The Putative Virulence Plasmid pYR4 of the Fish Pathogen Yersinia ruckeri Is Conjugative and Stabilized by a HigBA Toxin–Antitoxin System
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Bioinformatics
2.2. Bacterial Strains and Growth Conditions
2.3. Cloning
2.4. Creating a higB Knockout and Introducing a Selection Marker into pYR4
2.5. Plasmid Curing and Plasmid Loss Experiments
2.6. Conjugation
2.7. Toxicity Assays
2.8. Twitching Motility Assays
2.9. Galleria Infection Assays
2.10. Statistical Analysis
3. Results
3.1. pYR4 Carries Several Putative TA-Related Genes
3.2. pYR4 HigBA Is a Functional Post-Segregational Killing System
3.3. Qualitative Evidence of pYR4 Conjugation
3.4. Virulence-Related Phenotypes Encoded by pYR4
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A
Primer | Sequence (5′ → 3′) | Description |
---|---|---|
GMntu043 | GCATTTTTATCCATAAGATTAGCGG | pBAD33 MCS (forward) |
GMntu044 | GCGTTCTGATTTAATCTGTATCAGG | pBAD33 MCS (reverse) |
GMntu045 | CACCGTCATCACCGAAACG | pGM101 insert site (forward) |
GMntu046 | CTGTTTTATCAGACCGCTTCTGC | pGM101 insert site (reverse) |
GMmbiol057 | GGATCCTCTAGAGTCGACGCAAGGGACGAGCGTTAATGG | higB with pBAD33 overlap (forward) |
GMmbiol058 | AAGCTTGCATGCCTGCAGCGGGGGCTTGTTATTCGATTTG | higB with pBAD33 overlap (reverse) |
GMmbiol059 | AATCGAATAACAAGCCCCCGCTGCAGGCATGCAAGCTT | pBAD33 with higB overlap (forward) |
GMmbiol060 | CATTAACGCTCGTCCCTTGCGTCGACTCTAGAGGATCC | pBAD33 with higB overlap (reverse) |
GMmbiol061 | GCATGGCGGCTAAAGTTGTG | pYR4 repA (forward) |
GMmbiol062 | GCTAGATTATGCCTGCTCGC | pYR4 repA (reverse) |
GMmbiol063 | CCATGAGCAAGGGCGAATTGCT | pYR4 pilN (forward) |
GMmbiol064 | GCTTCAGTCATCACGCTGACAT | pYR4 pilN (reverse) |
GMmbiol065 | CCCGTATTTAGCAGGCGAAGAG | pYR4 higB (forward) |
GMmbiol066 | GCTGACTTATCGATTTCAGGAC | pYR4 higB (reverse) |
GMmbiol067 | CAGCGGAAAGTAGCTTG | Y. ruckeri 16S rDNA (forward) |
GMmbiol068 | TGTTCAGTGCTATTAACACTTAA | Y. ruckeri 16S rDNA (reverse) |
GMmbiol069 | CGCCCACAGGGTGCGCCGCTCGAAGCGGCATGCATTTACG | pGM101 with higBA overlap (forward) |
GMmbiol070 | TTGCACTTTTTGAGATGATTCCTTCGCGCGCGAATTGATC | pGM101 with higBA overlap (reverse) |
GMmbiol071 | GATCAATTCGCGCGCGAAGGAATCATCTCAAAAAGTGCAATTATTGC | higBA with pGM101 overlap (forward) |
GMmbiol072 | CGTAAATGCATGCCGCTTCGAGCGGCGCACCCTGTGG | higBA with pGM101 overlap (reverse) |
GMmbiol073 | TCAGCAAGGGACGAGCGTTACAAGCCCCCGCACTGCG | ΔhigB variant (forward) |
GMmbiol074 | CCCCGCAGTGCGGGGGCTTGTAACGCTCGTCCCTTGCTGA | ΔhigB variant (reverse) |
GMmbiol075 | GCAAGGGACGAGCGTTATACGAGTATCTAGAATTCATTGAG | higB mutation variant (forward) |
GMmbiol076 | ATGAATTCTAGATACTCGTATAACGCTCGTCCCTTGCTGACCG | higB mutation variant (reverse) |
MS103 | GGGAGAGCTCAAAAAAGCGACTTTAGCC | Upstream higBA (forward) |
MS104 | TGAGATGATTATGACAGGTTATGAATTGC | Upstream higBA (reverse) |
