AMARAL, A. M. do

BAPTISTA, J. C.

MACHADO, M. A.

TOLEDO, C. P.

English

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Transformation of Xanthomonas axonopodis pv. citri byElectroporationAlexandre M. do Amaral1,2, Cristiane P. Toledo2, Juliana C. Baptista2 & Marcos A. Machado21EMBRAPA Recursos Genéticos e Biotecnologia, aamaral@cenargen.embrapa.br; 2Centro APTA Citros “Sylvio Moreira”,Rod. Anhangüera, km 158, Cx. Postal 4, Cordeirópolis, SP, CEP 13490-970, fax (019) 3546-1399(Accepted for publication on 28/06/2004)Corresponding author: Alexandre Morais do AmaralMethods that facilitate the functional analysis of thegenome are essential for studies of the molecular interactionbetween microorganisms and the host plant. After thecomplete sequencing of various plant pathogenic bacteria,including the causal agent of the citrus (Citrus spp.) canker,Xanthomonas axonopodis pv. citri (Xac) (da Silva et al.,2002), protocols of such techniques have been even morenecessary.In general, the inactivation of a certain protein by thedisruption of its gene, i.e., the production of a mutant, is themain approach taken to initiate the study of its function(Dorsey et al., 2002; Serror et al., 2002). Taking intoconsideration that there will be no expression of the truncatedgene in the organism, it is possible to evaluate, at leastpartially, the consequences of a functional gene (protein) forthe survival of a pathogen and/or its capacity to colonize thehost plant. However, for such inactivation to take place, thefirst step is for a modified DNA harboring a fragment of thegene (“construct”) to enter the bacterial cell.Moreover, many reports have been published recentlywith the use of the technique to produce mutants of bacteria,including plant pathogens (Guilhabert et al., 2001; Monteiroet al., 2001; Serror et al., 2002; Brumbley et al., 2002).Although the causal agent of citrus canker is one of the mosteconomically important plant pathogens worldwide, there isno available literature on the electro transformation of thepathovar so far (Yang et al., 1991; Shaw & Khan, 1993; White& Gonzalez, 1995; Tung & Kuo, 1999). This indicates thatthe bacterium that causes citrus canker lacks either adescription of the methodology used for transformation byelectroporation or that the species shows a low level ofresponse to electroporation.DO AMARAL, A.M., TOLEDO, C.P., BAPTISTA, J.C. & MACHADO, M.A. Transformation of Xanthomonas axonopodispv. citri by electroporation. Fitopatologia Brasileira 30:292-294. 2005.ABSTRACTThis study describes the use of electroporation for transforming Xanthomonas axonopodis pv. citri (Xac), the causalagent of citrus (Citrus spp.) canker. It also evaluates the methodology used for this species under different electrical parameters.The bacterium used in the study (Xac 306) was the same strain used for recent complete sequencing of the organism. The use ofa plasmid (pUFR047, gentamycinr) is reported here to be able to replicate in cells of Xac. Following the preparation andresuspension of competent cells of Xac at a density of ~4 x 1010 cfu/ml, in 10% glycerol, and the addition of the replicativeplasmid, an electrical pulse was applied to each treatment. Selection of transformants showed a high efficiency of transformation(1.1 x 106 transformants/µg DNA), which indicates an effective, and inverse, combination between electrical resistance (50 Ω)and capacitance (50 µF) for this species, with an electrical field strength of 12.5 kV.cm-1 and 2.7-ms pulse duration. Besides thedescription of a method for electroporation of Xac 306, this study provides additional information for the use of the technique onstudies for production of mutants of this species.Additional key words: citrus canker, mutant, Citrus spp.RESUMOTransformação de Xanthomonas axonopodis pv. citri