Identification of Genes Involved in Antifungal Activity of Burkholderia seminalis Against Rhizoctonia solani Using Tn5 Transposon Mutation Method
Abstract
:1. Introduction
2. Results and Discussion
2.1. Construction of Tn5 Mutant Library
2.2. Location of Tn5 Inserted Sites
2.3. Functional Prediction of Antagonism-Related Genes
2.4. Verification by Constructing Deletion Mutants and Complements
2.5. Growth of Antagonism-Related Tn5 Transposon Mutants
2.6. Biofilm Formation
2.7. Motility
2.8. Tolerance to Hydrogen Peroxide (H2O2)
3. Materials and Methods
3.1. Bacterial Strains and Growth Conditions
3.2. Transposon Insertion of Tn5
3.3. Evaluation of Antagonistic Activity of Tn5 Mutants
3.4. Identifying the Location of Transposon Insertions
3.5. Generation of Deletion Mutants and Complemented Strains
3.6. Growth Measurement
3.7. Biofilm Formation Ability
3.8. Motility
3.9. Tolerance to H2O2
3.10. Statistical Analyses
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
BLAST | Basic local alignment search tool |
IR | Index of resistance |
PCR | Polymerase chain reaction |
PDA | Potato dextrose agar (medium) |
ShB | Rice sheath blight |
WT | Wild type |
References
- Singh, P.; Mazumdar, P.; Harikrishna, J.A.; Babu, S. Sheath blight of rice: A review and identification of priorities for future research. Planta 2019, 250, 1387–1407. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Dukare, A.S.; Paul, S.; Nambi, V.E.; Gupta, R.K.; Singh, R.; Sharma, K.; Vishwakarma, R.K. Exploitation of microbial antagonists for the control of postharvest diseases of fruits: A review. Crit. Rev. Food Sci. Nutr. 2019, 59, 1498–1513. [Google Scholar] [CrossRef] [PubMed]
- Agrawal, M.; Sunder, S. Effect of fungicides and non-conventional chemicals on Rhizoctonia solani AG-1 1A and sheath blight disease of rice. Plant Dis. Res. 2013, 28, 39–44. [Google Scholar]
- Luo, C.P.; Zhou, H.F.; Zou, J.C.; Wang, X.Y.; Zhang, R.S.; Xiang, Y.P.; Chen, Z.Y. Bacillomycin L and surfactin contribute synergistically to the phenotypic features of Bacillus subtilis 916 and the biocontrol of rice sheath blight induced by Rhizoctonia solani. Appl. Microbiol. Biotechnol. 2015, 99, 1897–1910. [Google Scholar] [CrossRef]
- Wang, Y.L.; Liu, S.Y.; Mao, X.Q.; Zhang, Z.; Jiang, H.; Chai, R.Y.; Qiu, H.P.; Wang, J.Y.; Du, X.F.; Li, B.; et al. Identification and characterization of rhizosphere fungal strain MF-91 antagonistic to rice blast and sheath blight pathogens. J. Appl. Microbiol. 2013, 114, 1480–1490. [Google Scholar] [CrossRef]
- Chen, L.H.; Zhang, J.; Shao, X.H.; Wang, S.S.; Miao, Q.S.; Mao, X.Y.; Zhai, Y.M.; She, D.L. Development and evaluation of Trichoderma asperellum preparation for control of sheath blight of rice (Oryza sativa L.). Biocontrol Sci. Technol. 2015, 25, 316–328. [Google Scholar] [CrossRef]
- Into, P.; Khunnamwong, P.; Jindamoragot, S.; Am-in, S.; Intanoo, W.; Limtong, S. Yeast associated with rice phylloplane and their contribution to control of rice sheath blight disease. Microorganisms 2020, 8, 21. [Google Scholar] [CrossRef] [Green Version]
- Li, B.; Liu, B.P.; Yu, R.R.; Lou, M.M.; Wang, Y.L.; Xie, G.L.; Li, H.Y.; Sun, G.C. Phenotypic and molecular characterization of rhizobacterium Burkholderia sp. strain R456 antagonistic to Rhizoctonia solani, sheath blight of rice. World J. Microbiol. Biotechnol. 2011, 27, 2305–2313. [Google Scholar] [CrossRef]
