Folia Microbiologica

, Volume 63, Issue 6, pp 789–802 | Cite as

Isolation, characterization, genomic sequencing, and GFP-marked insertional mutagenesis of a high-performance nitrogen-fixing bacterium, Kosakonia radicincitans GXGL-4A and visualization of bacterial colonization on cucumber roots

  • Shuaixin Sun
  • Yunpeng ChenEmail author
  • Jiejie Cheng
  • Qiongjie Li
  • Zhenchuan Zhang
  • Zhengliang Lan
Original Article


A gram-negative bacterium GXGL-4A was originally isolated from maize roots. It displayed nitrogen-fixing (NF) ability under nitrogen-free culture condition, and had a significant promotion effect on cucumber growth in the pot inoculation test. The preliminary physiological and biochemical traits of GXGL-4A were characterized. Furthermore, a phylogenetic tree was constructed based on 16S ribosomal DNA (rDNA) sequences of genetically related species. To determine the taxonomic status of GXGL-4A and further utilize its nitrogen-fixing potential, genome sequence was obtained using PacBio RS II technology. The analyses of average nucleotide identity based on BLAST+ (ANIb) and correlation indexes of tetra-nucleotide signatures (Tetra) showed that the NF isolate GXGL-4A is closely related to the Kosakonia radicincitans type strain DSM 16656. Therefore, the isolate GXGL-4A was eventually classified into the species of Kosakonia radicincitans and designated K. radicincitans GXGL-4A. A high consistency in composition and gene arrangement of nitrogen-fixing gene cluster I (nif cluster I) was found between K. radicincitans GXGL-4A and other Kosakonia NF strains. The mutants tagged with green fluorescence protein (GFP) were obtained by transposon Tn5 mutagenesis, and then, the colonization of gfp-marked K. radicincitans GXGL-4A cells on cucumber seedling root were observed under fluorescence microscopy. The preferential sites of the labeled GXGL-4A cell population were the lateral root junctions, the differentiation zone, and the elongation zone. All these results should benefit for the deep exploration of nitrogen fixation mechanism of K. radicincitans GXGL-4A and will definitely facilitate the genetic modification process of this NF bacterium in sustainable agriculture.



This work has been supported by the National Basic Research Program of China (No. 2015CB755702) and the National Training Program of Innovation and Entrepreneurship for Undergraduates (No. IPP13125).