através de eletroporaçãoÉ descrito o uso da eletroporação para a transformação de Xanthomonas axonopodis pv. citri (Xac), a agente causaldo cancro cítrico, e a avaliação da metodologia para esta espécie sob diferentes parâmetros elétricos. A bactéria utilizadaneste estudo (Xac 306) foi a mesma isolado usado recentemente no seqüenciamento completo do organismo. Aqui, é docu-mentado o uso do plasmídeo pUFR047 (gentamicinar) capaz de replicar em células de Xac. Após a preparação e re-suspensãodas células competentes de Xac na densidade de ~4 x 1010 ufc/ml, em 10% glycerol, e a adição do plasmídeo replicativo, umpulso elétrico foi aplicado a cada tratamento. A seleção dos transformantes mostrou uma alta eficiência de transformação (1,1x 106 transformantes/µg DNA), o que indica uma efetiva e inversa combinação entre a resistência elétrica (50 Ω) e acapacitância (50 µF) para esta espécie, com uma descarga elétrica de 12.5 kV.cm-1 e duração de pulso de 2,7 ms. Além dadescrição de método de eletroporação do isolado Xac 306, este estudo fornece informação adicional para o uso da técnica emestudos para a produção de mutantes da espécie.Palavras-chave adicionais: cancro cítrico, mutante, Citrus spp.292 Fitopatol. bras. 30(3), maio - jun 2005293Fitopatol. bras. 30(3), maio - jun 2005Transformation of Xanthomonas axonopodis pv. citri by electroporationBased on the fact that many species within the genusXanthomonas are electro-competents (White & Gonzalez,1991; Mongkolsuk et al., 1996; Mongkolsuk et al., 1998), thebacterium Xac is supposedly able to be transformed byelectroporation. This, however, remains to be determinedthrough a reliable protocol.The present report describes a simple and reliableprotocol  for the transformation of Xac by using a methodthat comprises the preparation of competent cells, theelectrical pulse, and selection of transformants.Preparation of competent cellsA small amount of the bacterium Xac 306, the samestrain used for the complete genome sequencing (Da Silva etal., 2002), was collected with wire loop from a -70 °C stockculture (stored in 15% glycerol) and spread onto culture plateswith NYG medium (5 g of peptone, 3  g of yeast extract, and20  g of glycerol per liter), without antibiotics. The plateswere incubated during 48 h at 28 °C. Xac colonies werescraped from plates and transferred to a 500-ml tubecontaining 200 ml of NB medium (8 g of nutrient broth and5 g of NaCl per liter, pH 7.0) for growth under rotary shaking(140 rpm) at 28 °C during approximately 4 h.Following growth in liquid medium until OD600 = 0.6[approximately 106 colony-forming units (cfu) per ml], whichcorresponds to the midlog-growth phase, bacterial cells werekept on ice during 1 h. Then, the cells were harvested andwashed by centrifugation steps (4000 xg), for 10 min, at 4 °Cwith resuspension in 1 and 0.5 volumes of 10 % cold sterileglycerol. The last washing was performed by addition of 1ml of 10 % cold sterile glycerol and centrifugation at 13,000xg, during 2 min and at 4 °C. The supernatant was partiallyremoved to allow enough liquid for resuspension of thebacterial cells and a final concentration of 4 x 1010 cfu/ml.Finally, the resulting competent cells were held on ice untilthe transformation (1-2 h).Preparation of replicative plasmidThe replicative plasmid pUFR047 (De Feyter et al.,1993) used for this study is 8.6 kb in size, has the replicationorigin of plasmid Sa (IncW replicon), and carries genes forresistance to ampicillin and gentamycin. The plasmid wastransformed into Escherichia coli and purified by usingQIAGEN Plasmid Mini Kit (Qiagen, Valencia, USA).A 3-µl aliquot of the plasmid suspension (30 ng/µl)was mixed to 40 µl of the electro competent cells thentransferred to a pre-chilled, sterile electroporation chamber(BIO-RAD, Hercules, USA) with 2-mm