- Zhu, B.; Ibrahim, M.; Cui, Z.Q.; Xie, G.L.; Jin, G.L.; Kube, M.; Li, B.; Zhou, X.P. Multi-omics analysis of niche specificity provides new insights into ecological adaptation in bacteria. ISME J. 2016, 10, 2072–2075. [Google Scholar] [CrossRef] [Green Version]
- Vanlaere, E.; LiPuma, J.J.; Baldwin, A.; Henry, D.; Brandt, E.D.; Mahenthiralingam, E.; Speert, D.; Dowson, C.; Vandamme, P. Burkholderia latens sp. nov., Burkholderia diffusa sp. nov., Burkholderia arboris sp. nov., Burkholderia seminalis sp. nov. and Burkholderia metallica sp. nov., novel species within the Burkholderia cepacia complex. Int. J. Syst. Evol. Microbiol. 2008, 58, 1580–1590. [Google Scholar] [CrossRef] [Green Version]
- Deveau, A.; Bonito, G.; Uehling, J.; Paoletti, M.; Becker, M.; Bindschedler, S.; Hacquard, S.; Herve, V.; Labbe, J.; Lastovetsky, O.A.; et al. Bacterial-fungal interactions: Ecology, mechanisms and challenges. FEMS Microbiol. Rev. 2018, 42, 335–352. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Fu, F.F.; Xue, H.W. Coexpression analysis identifies rice starch regulator1, a rice AP2/EREBP family transcription factor, as a novel rice starch biosynthesis regulator. Plant Physiol. 2010, 154, 927–938. [Google Scholar] [CrossRef] [PubMed]
- Teh, A.H.T.; Lee, S.M.; Dykes, G.A. Identification of potential Campylobacter jejuni genes involved in biofilm formation by EZ-Tn5 Transposome mutagenesis. BMC Res. Notes 2017, 10, 182. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Veeranagouda, Y.; Husain, F.; Wexler, H.M. Transposon mutagenesis of the anaerobic commensal, Bacteroides fragilis, using the EZ::TN5 transposome. FEMS Microbiol. Lett. 2012, 333, 94–100. [Google Scholar] [CrossRef] [Green Version]
- Luo, J.Y.; Qiu, W.; Chen, L.; Anjum, S.I.; Yu, M.H.; Shan, C.L.; Ilyas, M.; Li, B.; Wang, Y.L.; Sun, G.C. Identification of pathogenicity-related genes in biofilm-defective Acidovorax citrulli by transposon Tn5 mutagenesis. Int. J. Mol. Sci. 2015, 16, 28050–28062. [Google Scholar] [CrossRef] [Green Version]
- Dawoud, T.M.; Jiang, T.S.; Mandal, R.K.; Ricke, S.C.; Kwon, Y.M. Improving the efficiency of transposon mutagenesis in Salmonella Enteritidis by overcoming host-restriction barriers. Mol. Biotechnol. 2014, 56, 1004–1010. [Google Scholar] [CrossRef]
- Shehata, H.R.; Ettinger, C.L.; Eisen, J.A.; Raizada, M.N. Genes required for the anti-fungal activity of a bacterial endophyte isolated from a corn landrace grown continuously by subsistence farmers since 1000 BC. Front. Microbiol. 2016, 7, 13. [Google Scholar] [CrossRef] [Green Version]
- Parker, C.T.; Kloser, A.W.; Schnaitman, C.A.; Stein, M.A.; Gottesman, S.; Gibson, B.W. Role of the rfag and rfap genes in determining the lipopolysaccharide core structure and cell-surface properities of Escherichia-coli K-12. J. Bacteriol. 1992, 174, 2525–2538. [Google Scholar] [CrossRef] [Green Version]
- Chen, K.C.; Ravichandran, A.; Guerrero, A.; Deng, P.; Baird, S.M.; Smith, L.; Lu, S.E. The Burkholderia contaminans MS14 ocfC gene encodes a xylosyltransferase for production of the antifungal occidiofungin. Appl. Environ. Microbiol. 2013, 79, 2899–2905. [Google Scholar] [CrossRef] [Green Version]
- Tagai, C.; Morita, S.; Shiraishi, T.; Miyaji, K.; Iwamuro, S. Antimicrobial properties of arginine- and lysine-rich histones and involvement of bacterial outer membrane protease T in their differential mode of actions. Peptides 2011, 32, 2003–2009. [Google Scholar] [CrossRef]