  1. Baldani J, Caruso L, Baldani VLD, Goi SR, Döbereiner J (1997) Recent advances in BNF with non-legume plants. Soil Biol Biochem 29(5–6):911–922. CrossRefGoogle Scholar
  2. Beneduzi A, Peres D, Vargas LK, Bodanese-Zanettini MH, Passaglia LMP (2008) Evaluation of genetic diversity and plant growth promoting activities of nitrogen-fixing bacilli isolated from rice fields in South Brazil. Appl Soil Ecol 39(3):311–320. CrossRefGoogle Scholar
  3. Berg G (2009) Plant–microbe interactions promoting plant growth and health: perspectives for controlled use of microorganisms in agriculture. Appl Microbiol Biotechnol 84:11–18. CrossRefPubMedGoogle Scholar
  4. Berger B, Brock AK, Ruppel S (2013) Nitrogen supply influences plant growth and transcriptional responses induced by Enterobacter radicincitans in Solanum lycopersicum. Plant Soil 370(1–2):641–652. CrossRefGoogle Scholar
  5. Berger B, Wiesner M, Brock AK, Schreiner M, Ruppel S (2015) K. Radicincitans, a beneficial bacteria that promotes radish growth under field conditions. Agron Sustain Dev 35:1521–1528. CrossRefGoogle Scholar
  6. Bergottinia VM, Filippidoua S, Junierb T, Johnsonc S, Chainc PS, Otegui MB, Zapata PD, Junier P (2015) Genome sequence of Kosakonia radicincitans strain YD4, a plant growth-promoting rhizobacterium isolated from yerba mate (Ilex paraguariensis St. hill.). Genome Announc 3(2):e00239–e00215. CrossRefGoogle Scholar
  7. Bertalan M, Albano R, de Pádua V, Rouws L, Rojas C, Hemerly A, Teixeira K, Schwab S, Araujo J, Oliveira A, França L, Magalhães V, Alquéres S, Cardoso A, Almeida W, Loureiro M, Nogueira E, Cidade D, Oliveira D, Simão T, Macedo J, Valadão A, Dreschsel M, Freitas F, Vidal M, Guedes H, Rodrigues E, Meneses C, Brioso P, Pozzer L, Figueiredo D, Montano H, Junior J, de Souza Filho G, Martin Quintana Flores V, Ferreira B, Branco A, Gonzalez P, Guillobel H, Lemos M, Seibel L, Macedo J, Alves-Ferreira M, Sachetto-Martins G, Coelho A, Santos E, Amaral G, Neves A, Pacheco A, Carvalho D, Lery L, Bisch P, Rössle SC, Ürményi T, Rael Pereira A, Silva R, Rondinelli E, von Krüger W, Martins O, Baldani J, Ferreira PCG (2009) Complete genome sequence of the sugarcane nitrogen-fixing endophyte Gluconacetobacter diazotrophicus Pal5. BMC Genomics 10:450. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Bhattacharjee RB, Singh A (2008) Use of nitrogen-fixing bacteria as biofertiliser for non-legumes: prospects and challenges. Appl Microbiol Biotechnol 80:199–209. CrossRefPubMedGoogle Scholar
  9. Caicedo JC, Villamizar S, Ferro MIT, Kupper KC, Ferro JA (2016) Bacteria from the citrus phylloplane can disrupt cell–cell signalling in Xanthomonas citri and reduce citrus canker disease severity. Plant Pathol 65:782–791. CrossRefGoogle Scholar
  10. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL (2009) BLAST+: architecture and applications. BMC Bioinformatics 10:421. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Cao Y, Zhang ZH, Ling N, Yuan YJ, Zheng XY, Shen B, Shen QR (2011) Bacillus subtilis SQR 9 can control fusarium wilt in cucumber by colonizing plant roots. Biol Fertil Soils 47:495–506. CrossRefGoogle Scholar
  12. Cavalcante VA, Dobereiner J (1988) A new acid-tolerant nitrogen-fixing bacterium associated with sugarcane. Plant Soil 108(1):23–31. CrossRefGoogle Scholar
  13. Chen MY, Zhu B, Lin L, Yang LT, Li YR, An QL (2014) Complete genome sequence of Kosakonia sacchari type strain SP1T. Stand Genomic Sci 9:9031311. CrossRefGoogle Scholar
  14. Compant S, Clément C, Sessitsch A (2010) Plant growth-promoting bacteria in the rhizo- and endosphere of plants: their role, colonization, mechanisms involved and prospects for utilization. Soil Biol Biochem 42(5):669–678. CrossRefGoogle Scholar
  15. Ditta G, Stanfield S, Corbin D, Helinski DR (1980) Broad host range DNA cloning system for gram-negative bacteria: construction of gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A 77:7347–7351. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791. CrossRefPubMedGoogle Scholar
  17. Ferrarini M, Moretto M, Ward JA, Surbanovski N, Stevanovic V, Giongo L, Viola R, Cavalieri D, Velasco R, Cestaro A, Sargent D (2013) An evaluation of the PacBio RS platform for sequencing and de novo assembly of a chloroplast genome. BMC Genomics 14:670. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Franche C, Lindström K, Elmerich C (2009) Nitrogen-fixing bacteria associated with leguminous and non-leguminous plants. Plant Soil 321(1–2):35–59. CrossRefGoogle Scholar
