Identification of some genes involved in pathogenicity of an Erwinia amylovora native strain by random mutagenesis method

Document Type : Research Article

Authors

Abstract

Erwinia amylovora is a gram-negative bacterium that causes fire blight, an important disease in pome fruit trees. The aim of this study was to generate a transposon mutant library using mini-Tn5 lacZ1, in an Erwinia amylovora native isolate for identification of some virulence associated genes in this strain. Among 1500 mutated colonies, two colonies which indicated less virulence on immature pear fruits were selected for further analysis. The DNA sequencing results of the two selected mutants showed a transposon insertion in an araC family transcriptional regulator gene and an insertion in nonribosomal peptide synthetase (NRPS). This study demonstrated that the araC transcriptional regulator and the NRPS genes are required for full virulence of E. amylovora. More studies are necessary to identify the role of these genes in physiology and virulence of E. amylovora.

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Anderson E.S., Paulley J.T. and Roop R.M. 2008. The AraC-like transcriptional regulator DhbR is required for maximum expression of the 2,3-Dihydroxybenzoic Acid biosynthesis genes in Brucella abortus in response to iron deprivation. Journal of Bacteriology 190:1338-1342.
Arrebola E., Cazorla M.F., Romero D., Perez-Garcia A. and de Vicente A. 2007. A nonribosomal peptide synthetase gene (mgoA) of Pseudomonas syringae pv. syringae is involved in Mangotoxin biosynthesis and is required for full virulence. Molecular Plant and Microbe Interaction 20:500-509.
Bellemann P. and Geider K. 1992.  Localization of transposon insertions in pathogenicity mutants of Erwinia amylovora and their biochemical characterization. Journal of General Microbiology 138:931-940.
Bereswill S., Jock S., Aldridge P., Jansen J.D. and Geider K. 1997. Molecular characterization of natural Erwinia amylovora strains deficient in levan synthesis. Physiology and Molecular Plant Pathology 51:215–225.
Bereswill S., Pahl A., Bellemann P., Zeller W. and Geider K. 1992. Sensitive and species-specific detection of Erwinia amylovoraby polymerase chain reaction analysis. Applied and Environmental Microbiology 58:3522-3526.
Berry M.C., McGhee G.C., Zhao Y. and Sundin G.W. 2009. Effect of a waaL mutation on lipopolysaccharide composition, oxidative stress survival, and virulence in Erwinia amylovora. FEMS Microbiology 291:80-87.
Brook S.J. and Russell D.W. 2002. Molecular cloning: a laboratory manual. Science press, Beijing.
Eastgate J.A. 2000. Erwinia amylovora: the molecular basis of fire blight disease. Molecular Plant Pathology 1:325–329.
Etchegaraya A., Silva-Stenicoa M.E., Moona D.H. and Tsaia S.M. 2004. In silico analysis of nonribosomal peptide synthetases of Xanthomonas axonopodis pv. citri: identification of putative siderophore and lipopeptide biosynthetic genes. Microbiological research 159:425-437.
Ford S. and Olson B.H. 1998. Methos for detecting genetically engineered microorganisms in the environment. Advance Microbe Ecology 10:45-79.
Frota C.C., Papavinasasundaram K.G., Davis E.O. and Colston M.J. 2004. The AraC family transcriptional regulator Rv1931c plays a role in the virulence of Mycobacterium tuberculosis. Infection and Immunity 72:5483-5486.
Gross M., Geier G., Rudolph K. and Geider K. 1992. Levan and levansucrase synthesized by the fire blight pathogen Erwinia amylovora. Physiology and Molecular Plant Pathology 40:371–381.
Gross D.C., Lichens-Park A. and Kole C. 2015. Genomics of plant-associated bacteria. Springer press 238 pp.
Kim J.F. and Beer S.V. 2001. Molecular basis of the Hrp pathogenicity of the fire blight pathogen Erwinia amylovora: a type III protein secretion system encoded in a pathogenicity island. Plant Pathology Journal 17:77–82.
Lalitha S. 2000. Primer premier 5. Biotech Software and Internet Report 1:270-272.
 
