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Tn5 insertion in the tonB gene promoter affects iron-related phenotypes and increases extracellular siderophore levels in Gluconacetobacter diazotrophicus

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Abstract

TonB-dependent receptors in concert with the TonB–ExbB–ExbD protein complex are responsible for the uptake of iron and substances such as vitamin B12 in several bacterial species. In this study, Tn5 mutagenesis of the sugarcane endophytic bacterium Gluconacetobacter diazotrophicus led to the isolation of a mutant with a single Tn5-insertion in the promoter region of a tonB gene ortholog. This mutant, named Gdiaa31, displayed a reduced growth rate and a lack of response to iron availability when compared to the wild-type strain PAL5T. Several efforts to generate null-mutants for the tonB gene by insertional mutagenesis were without success. RT-qPCR analysis demonstrated reduced transcription of tonB in Gdiaa31 when compared to PAL5T. tonB transcription was inhibited in the presence of Fe3+ ions both in PAL5T and in Gdiaa31. In comparison with PAL5T, Gdiaa31 also demonstrated decreased nitrogenase activity and biofilm formation capability, two iron-requiring physiological characteristics of G. diazotrophicus. Additionally, Gdiaa31 accumulated higher siderophore levels in culture supernatant. The genetic complementation of the Gdiaa31 strain with a plasmid that carried the tonB gene including its putative promoter region (pP tonB ) restored nitrogenase activity and siderophore accumulation phenotypes. These results indicate that the TonB complex has a role in iron/siderophore transport and may be essential in the physiology of G. diazotrophicus.

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Acknowledgments

This work was financially supported by Embrapa (Process No. 02.09.06.001.00) and CNPq/INCT-FBN (Process No. 573828/2008-3). The first author thanks CAPES for a scholarship and fellowship, respectively. The author JIB also thanks CNPq for the fellowship.

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Authors and Affiliations

  1. Programa de Pós-graduação em Biotecnologia Vegetal, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Bloco K, Ilha do Fundão, Rio de Janeiro, RJ, 21941-590, Brazil

    Cleiton de Paula Soares

  2. Biochemistry and Biotechnology Department, Universidade Estadual de Londrina, PR-445, Km 380, Campus Universitário, PO Box 6001, Londrina, PR, 86051-970, Brazil

    Elisete Pains Rodrigues

  3. Agronomy Department, Universidade Federal Rural do Rio de Janeiro, Rodovia BR 465, Km 07, Seropédica, RJ, 23890-000, Brazil

    Jéssica de Paula Ferreira

  4. Genetics and Biochemistry Laboratory, Embrapa Agrobiologia, Rodovia BR 465, Km 07, Seropédica, RJ, 23890-000, Brazil

    Jean Luiz Simões Araújo, Luc Felicianus Marie Rouws, José Ivo Baldani & Marcia Soares Vidal

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  1. Cleiton de Paula Soares
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  2. Elisete Pains Rodrigues
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  3. Jéssica de Paula Ferreira
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Correspondence to Marcia Soares Vidal.

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Communicated by Erko Stackebrandt.

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203_2014_1045_MOESM1_ESM.doc

Fig. S1 Strategy of genetic complementation of the mutant Gdiaa31. a) Schematic drawing representing the tonB gene and putative promoter region cloned in pGEM®-T easy. The single crossing-over event with the complementation plasmid pPtonB is represented in the top image. The resultant cis-complemented chromosomal locus is represented in the lower image. P: predict promoter; The site of transposon insertion (1,221 bp) in tonB regulatory sequence is indicated (▼). Black arrows show the primers used to amplify the tonB gene with its predicted promoter region. b) PCR analysis of DNA of PAL5T, the mutant Gdiaa31, and the complemented mutant Gdiaa31 (pP tonB ) (DOC 118 kb)

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de Paula Soares, C., Rodrigues, E.P., de Paula Ferreira, J. et al. Tn5 insertion in the tonB gene promoter affects iron-related phenotypes and increases extracellular siderophore levels in Gluconacetobacter diazotrophicus . Arch Microbiol 197, 223–233 (2015). https://doi.org/10.1007/s00203-014-1045-4

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