Strategies to Investigate Membrane Damage, Nucleoid Condensation, and RNase Activity of Bacterial Toxin–Antitoxin Systems
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Bacterial Strains and Expression Vectors
2.3. Cloning, Transformation of E. coli Cells, and Culture Conditions
2.4. Instruments
2.5. Software
3. Results and Discussion
3.1. Strains and Vectors Suitable for Toxin Activity Studies
3.2. Production of Purified Toxins
3.3. Fluorescence Imaging to Monitor RNase Activity
3.4. Image Processing
3.5. Fluorescence Imaging to Monitor Membrane Permeabilization
3.6. Image Processing
3.7. DAPI/EB Staining to Assess Membrane Integrity in Bulk Culture Samples
3.8. Atomic Force Microscopy Imaging to Reveal Membrane Damages
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
- Yamaguchi, Y.; Park, J.H.; Inouye, M. Toxin-antitoxin systems in bacteria and archaea. Annu. Rev. Genet. 2011, 45, 61–79. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Christensen-Dalsgaard, M.; Jorgensen, M.G.; Gerdes, K. Three new RelE-homologous mRNA interferases of Escherichia coli differentially induced by environmental stresses. Mol. Microbiol. 2010, 75, 333–348. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Levante, A.; Folli, C.; Montanini, B.; Ferrari, A.; Neviani, E.; Lazzi, C. Expression of DinJ-YafQ System of Lactobacillus casei Group Strains in Response to Food Processing Stresses. Microorganisms 2019, 7, 438. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Fraikin, N.; Goormaghtigh, F.; Van Melderen, L. Type II Toxin-Antitoxin Systems: Evolution and Revolutions. J. Bacteriol. 2020, 202, e00763-19. [Google Scholar] [CrossRef] [Green Version]
- LeRoux, M.; Culviner, P.H.; Liu, Y.J.; Littlehale, M.L.; Laub, M.T. Stress Can Induce Transcription of Toxin-Antitoxin Systems without Activating Toxin. Mol. Cell 2020, 79, 280–292.e288. [Google Scholar] [CrossRef]
- Brielle, R.; Pinel-Marie, M.L.; Felden, B. Linking bacterial type I toxins with their actions. Curr. Opin. Microbiol. 2016, 30, 114–121. [Google Scholar] [CrossRef]
- Song, S.; Wood, T.K. Toxin/Antitoxin System Paradigms: Toxins Bound to Antitoxins Are Not Likely Activated by Preferential Antitoxin Degradation. Adv. Biosyst. 2020, 4, e1900290. [Google Scholar] [CrossRef]
- Guo, Y.; Quiroga, C.; Chen, Q.; McAnulty, M.J.; Benedik, M.J.; Wood, T.K.; Wang, X. RalR (a DNase) and RalA (a small RNA) form a type I toxin-antitoxin system in Escherichia coli. Nucleic Acids Res. 2014, 42, 6448–6462. [Google Scholar] [CrossRef] [Green Version]
- Kawano, M.; Aravind, L.; Storz, G. An antisense RNA controls synthesis of an SOS-induced toxin evolved from an antitoxin. Mol. Microbiol. 2007, 64, 738–754. [Google Scholar] [CrossRef] [Green Version]
- Patel, S.; Weaver, K.E. Addiction toxin Fst has unique effects on chromosome segregation and cell division in Enterococcus faecalis and Bacillus subtilis. J. Bacteriol. 2006, 188, 5374–5384. [Google Scholar] [CrossRef] [Green Version]
- 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] [Green Version]
- Wang, X.; Lord, D.M.; Cheng, H.Y.; Osbourne, D.O.; Hong, S.H.; Sanchez-Torres, V.; Quiroga, C.; Zheng, K.; Herrmann, T.; Peti, W.; et al. A new type V toxin-antitoxin system where mRNA for toxin GhoT is cleaved by antitoxin GhoS. Nat. Chem. Biol. 2012, 8, 855–861. [Google Scholar] [CrossRef] [Green Version]
- Aakre, C.D.; Phung, T.N.; Huang, D.; Laub, M.T. A bacterial toxin inhibits DNA replication elongation through a direct interaction with the beta sliding clamp. Mol. Cell 2013, 52, 617–628. [Google Scholar] [CrossRef] [Green Version]