MS105 | AACCTGTCATAATCATCTCAAAAAGTGCAATTATTGCACTATTTTATATTTTTATTTAGCGAGCGTATACC | ΔhigA (forward) |
MS106 | TAATTCCCATGAGCGGCGCACCCTGTGG | ΔhigA (reverse) |
MS107 | TGCGCCGCTCATGGGAATTAGCCATGGTCC | Chloramphenicol cassette (forward) |
MS108 | TGGCCAGTAAGTGTAGGCTGGAGCTGCTTC | Chloramphenicol cassette (reverse) |
MS109 | CAGCCTACACTTACTGGCCACTTCCGTG | Downstream higBA (forward) |
MS110 | TACCGCATGCAGCCGAAGCATATGTTTTG | Downstream higBA (reverse) |
References
- FAO. Fishery and Aquaculture Statistics—Yearbook 2021; Food and Agriculture Organization: Rome, Italy, 2024. [Google Scholar]
- OECD/FAO. Fish. In OECD-FAO Agricultural Outlook 2023–2032; OECD Publishing: Paris, France, 2023; pp. 214–224. [Google Scholar]
- Segner, H.; Sundh, H.; Buchmann, K.; Douxfils, J.; Sundell, K.S.; Mathieu, C.; Ruane, N.; Jutfelt, F.; Toften, H.; Vaughan, L. Health of farmed fish: Its relation to fish welfare and its utility as welfare indicator. Fish Physiol. Biochem. 2011, 38, 85–105. [Google Scholar] [CrossRef]
- Stentiford, G.D.; Sritunyalucksana, K.; Flegel, T.W.; Williams, B.A.P.; Withyachumnarnkul, B.; Itsathitphaisarn, O.; Bass, D. New paradigms to help solve the global aquaculture disease crisis. PLoS Pathog. 2017, 13, e1006160. [Google Scholar] [CrossRef] [PubMed]
- Kumar, G.; Menanteau-Ledouble, S.; Saleh, M.; El-Matbouli, M. Yersinia ruckeri, the causative agent of enteric redmouth disease in fish. Vet. Res. 2015, 46, 103. [Google Scholar] [CrossRef] [PubMed]
- Fernandez-Espinel, C.; Medina-Morillo, M.; Irgang, R.; Sotil, G.; Araya-León, H.; Flores-Dominick, V.; Romalde, J.L.; Avendaño-Herrera, R.; Yunis-Aguinaga, J. Co-existence of two Yersinia ruckeri biotypes and serotype O1a retrieved from rainbow trout (Oncorhynchus mykiss) farmed in Puno, Peru. J. Fish Dis. 2022, 46, 157–163. [Google Scholar] [CrossRef] [PubMed]
- Riborg, A.; Colquhoun, D.J.; Gulla, S. Biotyping reveals loss of motility in two distinct Yersinia ruckeri lineages exclusive to Norwegian aquaculture. J. Fish Dis. 2022, 45, 641–653. [Google Scholar] [CrossRef]
- Fouz, B.; Zarza, C.; Amaro, C. First description of non-motile Yersinia ruckeri serovar I strains causing disease in rainbow trout, Oncorhynchus mykiss (Walbaum), cultured in Spain. J. Fish Dis. 2006, 29, 339–346. [Google Scholar] [CrossRef]
- Wrobel, A.; Leo, J.C.; Linke, D. Overcoming fish defences: The virulence factors of Yersinia ruckeri. Genes 2019, 10, 700. [Google Scholar] [CrossRef]
- Carniel Arniel, E. Plasmids and Pathogenicity Islands of Yersinia. Curr. Top. Microbiol. Immunol. 2002, 264, 89–108. [Google Scholar] [CrossRef]
- Andreopoulos, W.B.; Geller, A.M.; Lucke, M.; Balewski, J.; Clum, A.; Ivanova, N.N.; Levy, A. Deeplasmid: Deep learning accurately separates plasmids from bacterial chromosomes. Nucleic Acids Res. 2021, 50, e17. [Google Scholar] [CrossRef] [PubMed]