- David, M.W. Pseudomonas biocontrol agents of soilborne pathogens: Looking back over 30 years. Phytopathology 2007, 2, 250–256. [Google Scholar]
- Cameron, D.M.; Gregory, S.T.; Thompson, J.; Suh, M.J.; Limbach, P.A.; Dahlberg, A.E. Thermus thermophilus L11 methyltransferase, PrmA, is dispensable for growth and preferentially modifies free ribosomal protein L11 prior to ribosome assembly. J. Bacteriol. 2004, 186, 5819–5825. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Gupta, R.; Gobble, T.R.; Schuster, M. GidA posttranscriptionally regulates rhl quorum sensing in Pseudomonas aeruginosa. J. Bacteriol. 2009, 191, 5785–5792. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Xu, X.Q.; Pan, S.Q. An agrobacterium catalase is a virulence factor involved in tumorigenesis. Mol. Microbiol. 2000, 35, 407–414. [Google Scholar] [CrossRef]
- Mikani, A.; Etebarian, H.R.; Aminian, H. Changes in peroxidase and phenols activity in apple fruit inoculated with antagonistic Pseudomonas fluorescens isolates and Botrytis mali. Pak. J. Biol. Sci. 2011, 14, 854–861. [Google Scholar] [CrossRef]
- Shishodia, S.K.; Shankar, J. Proteomic analysis revealed ROS-mediated growth inhibition of Aspergillus terreus by shikonin. J. Proteom. 2020, 224, 12. [Google Scholar] [CrossRef]
- Zheng, C.L.; Nie, L.; Qian, L.; Wang, Z.L.; Liu, G.Z.; Liu, J.S. K30, H150, and H168 are essential residues for coordinating pyridoxal 5’-phosphate of o-acetylserine sulfhydrylase from Acidithiobacillus ferrooxidans. Curr. Microbiol. 2010, 60, 461–465. [Google Scholar] [CrossRef] [Green Version]
- Liu, J.; Sui, Y.; Wisniewski, M.; Droby, S.; Liu, Y.S. Review: Utilization of antagonistic yeasts to manage postharvest fungal diseases of fruit. Int. J. Food Microbiol. 2013, 167, 153–160. [Google Scholar] [CrossRef]
- Nakayama, T.; Zhang-Akiyama, Q.M. pqiABC and yebST, Putative mce operons of Escherichia coli, encode transport pathways and contribute to membrane integrity. J. Bacteriol. 2017, 199, 13. [Google Scholar] [CrossRef] [Green Version]
- Chavez-Ramirez, B.; Kerber-Diaz, J.C.; Acoltzi-Conde, M.C.; Ibarra, J.A.; Vasquez-Murrieta, M.S.; Estrada-de los Santos, P. Inhibition of Rhizoctonia solani RhCh-14 and Pythium ultimum PyFr-14 by Paenibacillus polymyxa NMA1017 and Burkholderia cenocepacia CACua-24: A proposal for biocontrol of phytopathogenic fungi. Microbiol. Res. 2020, 230, 10. [Google Scholar] [CrossRef]
- Coplin, D.L. Plasmids and their role in the evolution of plant pathogenic bacteria. Annu. Rev. Phytopathol. 1989, 27, 187–212. [Google Scholar] [CrossRef]
- Kaldhone, P.R.; Han, J.; Deck, J.; Khajanchi, B.; Nayak, R.; Foley, S.L.; Ricke, S.C. Evaluation of the genetics and functionality of plasmids in incompatibility group I1-positive Salmonella enterica. Foodborne Pathog. Dis. 2018, 15, 168–176. [Google Scholar] [CrossRef] [PubMed]
- Devi, S.M.; Halami, P.M. Conjugal transfer of bacteriocin plasmids from different genera of lactic acid bacteria into Enterococcus faecalis JH2-2. Ann. Microbiol. 2013, 63, 1611–1617. [Google Scholar] [CrossRef]
- Araujo, W.L.; Creason, A.L.; Mano, E.T.; Camargo-Neves, A.A.; Minami, S.N.; Chang, J.H.; Loper, J.E. Genome sequencing and transposon mutagenesis of Burkholderia seminalis TC3.4.2R3 identify genes contributing to suppression of orchid necrosis caused by B-gladioli. Mol. Plant Microbe Interact. 2016, 29, 435–446. [Google Scholar] [CrossRef] [Green Version]