gap.Electroporation and selectionA single high-voltage pulse was applied to eachtreatment, which was represented by the combination betweenresistance (50, 100, 200, and 600 Ω) and capacitance (50,25, 10, and 3 µF). The resulting pulse duration was theresponse of those two parameters. Each sample was submittedto the same electric field strength (12.5 kV.cm-1). Followingthe pulse, 1 ml of NBY medium [0.8% nutrient broth (Difco,Erembodegem, Belgium), 0.2% yeast extract, 0.2% K2HPO4,0.05% KH2PO4, 0.25% glucose] supplemented with 20 ml ofglucose (10%) and 1 ml of 1 M MgSO4 per liter, withoutantibiotics, was immediately added to the cells. Then, thesuspension was transferred to a tube and placed in a rotaryshaker (140 rpm), at 28 ºC for 1 h, to allow expression ofantibiotic resistance. After incubation, the cells were selectedon plates with Nutrient Agar (3 g beef extract, 5 g peptone, 8g sodium chloride and 15 g agar per liter) supplemented withgentamycin (5 µg/ml). Plates with Xac were placed at 28 ºCduring 48-72 h for selection of transformants. The assay wasreplicated three times.Time of pulse is a result of the combination betweenresistance and capacitance parameters applied to Xac cells(Table 1). Accordingly, there was a reasonable ratio betweenthe two parameters to allow an optimum rate of transformationand avoid arcing of samples, which is one of the main sourcesof failure in recovering transformed cells. In general, the bestresults for transformation of Xac 306 were obtained usinglow levels of resistance (50 Ω) and capacitance (50 µF), whosevalues are quite different from those used for maximumtransformation efficiency of Xylella fastidiosa Wells (4 Ω and300 µF) (Guilhabert et al., 2001), although both experimentshad different objectives. In general, there was an average offrequency (transformants/survivors) of nearly 2.5 x 10-6.It is noteworthy that both, the species and the strain ofmost bacteria, influence over their capacity of beingtransformed (Guilhabert et al., 2001; Brumbley et al., 2002).For instance, X. fastidiosa, the causal agent of citrusvariegated chlorosis (CVC), shows a considerable variationamong isolates for transformation by electroporation. Actually,some important strains, such as the strain used for thecomplete genome sequencing, have shown no competencefor transformation by electroporation so far. Other strains ofthe same bacterium, however, are electro-transformable(Monteiro et al., 2001).In fact, in Xanthomonas spp., a wide range of responsesto electroporation is expected according to the strain (White& Gonzalez, 1991), which indicates that the isolate has to betaken into consideration when use of the transformationtechnique is intended.TABLE 1 - Electroporation parameters used for transformation ofcompetent cells of Xanthomonas axonopodis pv. citri with thereplicative plasmid pUFR0471Each value of transformation efficiency corresponds to the highest value foundin each treatment.294 Fitopatol. bras. 30(3), maio - jun 2005A.M. Amaral et al.The intermediate efficiency identified in Xac fortransformation when compared to other xanthomonads maybe a result of various aspects of its biology, besides the strainper se. For instance, the high content of polysaccharidespresent in cells, which is related to the species as well as tothe growth phase, is a critical component for the success ofelectrical shock (Murooka et al., 1987). Other aspects to betaken into account for the rate of transformation of Xac arethe preparation of competent cells and the recovery oftransformants. Although they are obviously variable fordifferent species of xanthomonads, it is an open question ifmodifications in the protocols used in this study may somehowoptimize the technique. In future assays, more isolates couldbe added in studies that aim to verify optimum