- Riborg, A.; Gulla, S.; Fiskebeck, E.Z.; Ryder, D.; Verner-Jeffreys, D.W.; Colquhoun, D.J.; Welch, T.J. Pan-genome survey of the fish pathogen Yersinia ruckeri links accessory- and amplified genes to virulence. PLoS ONE 2023, 18, e0285257. [Google Scholar] [CrossRef]
- Wrobel, A.; Ottoni, C.; Leo, J.C.; Linke, D. pYR4 from a Norwegian isolate of Yersinia ruckeri is a putative virulence plasmid encoding both a type IV pilus and a type IV secretion system. Front. Cell. Infect. Microbiol. 2018, 8, 373. [Google Scholar] [CrossRef]
- Pelicic, V. Type IV pili: E pluribus unum? Mol. Microbiol. 2008, 68, 827–837. [Google Scholar] [CrossRef]
- Costa, T.R.D.; Patkowski, J.B.; Macé, K.; Christie, P.J.; Waksman, G. Structural and functional diversity of type IV secretion systems. Nat. Rev. Microbiol. 2023, 22, 170–185. [Google Scholar] [CrossRef] [PubMed]
- Méndez, J.; Fernández, L.; Menéndez, A.; Reimundo, P.; Pérez-Pascual, D.; Navais, R.; Guijarro, J.A. A chromosomally located traHIJKCLMN operon encoding a putative type IV secretion system is involved in the virulence of Yersinia ruckeri. Appl. Environ. Microbiol. 2009, 75, 937–945. [Google Scholar] [CrossRef] [PubMed]
- Qiu, J.; Zhai, Y.; Wei, M.; Zheng, C.; Jiao, X. Toxin–antitoxin systems: Classification, biological roles, and applications. Microbiol. Res. 2022, 264, 127159. [Google Scholar] [CrossRef] [PubMed]
- Harms, A.; Brodersen, D.E.; Mitarai, N.; Gerdes, K. Toxins, targets, and triggers: An overview of toxin-antitoxin biology. Mol. Cell 2018, 70, 768–784. [Google Scholar] [CrossRef] [PubMed]
- Chan, W.T.; Espinosa, M.; Yeo, C.C. Keeping the wolves at bay: Antitoxins of prokaryotic type II toxin-antitoxin systems. Front. Mol. Biosci. 2016, 3, 9. [Google Scholar] [CrossRef]
- Tian, Q.B.; Ohnishi, M.; Murata, T.; Nakayama, K.; Terawaki, Y.; Hayashi, T. Specific protein–DNA and protein–protein interaction in the hig gene system, a plasmid-borne proteic killer gene system of plasmid Rts1. Plasmid 2001, 45, 63–74. [Google Scholar] [CrossRef]
- Christensen-Dalsgaard, M.; Gerdes, K. Two higBA loci in the Vibrio cholerae superintegron encode mRNA cleaving enzymes and can stabilize plasmids. Mol. Microbiol. 2006, 62, 397–411. [Google Scholar] [CrossRef]
- Altschul, S.F.; Madden, T.L.; Schaffer, A.A.; Zhang, J.H.; Zhang, Z.; Miller, W.; Lipman, D.J. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 1997, 25, 3389–3402. [Google Scholar] [CrossRef]
- Solovyev, V.; Salamov, A. Automatic annotation of microbial genomes and metagenomic sequences. In Metagenomics and Its Applications in Agriculture, Biomedicine and Environmental Studies; Li, R.W., Ed.; Nova Science Publishers, Incorporated: Hauppauge, NY, USA, 2011; pp. 61–78. [Google Scholar]