- Wang, X.; Yao, J.; Sun, Y.C.; Wood, T.K. Type VII Toxin/Antitoxin Classification System for Antitoxins that Enzymatically Neutralize Toxins. Trends Microbiol. 2021, 29, 388–393. [Google Scholar] [CrossRef]
- Choi, J.S.; Kim, W.; Suk, S.; Park, H.; Bak, G.; Yoon, J.; Lee, Y. The small RNA, SdsR, acts as a novel type of toxin in Escherichia coli. RNA Biol. 2018, 15, 1319–1335. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Fozo, E.M.; Makarova, K.S.; Shabalina, S.A.; Yutin, N.; Koonin, E.V.; Storz, G. Abundance of type I toxin-antitoxin systems in bacteria: Searches for new candidates and discovery of novel families. Nucleic Acids Res. 2010, 38, 3743–3759. [Google Scholar] [CrossRef]
- Ferrari, A.; Maggi, S.; Montanini, B.; Levante, A.; Lazzi, C.; Yamaguchi, Y.; Rivetti, C.; Folli, C. Identification and first characterization of DinJ-YafQ toxin-antitoxin systems in Lactobacillus species of biotechnological interest. Sci. Rep. 2019, 9, 7645. [Google Scholar] [CrossRef] [PubMed]
- Folli, C.; Levante, A.; Percudani, R.; Amidani, D.; Bottazzi, S.; Ferrari, A.; Rivetti, C.; Neviani, E.; Lazzi, C. Toward the identification of a type I toxin-antitoxin system in the plasmid DNA of dairy Lactobacillus rhamnosus. Sci. Rep. 2017, 7, 12051. [Google Scholar] [CrossRef] [Green Version]
- Levante, A.; Lazzi, C.; Vatsellas, G.; Chatzopoulos, D.; Dionellis, V.S.; Makrythanasis, P.; Neviani, E.; Folli, C. Genome Sequencing of five Lacticaseibacillus Strains and Analysis of Type I and II Toxin-Antitoxin System Distribution. Microorganisms 2021, 9, 648. [Google Scholar] [CrossRef] [PubMed]
- Maggi, S.; Yabre, K.; Ferrari, A.; Lazzi, C.; Kawano, M.; Rivetti, C.; Folli, C. Functional characterization of the type I toxin Lpt from Lactobacillus rhamnosus by fluorescence and atomic force microscopy. Sci. Rep. 2019, 9, 15208. [Google Scholar] [CrossRef] [PubMed]
- Luo, X.; Lin, J.; Yan, J.; Kuang, X.; Su, H.; Lin, W.; Luo, L. Characterization of DinJ-YafQ toxin-antitoxin module in Tetragenococcus halophilus: Activity, interplay, and evolution. Appl. Microbiol. Biotechnol. 2021, 105, 3659–3672. [Google Scholar] [CrossRef] [PubMed]
- Novagen. pET System Manual; Novagen: New York, NY, USA, 1999. [Google Scholar]
- Moffatt, B.A.; Studier, F.W. T7 lysozyme inhibits transcription by T7 RNA polymerase. Cell 1987, 49, 221–227. [Google Scholar] [CrossRef]
- Schlegel, S.; Genevaux, P.; de Gier, J.W. De-convoluting the Genetic Adaptations of E. coli C41(DE3) in Real Time Reveals How Alleviating Protein Production Stress Improves Yields. Cell Rep. 2015, 10, 1758–1766. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Khan, S.R.; Gaines, J.; Roop, R.M.; Farrand, S.K. Broad-host-range expression vectors with tightly regulated promoters and their use to examine the influence of TraR and TraM expression on Ti plasmid quorum sensing. Appl. Environ. Microbiol. 2008, 74, 5053–5062. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Fernandez-Garcia, L.; Kim, J.S.; Tomas, M.; Wood, T.K. Toxins of toxin/antitoxin systems are inactivated primarily through promoter mutations. J. Appl. Microbiol. 2019, 127, 1859–1868. [Google Scholar] [CrossRef]
- Maehigashi, T.; Ruangprasert, A.; Miles, S.J.; Dunham, C.M. Molecular basis of ribosome recognition and mRNA hydrolysis by the E. coli YafQ toxin. Nucleic Acids Res. 2015, 43, 8002–8012. [Google Scholar] [CrossRef] [Green Version]
- Ruangprasert, A.; Maehigashi, T.; Miles, S.J.; Giridharan, N.; Liu, J.X.; Dunham, C.M. Mechanisms of toxin inhibition and transcriptional repression by Escherichia coli DinJ-YafQ. J. Biol. Chem. 2014, 289, 20559–20569. [Google Scholar] [CrossRef] [Green Version]