  19. Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM (2007) DNA-DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol 57:81–91. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Götz M, Gomes NCM, Dratwinski A, Costa R, Berg G, Peixoto R, Hagler LM, Smalla K (2006) Survival of gfp-tagged antagonistic bacteria in the rhizosphere of tomato plants and their effects on the indigenous bacterial community. FEMS Microbiol Ecol 56:207–218. CrossRefPubMedGoogle Scholar
  21. Gray KM, Pearson JP, Downie JA, Boboye BE, Greenberg EP (1996) Cell-to-cell signaling in the symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum: autoinduction of a stationary phase and rhizosphere-expressed genes. J Bacteriol 178(2):372–376. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Hallmann J, Hallmann AQ, Miller WG, Sikora RA, Lindow SE (2001) Endophytic colonization of plants by the biocontrol agent Rhizobium etli G12 in relation to Meloidogyne incognita infection. Phytopathology 91(4):415–422. CrossRefPubMedGoogle Scholar
  23. Hardy RWF, Burns RC, Holsten RD (1973) Applications of the acetylene-ethylene assay for measurement of nitrogen fixation. Soil Biol Biochem 5:47–81. CrossRefGoogle Scholar
  24. Iniguez AL, Dong YM, Triplett EW (2004) Nitrogen fixation in wheat provided by Klebsiella pneumoniae 342. Mol Plant Microbe Interact 17(10):1078–1085. CrossRefPubMedGoogle Scholar
  25. Kennedy IR, Choudhury ATM, Kecskés ML (2004) Non-symbiotic bacterial diazotrophs in crop-farming systems: can their potential for plant growth promotion be better exploited? Soil Biol Biochem 36(8):1229–1244. CrossRefGoogle Scholar
  26. Khammas KM, Ageron E, Grimont PAD, Kaiser P (1989) Azospirillum irakense sp. nov., a nitrogen-fixing bacterium associated with rice roots and rhizosphere soil. Res Microbiol 140(9):679–693. CrossRefPubMedGoogle Scholar
  27. Kim CW, Kecskés ML, Deaker RJ, Gilchrist K, New PB, Kennedy IR, Kim S, Sa TM (2005) Wheat root colonization and nitrogenase activity by Azospirillum isolates from crop plants in Korea. Can J Microbiol 51(11):948–956. CrossRefPubMedGoogle Scholar
  28. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874. CrossRefPubMedGoogle Scholar
  29. Li YY, Li SY, Chen MY, Peng GX, Tan ZY, An QL (2017) Complete genome sequence of Kosakonia oryzae type strain Ola 51T. Stand Genomic Sci 12:28. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Mehnaz S, Weselowski B, Lazarovits G (2007) Azospirillum canadense sp. nov., a nitrogen-fixing bacterium isolated from corn rhizosphere. Int J Syst Evol Microbiol 57:620–624. CrossRefPubMedGoogle Scholar
  31. Meneses CHSG, Rouws LFM, Simões-Araújo JL, Vidal MS, Baldani JI (2011) Exopolysaccharide production is required for biofilm formation and plant colonization by the nitrogen-fixing endophyte Gluconacetobacter diazotrophicus. Mol Plant-Microbe Interact 24(12):1448–1458. CrossRefPubMedGoogle Scholar
  32. Meng XF, Bertani I, Abbruscato P, Piffanelli P, Licastro D, Wang CH, Venturi V (2015) Draft genome sequence of rice endophyte-associated isolate Kosakonia oryzae KO348. Genome Announc 3(3):e00594–e00515. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Meyer J, Iida S, Arber W (1980) Does the insertion element IS1 transpose preferentially into A+T-rich DNA segments? Mol Gen Genet 178:471–473. CrossRefPubMedGoogle Scholar
  34. Nogales J, Campos R, BenAbdelkhalek H, Olivares J, Lluch C, Sanjuan J (2002) Rhizobium tropici genes involved in free-living salt tolerance are required for the establishment of efficient nitrogen-fixing symbiosis with Phaseolus vulgaris. Mol Plant Microbe Interact 15(3):225–232. CrossRefPubMedGoogle Scholar
  35. Ozturk A, Caglar O, Sahin F (2003) Yield response of wheat and barley to inoculation of plant growth promoting rhizobacteria at various levels of nitrogen fertilization. J Plant Nutr Soil Sci 166(2):262–266. CrossRefGoogle Scholar
  36. Ramos HJO, Roncato-Maccari LDB, Souza EM, Soares-Ramos JRL, Hungria M, Pedrosa FO (2002) Monitoring Azospirillum-wheat interactions using the gfp and gusA genes constitutively expressed from a new broad-host range vector. J Biotechnol 97(3):243–252. CrossRefPubMedGoogle Scholar
  37. Rediers H, Bonnecarrère V, Rainey PB, Hamonts K, Vanderleyden J, Mot RD (2003) Development and application of a dapB-based in vivo expression technology system to study colonization of rice by the endophytic nitrogen-fixing bacterium Pseudomonas stutzeri A15. Appl Environ Microbiol 69(11):6864–6874. CrossRefPubMedPubMedCentralGoogle Scholar
  38. Rejesus RM, Hornbaker RH (1999) Economic and environmental evaluation of alternative pollution-reducing nitrogen management practices in Central Illinois. Agric Ecosyst Environ 75:41–53. CrossRefGoogle Scholar