- Robertson, J.; Nash, J.H.E. MOB-suite: Software tools for clustering, reconstruction and typing of plasmids from draft assemblies. Microb. Genom. 2018, 4, e000206. [Google Scholar] [CrossRef]
- Wrobel, A.; Ottoni, C.; Leo, J.C.; Gulla, S.; Linke, D. The repeat structure of two paralogous genes, Yersinia ruckeri invasin (yrInv) and a “Y. ruckeri invasin-like molecule”, (yrIlm) sheds light on the evolution of adhesive capacities of a fish pathogen. J. Struct. Biol. 2018, 201, 171–183. [Google Scholar] [CrossRef]
- Wasteson, U.; Hvaal, A.; Serum, H.; Myhr, E.; Fossum, K. Antibacterial spectrum and some other characteristics of an antimicrobial factor produced by Yersinia ruckeri. Acta Vet. Scand. 1989, 30, 253–257. [Google Scholar] [CrossRef] [PubMed]
- Bertani, G. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacteriol. 1951, 62, 293–300. [Google Scholar] [CrossRef] [PubMed]
- Anonymous. M9 minimal medium (standard). Cold Spring Harb. Protoc. 2010, 2010, pdb.rec12295. [Google Scholar] [CrossRef]
- Gibson, D.G.; Chuang, R.; Hutchison, C.A.; Venter, J.C.; Smith, H.O.; Young, L. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 2009, 6, 343–345. [Google Scholar] [CrossRef]
- Guzman, L.M.; Belin, D.; Carson, M.J.; Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 1995, 177, 4121–4130. [Google Scholar] [CrossRef] [PubMed]
- Mcvicker, G.; Tang, C.M. Deletion of toxin–antitoxin systems in the evolution of Shigella sonnei as a host-adapted pathogen. Nat. Microbiol. 2016, 2, 16204. [Google Scholar] [CrossRef]
- Datsenko, K.A.; Wanner, B.L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 2000, 97, 6640–6645. [Google Scholar] [CrossRef]
- Hossain, M.J.; Thurlow, C.M.; Sun, D.; Nasrin, S.; Liles, M.R. Genome modifications and cloning using a conjugally transferable recombineering system. Biotechnol. Rep. 2015, 8, 24–35. [Google Scholar] [CrossRef]
- Turnbull, L.; Whitchurch, C.B. Motility assay: Twitching motility. Methods Mol. Biol. 2014, 1149, 73–86. [Google Scholar] [CrossRef] [PubMed]
- Serrano, I.; Verdial, C.; Tavares, L.; Oliveira, M. The virtuous Galleria mellonella model for scientific experimentation. Antibiotics 2023, 12, 505. [Google Scholar] [CrossRef]
- Schureck, M.A.; Meisner, J.; Hoffer, E.D.; Wang, D.; Onuoha, N.; Ei Cho, S.; Lollar, P.; Dunham, C.M. Structural basis of transcriptional regulation by the HigA antitoxin. Mol. Microbiol. 2019, 111, 1449–1462. [Google Scholar] [CrossRef]
- Overgaard, M.; Borch, J.; Jørgensen, M.G.; Gerdes, K. Messenger RNA interferase RelE controls relBE transcription by conditional cooperativity. Mol. Microbiol. 2008, 69, 841–857. [Google Scholar] [CrossRef] [PubMed]
- Tian, Q.B.; Ohnishi, M.; Tabuchi, A.; Terawaki, Y. A new plasmid-encoded proteic killer gene system: Cloning, sequencing, and analyzing hig locus of plasmid Rts1. Biochem. Biophys. Res. Commun. 1996, 220, 280–284. [Google Scholar] [CrossRef] [PubMed]