- Jin, C.; Kang, S.-M.; Kim, D.-H.; Lee, B.-J. Structural and functional analysis of the Klebsiella pneumoniae MazEF toxin-antitoxin system. IUCrJ 2021, 8, 362–371. [Google Scholar] [CrossRef]
- Liang, Y.; Gao, Z.; Wang, F.; Zhang, Y.; Dong, Y.; Liu, Q. Structural and functional characterization of Escherichia coli toxin-antitoxin complex DinJ-YafQ. J. Biol. Chem. 2014, 289, 21191–21202. [Google Scholar] [CrossRef] [Green Version]
- Armalyte, J.; Jurenaite, M.; Beinoraviciute, G.; Teiserskas, J.; Suziedeliene, E. Characterization of Escherichia coli dinJ-yafQ toxin-antitoxin system using insights from mutagenesis data. J. Bacteriol. 2012, 194, 1523–1532. [Google Scholar] [CrossRef] [Green Version]
- Zhang, S.-P.; Wang, Q.; Quan, S.-W.; Yu, X.-Q.; Wang, Y.; Guo, D.-D.; Peng, L.; Feng, H.-Y.; He, Y.-X. Type II toxin–antitoxin system in bacteria: Activation, function, and mode of action. Biophys. Rep. 2020, 6, 68–79. [Google Scholar] [CrossRef]
- Sugimoto, S.; Arita-Morioka, K.; Mizunoe, Y.; Yamanaka, K.; Ogura, T. Thioflavin T as a fluorescence probe for monitoring RNA metabolism at molecular and cellular levels. Nucleic Acids Res. 2015, 43, e92. [Google Scholar] [CrossRef] [Green Version]
- Berney, M.; Hammes, F.; Bosshard, F.; Weilenmann, H.U.; Egli, T. Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight Kit in combination with flow cytometry. Appl. Environ. Microbiol. 2007, 73, 3283–3290. [Google Scholar] [CrossRef] [Green Version]
- Jernaes, M.W.; Steen, H.B. Staining of Escherichia coli for flow cytometry: Influx and efflux of ethidium bromide. Cytometry 1994, 17, 302–309. [Google Scholar] [CrossRef]
- Herrera, G.; Martinez, A.; Blanco, M.; O’Connor, J.E. Assessment of Escherichia coli B with enhanced permeability to fluorochromes for flow cytometric assays of bacterial cell function. Cytometry 2002, 49, 62–69. [Google Scholar] [CrossRef]
- Meincken, M.; Holroyd, D.L.; Rautenbach, M. Atomic force microscopy study of the effect of antimicrobial peptides on the cell envelope of Escherichia coli. Antimicrob. Agents Chemother. 2005, 49, 4085–4092. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Rakowska, P.D.; Jiang, H.; Ray, S.; Pyne, A.; Lamarre, B.; Carr, M.; Judge, P.J.; Ravi, J.; Gerling, U.I.; Koksch, B.; et al. Nanoscale imaging reveals laterally expanding antimicrobial pores in lipid bilayers. Proc. Natl. Acad. Sci. USA 2013, 110, 8918–8923. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Mularski, A.; Wilksch, J.J.; Hanssen, E.; Strugnell, R.A.; Separovic, F. Atomic force microscopy of bacteria reveals the mechanobiology of pore forming peptide action. Biochim. Biophys. Acta 2016, 1858, 1091–1098. [Google Scholar] [CrossRef]
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2021 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
Maggi, S.; Ferrari, A.; Yabre, K.; Bonini, A.A.; Rivetti, C.; Folli, C. Strategies to Investigate Membrane Damage, Nucleoid Condensation, and RNase Activity of Bacterial Toxin–Antitoxin Systems. Methods Protoc. 2021, 4, 71. https://doi.org/10.3390/mps4040071
Maggi S, Ferrari A, Yabre K, Bonini AA, Rivetti C, Folli C. Strategies to Investigate Membrane Damage, Nucleoid Condensation, and RNase Activity of Bacterial Toxin–Antitoxin Systems. Methods and Protocols. 2021; 4(4):71. https://doi.org/10.3390/mps4040071
Chicago/Turabian StyleMaggi, Stefano, Alberto Ferrari, Korotoum Yabre, Aleksandra Anna Bonini, Claudio Rivetti, and Claudia Folli. 2021. "Strategies to Investigate Membrane Damage, Nucleoid Condensation, and RNase Activity of Bacterial Toxin–Antitoxin Systems" Methods and Protocols 4, no. 4: 71. https://doi.org/10.3390/mps4040071