conditions forthis same species.However, as a first approach for the transformation ofXac 306 by electroporation, the methodology and theparameters used in this study show that this technique canserve in studies that emphasize the genetics and the mutationalanalysis of this bacterium. Furthermore, this tool can help inusing the enormous amount of data from the complete genomeof the causal agent of citrus canker.ACKNOWLEDGEMENTSWe wish to thank Ricardo Harakava, on behalf of DeanW. Gabriel, for providing the replicative plasmid pUFR047.LITERATURE CITEDBRUMBLEY, S.M., PETRASOVITS, L.A., BIRCH, R.G. &TAYLOR, P.W.J. Transformation and transposon mutagenesis ofLeifsonia xyli subsp. xyli, causal organism of ratoon stuntingdisease of sugarcane. Molecular Plant-Microbe Interaction 15:262-268. 2002.Da SILVA, A.C.R et al. Comparison of the genomes of twoXanthomonas pathogens with differing host specificities. Nature417:459-463. 2002.De FEYTER, R., YAN, Y. & GABRIEL, D.W. Gene for genesinteractions between cotton R genes and Xanthomonas campestrispv. malvacearum avr genes. Molecular Plant-Microbe Interaction6:225-237. 1993.DORSEY, C.W., TOMARAS, A.P. & ACTIS, L.A. Genetic andphenotypic analysis of Acinetobacter baumannii  insertionderivatives generated with a transposome system. Applied andEnvironmental Microbiology 68:6353-6360. 2002.GUILHABERT, M.R., HOFFMAN, L.M., MILLS, D.A. &KIRKPATRICK, B.C. Transposon mutagenesis of Xylella fastidiosaby electroporation of Tn5 synaptic complexes Molecular Plant-Microbe Interaction 14:701-706. 2001.MONGKOLSUK, S., LOPRASERT, S., VATTANAVIBOON, P.,CHANVANICHAYACHAI, C., CHAMNONGPOL, S. &SUPSAMRAN, N. Heterologous growth phase- and temperature-dependent expression and H2O2 toxicity protection of a superoxide-inducible monofunctional catalase gene from Xanthomonas oryzaepv. oryzae. Journal of Bacteriology 178:3578-3584. 1996.MONGKOLSUK, S., SUKCHAWALIT, R., LOPRASERT, S.,PRAITUAN, W. & UPAICHIT, A. Construction and physiologicalanalysis of a Xanthomonas mutant to examine the role of the oxyRgene in oxidant-induced protection against peroxide killing. Journalof Bacteriology 180:3988-3991. 1998.MONTEIRO, P.B., TEIXEIRA, D.C., PALMA, R. R., GARNIER,M., BOVÉ, J.M. & RENAUDIN, J. Stable transformation of theXylella fastidiosa  citrus variegated chlorosis strain with oriCplasmids. Applied and Environmental Microbiology 67:2263-2269.2001.MUROOKA, Y., IWAMOTO, H., HAMAMOTO, A. &YAMAUCHI, T. Efficient transformation of phytopathogenic strainsof Xanthomonas species. Journal of Bacteriology 169:4406-4409.1987.SERROR, P., SASAKI, T., DUSKO EHRLICH, S. & MAGUIN,E. Electrotransformation of Lactobacillus delbrueckii subsp.bulgaricus and L. delbrueckii subsp. lactis with various plasmids.Applied and Environmental Microbiology 68:46-52. 2002.SHAW, J.J. & KHAN, I. Efficient transposon mutagenesis ofXanthomonas campestris  pv.  campestris  by high-voltageelectroporation. Biotechniques 14:556-558. 1993.TUNG, S.Y. & KUO, T.T. Requirement for phosphoglucoseisomerase of Xanthomonas campestris in pathogenesis of citruscanker. Applied and Environmental Microbiology 65:5564-5570.1999.WHITE, T.J. & GONZALEZ, C.F. Application of electroporationfor efficient transformation of Xanthomonas campestris pv. oryzae.Phytopathology 81:521-524. 1991.WHITE, T.J. & GONZALEZ, C.F. Electroporation ofXanthomonas. Methods in Molecular Biology 47:135-141. 1995.YANG, M.K., SU, W.C. & KUO, T.T. Highly effecient transfectionof Xanthomonas campestris by electroporation. Botanical Bulletinof Academia Sinica 32:197-203. 1991.03103

Transformation of Xanthomonas axonopodis pv. citri by electroporation.

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Transformation of Xanthomonas axonopodis pv. citri by electroporation.

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