- Kristich, C.J.; Nguyen, V.T.; Le, T.; Barnes, A.M.T.; Grindle, S.; Dunny, G.M. Development and use of an efficient system for random mariner transposon mutagenesis to identify novel genetic determinants of biofilm formation in the core Enterococcus faecalis genome. Appl. Environ. Microbiol. 2008, 74, 3377–3386. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Tala, L.; Fineberg, A.; Kukura, P.; Persat, A. Pseudomonas aeruginosa orchestrates twitching motility by sequential control of type IV pili movements. Nat. Microbiol. 2019, 4, 774–780. [Google Scholar] [CrossRef]
- Van Alst, N.E.; Picardo, K.F.; Iglewski, B.H.; Haidaris, C.G. Nitrate sensing and metabolism modulate motility, biofilm formation, and virulence in Pseudomonas aeruginosa. Infect. Immun. 2007, 75, 3780–3790. [Google Scholar] [CrossRef] [Green Version]
- Rajagopala, S.V.; Titz, B.; Goll, J.; Parrish, J.R.; Wohlbold, K.; McKevitt, M.T.; Palzkill, T.; Mori, H.; Finley, R.L.; Uetz, P. The protein network of bacterial motility. Mol. Syst. Biol. 2007, 3, 13. [Google Scholar] [CrossRef] [Green Version]
- Haiko, J.; Westerlund-Wikstrom, B. The role of the bacterial flagellum in adhesion and virulence. Biology 2013, 2, 1242–1267. [Google Scholar] [CrossRef] [Green Version]
- Spadaro, D.; Droby, S. Development of biocontrol products for postharvest diseases of fruit: The importance of elucidating the mechanisms of action of yeast antagonists. Trends Food Sci. Technol. 2016, 47, 39–49. [Google Scholar] [CrossRef]
- Sambrook, J.; Russell, D.W. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, USA, 2001; pp. 1803–12500. [Google Scholar]
- Balouiri, M.; Sadiki, M.; Ibnsouda, S.K. Methods for in vitro evaluating antimicrobial activity: A review. J. Pharm. Anal. 2016, 6, 71–79. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Liu, H.; Tian, W.X.; Ibrahim, M.; Li, B.; Zhang, G.Q.; Zhu, B.; Xie, G.L. Characterization of pilP, a gene required for twitching motility, pathogenicity, and biofilm formation of Acidovorax avenae subsp. avenae RS-1. Eur. J. Plant Pathol. 2012, 134, 551–560. [Google Scholar] [CrossRef]
- Ogunyemi, S.O.; Fang, Y.S.; Qiu, W.; Li, B.; Chen, J.; Yang, M.; Hong, X.X.; Luo, J.Y.; Wang, Y.L.; Sun, G.C. Role of type IV secretion system genes in virulence of rice bacterial brown stripe pathogen Acidovorax oryzae strain RS-2. Microb. Pathog. 2019, 126, 343–350. [Google Scholar] [CrossRef] [PubMed]
- Coenye, T.; Peeters, E.; Nelis, H.J. Biofilm formation by Propionibacterium acnes is associated with increased resistance to antimicrobial agents and increased production of putative virulence factors. Res. Microbiol. 2007, 158, 386–392. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Bahar, O.; Goffer, T.; Burdman, S. Type IV pili are required for virulence, twitching motility, and biofilm formation of Acidovorax avenae subsp. citrulli. Mol. Plant Microbe Interact. 2009, 22, 909–920. [Google Scholar] [CrossRef] [Green Version]
- Babaei-Bondarti, Z.; Shahpiri, A. A metallothionein type 2 from Avicennia marina binds to iron and mediates hydrogen peroxide balance by activation of enzyme catalase. Phytochemistry 2020, 176, 8. [Google Scholar] [CrossRef]
- Pan, X.W.; Sun, C.H.; Tang, M.; You, J.J.; Osire, T.; Zhao, Y.X.; Xu, M.J.; Zhang, X.; Shao, M.L.; Yang, S.T.; et al. LysR-type transcriptional regulator MetR controls prodigiosin production, methionine biosynthesis, cell Motility, H2O2 tolerance, heat tolerance, and exopolysaccharide synthesis in Serratia marcescens. Appl. Environ. Microbiol. 2020, 86, 18. [Google Scholar] [CrossRef]