Layer A., Barbour E., Azhar E., El Sahabi A.A. and Qadri I. 2013. Transposable elements in Escherichia coli antimicrobial resistance. Advances in Bioscience and Biotechnology 4:415-423.
Leach J.E., White F.F., Rhoads M.L. and Leung H. 1990. A repetitive DNA sequence differentiates Xanthomonas campestris pv. oryzae from other pathovars of Xanthomonas campestris. MolecularPlantandMicrobeInteraction 3:238-246.
Lorenzo V.D., Herrero M., Jakubzik U. and Timmis K.M. 1990. Mini-Tn5 tronsposon derivatives for insertion mutagenesis promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. JournalofBacteriology 172:6568-6572.
Martiel J.L. 2002. Transposable elements and fitness of bacteria. Theoretical Population Biology 61:509-518.
Martin J.J.V. and Mohn W.W. 1999. An alternative inverse PCR (IPCR) method to amplify DNA sequences flanking Tn5 transposon insertions. Journal of Microbiological Methods 35:163-166.
McGinnis S. and Madden T.L. 2004. BLAST: at the core of a powerful and diverse set of sequence analysis tools. Nucleic Acids Research 32:20-25.
Mukherjee P.K., Buensanteai N., Moran-Diez M.E., Druzhinina I.S. and Kenerley C.M. 2012. Functional analysis of non-ribosomal peptide synthetases (NRPSs) in Trichoderma virens reveals a polyketide synthase (PKS)/NRPS hybrid enzyme involved in the induced systemic resistance response in maize. Microbiology 158:155-165.
Norelli J.L., Jones A.L. and Aldwinckle H.S. 2003. Fire blight management in the twenty-first century: using new technologies that enhance host resistance in apple. Plant Disease 87:756–765.
Ochman H., Gerber A.S. and Hartl D.L. 1988. Genetic applications of an inverse polymerase chain reaction. Genetics 120:621–625.
Oh C.S., Kim J.F. and Beer S.V. 2005. The Hrp pathogenicity island of Erwinia amylovora and identification of three novel genes required for systemic infection. Molecular Plant Pathology 6:125–138.
Oide S., Moeder W., Krasnoff S., Gibson D., Haas H., Yoshioka K. and Turgeona B.G. 2006. NPS6, encoding a nonribosomal peptide synthetase involved in siderophore-mediated iron metabolism, is a conserved virulence determinant of plant pathogenic Ascomycetes. The Plant Cell 18:2836-2853.
Ordax M., Marco-Noales E., Lopez M.M. and Biosca E.G. 2010. Exopolysaccharides favor the survival of Erwinia amylovora under copper stress through different strategies. Research in Microbiology 161:549–555.
Pique N., Galbis-Minana D., Merino S. and Tomas J.M. 2015. Virulence factors of Erwinia amylovora: a review. International Journal Molecular Sciences 16:12836-12854.
Pletzer D., Schweizer G. and Weingarta H. 2014. AraC/XylS family stress response regulators Rob, SoxS, PliA, and OpiA in the fire blight pathogen Erwinia amylovora. Journal of Bacteriology 17:3098-3110.
Ramos L.S., Lehman B.L., Peter K.A. and McNellisa W.T. 2014. Mutation of the Erwinia amylovora argD gene causes arginine auxotrophy, nonpathogenicity in apples, and reduced virulence in pears. Applied and Environmental Microbiology 80:6739-6749.
Ramos L.S., Lehman B.L., Sinn J.P., Pfeufer E.E., Halbrendt N.O. and Mcnellis T.W. 2013. The fire blight pathogen Erwinia amylovora requires the rpoN gene for pathogenicity in apple. Molecular Plant Pathology 14:838-843.
Sambrook J. and Russell D.W. 2002. Molecular cloning: a laboratory manual. 3rd., Science press, Beijing. 461-471.
Thomson S.V. 1986. The role of the stigma in fire blight infections. Phytopathology 76:476–482.
Van der Zwet T. and Keil H.L. 1979. Fire blight: a bacterial disease of rosaceous species. United States Department of Agriculture Handbook. 510 pp.
Wang H., Fewer D.P., Holm L., Rouhiainen L. and Sivonen K. 2014. Atlas ofnonribosomal peptide and polyketide biosynthetic pathways revealscommon occurrence of nonmodular enzymes. PNAS 111:9259–9264.
Yang J., Tauschek M. and Robins-Browne R.M. 2011. Control of bacterial virulence by AraC-like regulators that respond to chemical signals. Cell press 19:128-135.
Zhao Y., Wang D., Nakka S., Sundin G.W. and Korban S.S. 2009. Systems level analysis of two-component signal transduction systems in Erwinia amylovora: role in virulence, regulation of amylovoran biosynthesis and swarming motility. BMC Genomics 10:245. 1:16.
Zheng R., Feng X., Wei X., Pan X., Liu C., Song R., Jin Y., Bai F., Jin S., Wu W. and Cheng Z. 2018. PutA is required for virulence and regulated by PruR in Pseudomonas aeruginosa. Frontiers in Microbiology 9: 1-12.