- Pandey, D.P.; Gerdes, K. Toxin-antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes. Nucleic Acids Res. 2005, 33, 966–976. [Google Scholar] [CrossRef]
- Yang, Y.; Mei, L.; Chen, J.; Chen, X.; Wang, Z.; Liu, L.; Yang, A. Legionella pneumophila-mediated host posttranslational modifications. J. Mol. Cell Biol. 2023, 15, mjad032. [Google Scholar] [CrossRef]
- Allard, N.; Neil, K.; Grenier, F.; Rodrigue, S. The type IV pilus of plasmid TP114 displays adhesins conferring conjugation specificity and is important for DNA transfer in the mouse gut microbiota. Microbiol. Spectr. 2022, 10, e0230321. [Google Scholar] [CrossRef]
- Carter, M.Q.; Chen, J.; Lory, S. The Pseudomonas aeruginosa pathogenicity island PAPI-1 is transferred via a novel type IV pilus. J. Bacteriol. 2010, 192, 3249–3258. [Google Scholar] [CrossRef]
- Ishiwa, A.; Komano, T. PilV adhesins of plasmid R64 thin pili specifically bind to the lipopolysaccharides of recipient cells. J. Mol. Biol. 2004, 343, 615–625. [Google Scholar] [CrossRef]
- Guijarro, J.A.; Cascales, D.; García-Torrico, A.I.; García-Domínguez, M.; Méndez, J. Temperature-dependent expression of virulence genes in fish-pathogenic bacteria. Front. Microbiol. 2015, 6, 700. [Google Scholar] [CrossRef] [PubMed]
- Mendez, J.; Cascales, D.; Garcia-Torrico, A.I.; Guijarro, J.A. Temperature-dependent gene expression in Yersinia ruckeri: Tracking specific genes by bioluminescence during in vivo colonization. Front. Microbiol. 2018, 9, 1098. [Google Scholar] [CrossRef]
- Wrobel, A.; Saragliadis, A.; Pérez-Ortega, J.; Sittman, C.; Göttig, S.; Liskiewicz, K.; Spence, M.H.; Schneider, K.; Leo, J.C.; Arenas, J.; et al. The inverse autotransporters of Yersinia ruckeri, YrInv and YrIlm, contribute to biofilm formation and virulence. Environ. Microbiol. 2020, 22, 2939–2955. [Google Scholar] [CrossRef] [PubMed]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Floras, F.; Mawere, C.; Singh, M.; Wootton, V.; Hamstead, L.; McVicker, G.; Leo, J.C. The Putative Virulence Plasmid pYR4 of the Fish Pathogen Yersinia ruckeri Is Conjugative and Stabilized by a HigBA Toxin–Antitoxin System. Biology 2024, 13, 652. https://doi.org/10.3390/biology13090652
Floras F, Mawere C, Singh M, Wootton V, Hamstead L, McVicker G, Leo JC. The Putative Virulence Plasmid pYR4 of the Fish Pathogen Yersinia ruckeri Is Conjugative and Stabilized by a HigBA Toxin–Antitoxin System. Biology. 2024; 13(9):652. https://doi.org/10.3390/biology13090652
Chicago/Turabian StyleFloras, Fisentzos, Chantell Mawere, Manvir Singh, Victoria Wootton, Luke Hamstead, Gareth McVicker, and Jack C. Leo. 2024. "The Putative Virulence Plasmid pYR4 of the Fish Pathogen Yersinia ruckeri Is Conjugative and Stabilized by a HigBA Toxin–Antitoxin System" Biology 13, no. 9: 652. https://doi.org/10.3390/biology13090652