Mutants | Locus_Tag | Enzyme Site | Sequence Length (bp) | Function Prediction |
---|---|---|---|---|
Tn5-30 | BsemR456_1725 | SalI | 861 | glycosyl transferase |
Tn5-45 | BsemR456_1417 | PaeI | 203 | multispecies: histone H1 |
Tn5-63 | BsemR456_2057 | SalI | 479 | nonribosomal peptide synthetase |
Tn5-145 | BsemR456_1423 | SalI | 55 | 50S ribosomal protein L11 methyltransferase |
Tn5-146 | BsemR456_6327 | SalI | 150 | Plasmid, membrane integrity-associated transporter subunit PqiC |
Tn5-158 | BsemR456_6210 | SalI | 452 | Plasmid, hypothetical protein |
Tn5-216 | BsemR456_1157 | SalI | 765 | tRNA uridine-5-carboxymethylaminomethyl (34) synthesis enzyme MnmG. |
Tn5-225 | BsemR456_2679 | PaeI | 33 | YeiH family putative sulfate export transporter |
Tn5-273 | BsemR456_1556 | SalI | 281 | catalase/peroxidase HPI |
Tn5-331 | BsemR456_315 | SalI | 1173 | sulfate adenylyltransferase subunit CysD |
Tn5-355 | BsemR456_6210 | SalI | 452 | Plasmid, hypothetical protein |
Bacterial Strains | Incubation Time (h) | |||
---|---|---|---|---|
6 | 12 | 18 | 24 | |
Wild type | 0.50 ± 0.03 ab | 0.90 ± 0.00 c | 1.12 ± 0.00 bc | 1.31 ± 0.00 ab |
Tn5-30 | 0.34 ± 0.00 d | 0.90 ± 0.08 c | 1.14 ± 0.02 b | 1.25 ± 0.04 b |
Tn5-45 | 0.53 ± 0.01 ab | 0.93 ± 0.05 bc | 1.09 ± 0.02 c | 1.27 ± 0.02 b |
Tn5-63 | 0.40 ± 0.02 c | 0.81 ± 0.03 d | 1.07 ± 0.02 c | 1.28 ± 0.01 b |
Tn5-145 | 0.56 ± 0.06 a | 1.06 ± 0.02 a | 1.19 ± 0.03 a | 1.36 ± 0.02 a |
Tn5-146 | 0.47 ± 0.08 b | 0.99 ± 0.04 ab | 1.12 ± 0.03 bc | 1.31 ± 0.00 ab |
Tn5-158 | 0.49 ± 0.03 b | 0.99 ± 0.03 b | 1.12 ± 0.04 bc | 1.28 ± 0.08 b |
Tn5-216 | 0.30 ± 0.01 d | 0.73 ± 0.03 e | 0.90 ± 0.03 e | 1.10 ± 0.06 c |
Tn5-225 | 0.47 ± 0.04 b | 0.98 ± 0.06 b | 1.05 ± 0.01 d | 1.04 ± 0.00 c |
Tn5-273 | 0.46 ± 0.03 bc | 0.95 ± 0.04 bc | 1.10 ± 0.02 c | 1.32 ± 0.03 ab |
Tn5-331 | 0.44 ± 0.01 bc | 0.93 ± 0.01 bc | 1.11 ± 0.03 bc | 1.28 ± 0.05 b |
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Zhang, M.; Wang, X.; Ahmed, T.; Liu, M.; Wu, Z.; Luo, J.; Tian, Y.; Jiang, H.; Wang, Y.; Sun, G.; et al. Identification of Genes Involved in Antifungal Activity of Burkholderia seminalis Against Rhizoctonia solani Using Tn5 Transposon Mutation Method. Pathogens 2020, 9, 797. https://doi.org/10.3390/pathogens9100797
Zhang M, Wang X, Ahmed T, Liu M, Wu Z, Luo J, Tian Y, Jiang H, Wang Y, Sun G, et al. Identification of Genes Involved in Antifungal Activity of Burkholderia seminalis Against Rhizoctonia solani Using Tn5 Transposon Mutation Method. Pathogens. 2020; 9(10):797. https://doi.org/10.3390/pathogens9100797
Chicago/Turabian StyleZhang, Muchen, Xiaoxuan Wang, Temoor Ahmed, Mengju Liu, Zhifeng Wu, Jinyan Luo, Ye Tian, Hubiao Jiang, Yanli Wang, Guochang Sun, and et al. 2020. "Identification of Genes Involved in Antifungal Activity of Burkholderia seminalis Against Rhizoctonia solani Using Tn5 Transposon Mutation Method" Pathogens 9, no. 10: 797. https://doi.org/10.3390/pathogens9100797
APA StyleZhang, M., Wang, X., Ahmed, T., Liu, M., Wu, Z., Luo, J., Tian, Y., Jiang, H., Wang, Y., Sun, G., & Li, B. (2020). Identification of Genes Involved in Antifungal Activity of Burkholderia seminalis Against Rhizoctonia solani Using Tn5 Transposon Mutation Method. Pathogens, 9(10), 797. https://doi.org/10.3390/pathogens9100797