D.S. acquired the funding and supervised the work. E.B. and D.S. wrote the manuscript draft, and all authors edited and approved the paper.

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Correspondence to Eran Bosis, Udi Qimron or Dor Salomon.

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Nature Microbiology thanks Frédérique Le Roux, François Rousset and Lauren Speare 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 Closely related GmtY proteins are found in diverse bacterial orders.

Phylogenetic distribution of GmtY proteins encoded within GMT islands for which an insertion site was identified. The bacterial order and the type of predicted GMT island insertion site are denoted. Blue bars denote the GMT island cargo length. The evolutionary history was inferred by using the Maximum Likelihood method and LG+G+I model. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA X.

Extended Data Fig. 2 GMT islands with a predicted intragenic insertion site contain a homolog of the disrupted gene.

Representative GMT islands (cyan rectangles) predicted to disrupt a gene upon insertion into the bacterial genome are shown above a predicted naïve insertion site in a closely related Vibrio strain. The disrupted and the homologous genes are denoted with the same color. Gray rectangles denote homologous regions. The amino acid identity percentage between the protein encoded by the gene in which the predicted naïve insertion site is found and the homolog within the GMT island are denoted in blue rectangles. The strain names and the RefSeq accession numbers are provided.

Extended Data Fig. 3. The insertion site spacer sequence is important to facilitate GMT island insertion.

(a) Agarose gel electrophoresis analysis of the indicated amplicons. The total DNA isolated from wild-type V. parahaemolyticus RIMD 2210633 cells conjugated with an empty plasmid (pEmpty), a plasmid containing a predicted 30 bp-long naïve insertion site for VPaI-6 (pNISVPaI-6), or a plasmid containing a mutated version of the insertion site in which the spacer between the inverted repeats was modified (pNISVPaI-6-Mut), was used as a template. Arrows denote the positions of primers used for each amplicon; the expected amplicon size is denoted in gray. cat, chloramphenicol resistance gene found on the plasmids. A representative result out of at least three independent experiments is shown. (b) The sequences of the natural VPaI-6 naïve insertion site (NISVPaI-6) and its mutated form (NISVPaI-6-Mut) used in (a).

Source data

Extended Data Fig. 4 The GMT island in V. parahaemolyticus 04.2548 is a functional mobile element.

(a) Schematic representation of the GMT island in V. parahaemolyticus 04.2548 (cyan rectangle). A predicted intragenic (tRNA-Gly), inverted repeat-containing (pink and purple-colored sequences) naïve insertion site identified in V. parahaemolyticus 20140829008-1 is shown below. Inverted repeat sequences flanking the GMT island are denoted. GenBank accession numbers are provided. (b) Agarose gel electrophoresis analysis of the indicated amplicons. The total DNA isolated from wild-type V. parahaemolyticus 04.2548 cells conjugated with an empty plasmid (pEmpty), a plasmid containing a predicted naïve insertion site for VPaI-6 (pNISVPaI-6), or a plasmid containing a predicted naïve insertion site for the 04.2548 GMT island (pNIS04.2548), was used as a template. Arrows denote the positions of primers used for each amplicon; the expected amplicon size is denoted in gray. cat, chloramphenicol resistance gene found on the plasmids. A representative result out of at least three independent experiments is shown.

Source data

Extended Data Fig. 5 GAPS1 homologues are widespread in bacteria.

Phylogenetic distribution of GAPS1 homologues. The bacterial order is denoted by color. Blue bars denote the number of genomes in which each protein accession was identified (Log10 scale). The evolutionary history was inferred by using the Maximum Likelihood method and LG+G+I+F model. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA X.

Extended Data Fig. 6 Closely positioned residues in the T7 phage capsid protein are mutated in GAPS1 escape mutants.

The position of glutamic acid 183 (yellow), isoleucine 217 (green), and valine 247 (magenta) in the solved structure of the T7 phage capsid protein Gp10 (PDB:3j7x chain G).

Extended Data Fig. 7 GAPS1 homologues in E. coli.

Multiple sequence alignment for the indicated protein sequences was performed with Clustal W using Mega X. Similarity (red letter) and identity (red background) shading were done in ESPript 3.0. The accession number of GAPS1 is WP_005477165.1.

Extended Data Fig. 8 A proposed model for GMT island replicative transfer.

A yet-unknown event or process leads to the excision and circularization of one of the GMT island strands without the flanking inverted repeat sequences; base pairing between the inverted repeat sequences may play a role in the process. A complementary strand is polymerized, resulting in a double-stranded circular form of the GMT island. The GMT system proteins, which are required for the excision and circularization, possibly remain bound to the junction between the ends of the GMT island and mediate recognition of a naïve insertion site containing a specific inverted repeat sequence. The naïve insertion site is cleaved within the spacer sequence found between the inverted repeats, and the GMT island is inserted into the recipient site. In parallel, the cell replaces the excised strand in the donor DNA using the remaining strand as a template. This process results in two identical GMT islands.

Extended Data Fig. 9 Identical GMT islands on the chromosome and on a plasmid have similar predicted insertion sites.

(a-b) The nucleotide sequences flanking identical GMT islands on the chromosome (a) and on a plasmid (b) in Vibrio alginolyticus, and their predicted naïve insertion sites (shown below). Inverted repeat sequences are denoted in blue and green; the conserved repeat core found in both predicted insertion sites is denoted in bold. Direct repeat sequences resulting from apparent spacer duplication upon GMT island insertion into the chromosomal site are denoted in pink and purple (a). Gray rectangles denote identical sequences. The strain names and the GenBank accession numbers are provided.

Extended Data Fig. 10 Identification of GMT islands.

(a) Criteria for the identification of genomic accessions in closely related genomes that are homologous to the sequences flanking the GMT system. Thick lines represent the alignments between query and subject accessions. (b) Sequences upstream and downstream of the GMT islands should contain sequence alignments to the subject accessions in at least 4 kbp out of 10 kbp upstream and downstream sequences. (c) Grouping of alignments meeting all the requirements to determine the 5′ and 3′ borders of GMT islands. (d) Intragenic and intergenic insertion sites. (e) Analysis of sequences surrounding a predicted insertion site to identify direct and inverted repeats. Bold letters denote an inverted repeat found within the sequence example.

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2 and references (for citations found in supplementary tables).

Reporting Summary

Peer Review File

Supplementary Tables

Table 1: Mutations found in GAPS1 T7 phage escape mutants. Table 2: List of bacterial strains used in this study. Table 3: List of gene fragments that were commercially synthesized for this study. Table 4: List of plasmids used in this study. Table 5: List of primers used in this study. Table 6: List of bacteriophages used in this study.

Supplementary Data 1

List of GMT proteins and adjacently encoded proteins.

Supplementary Data 2

The borders of GMT islands found within complete genomes.

Supplementary Data 3

Anti-phage defence systems and antibacterial T6SS effectors identified in GMT island cargoes and the presence of a T6SS in these strains.

Supplementary Data 4

List of GAPS1 homologues.

Source data

Source Data Fig. 1

Unprocessed gels.

Source Data Fig. 2

Unprocessed gels.

Source Data Figs. 2–6

Graph and statistical source data for Figs. 2–6.

Source Data Extended Data Fig. 3

Unprocessed gels.

Source Data Extended Data Fig. 4

Unprocessed gels.

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Mahata, T., Kanarek, K., Goren, M.G. et al. Gamma-Mobile-Trio systems are mobile elements rich in bacterial defensive and offensive tools. Nat Microbiol 9, 3268–3283 (2024). https://doi.org/10.1038/s41564-024-01840-5

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