  39. Richter M, Rosselló-Móra R (2009) Shifting the genome gold standard for the prokaryotic species definition. Proc Natl Acad Sci U S A 106:19126–19131. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Richter M, Rosselló-Móra R, Glöckner FO, Peplies J (2015) JSpeciesWS: a web server for prokaryotic species circumscription based on pairwise genome comparison. Bioinformatics 32(6):929–931. CrossRefPubMedPubMedCentralGoogle Scholar
  41. Rouws LFM, Meneses CHSG, Guedes HV, Vidal MS, Baldani JI, Schwab S (2010) Monitoring the colonization of sugarcane and rice plants by the endophytic diazotrophic bacterium Gluconacetobacter diazotrophicus marked with gfp and gusA reporter genes. Lett Appl Microbiol 51(3):325–330. CrossRefPubMedGoogle Scholar
  42. Ruppel S, Rühlmann J, Merbach W (2006) Quantification and localization of bacteria in plant tissues using quantitative real-time PCR and online emission fingerprinting. Plant Soil 286(1–2):21–35. CrossRefGoogle Scholar
  43. Ruvkun GB, Sundaresan V, Ausubel FM (1982) Directed transposon Tn5 mutagenesis and complementation analysis of Rhizobium meliloti symbiotic nitrogen fixation genes. Cell 29(2):551–559. CrossRefPubMedGoogle Scholar
  44. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425. CrossRefPubMedPubMedCentralGoogle Scholar
  45. Shahbaz M, Zia B (2011) Does exogenous application of glycinebetaine through rooting medium alter rice (Oryza sativa L.) mineral nutrient status under saline conditions? J Appl Bot 84:54–60Google Scholar
  46. Shen XQ, Chen YP, Liu T, Hu XL, Gu ZF (2003) Development of a high-efficient transformation system of Bacillus pumilus strain DX01 to facilitate gene isolation via gfp-tagged insertional mutagenesis and visualize bacterial colonization of rice roots. Folia Microbiol 58:409–417. CrossRefGoogle Scholar
  47. Shinjo R, Uesaka K, Ihara K, Loshakova K, Mizuno Y, Yano K, Tanaka A (2016) Complete genome sequence of Kosakonia sacchari strain BO-1, an endophytic diazotroph isolated from a sweet potato. Genome Announc 4(5):e00868–e00816. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Steenhoudt O, Vanderleyden J (2000) Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects. FEMS Microbiol Rev 24(4):487–506. CrossRefPubMedGoogle Scholar
  49. Tamura K, Nei M, Kumar S (2004) Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci U S A 101:11030–11035. CrossRefPubMedPubMedCentralGoogle Scholar
  50. Taylor WI, Achanzar D (1972) Catalase test as an aid to the identification of Enterobacteriaceae. Appl Environ Microbiol 24(1):58–61Google Scholar
  51. Vanstockem M, Michiels K, Vanderleyden J, Gool APV (1987) Transposon mutagenesis of Azospirillum brasilense and Azospirillum lipoferum: physical analysis of Tn5 and Tn5-mob insertion mutants. Appl Environ Microbiol 53(2):410–415PubMedPubMedCentralGoogle Scholar
  52. Verma SC, Ladha JK, Tripathi AK (2001) Evaluation of plant growth promoting and colonization ability of endophytic diazotrophs from deep water rice. J Biotechnol 91(2–3):127–141. CrossRefPubMedGoogle Scholar
  53. Wei CY, Lin L, Luo LJ, Xing YX, Hu CJ, Yang LT, Li YR, An QL (2014) Endophytic nitrogen-fixing Klebsiella variicola strain DX120E promotes sugarcane growth. Biol Fertil Soils 50(4):657–666. CrossRefGoogle Scholar
  54. Witzel K, Gwinn-Giglio M, Nadendla S, Shefchek K, Ruppel S (2012) Genome sequence of Enterobacter radicincitans DSM16656T, a plant growth-promoting endophyte. J Bacteriol 194:5469. CrossRefPubMedPubMedCentralGoogle Scholar
  55. Yan YL, Yang J, Dou YT, Chen M, Ping SZ, Peng JP, Lu W, Zhang W, Yao Z, Li H, Liu W, He S, Geng L, Zhang X, Yang F, Yu H, Zhan Y, Li D, Lin Z, Wang Y, Elmerich C, Lin M, Jin Q (2008) Nitrogen fixation island and rhizosphere competence traits in the genome of root-associated pseudomonas stutzeri A1501. Proc Natl Acad Sci U S A 105:7564–7569. CrossRefPubMedPubMedCentralGoogle Scholar
  56. Zakria M, Ohsako A, Saeki Y, Yamamoto A, Akao S (2008) Colonization and growth promotion characteristics of Enterobacter sp. and Herbaspirillum sp. on Brassica oleracea. Soil Sci Plant Nutr 54(4):507–516. CrossRefGoogle Scholar

Copyright information

© Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2018

Authors and Affiliations

  • Shuaixin Sun
    • 1
  • Yunpeng Chen
    • 1
    Email author
  • Jiejie Cheng
    • 1
  • Qiongjie Li
    • 1
  • Zhenchuan Zhang
    • 1
  • Zhengliang Lan
    • 1
  1. 1.Key Laboratory of Urban Agriculture (South) of Ministry of Agriculture, School of Agriculture and BiologyShanghai Jiaotong UniversityShanghaiPeople’s Republic of China

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