title: Tn5 insertion in the tonB gene promoter affects iron-related phenotypes and increases extracellular siderophore levels in Gluconacetobacter diazotrophicus.
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 PAL5(T). 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 PAL5(T). tonB transcription was inhibited in the presence of Fe(3+) ions both in PAL5(T) and in Gdiaa31. In comparison with PAL5(T), 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.
authors.affiliation: 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.
authors.forename: Cleiton
authors.initials: C
authors.lastname: de Paula Soares
authors.forename: Elisete Pains
authors.initials: EP
authors.lastname: Rodrigues
authors.forename: Jéssica
authors.initials: J
authors.lastname: de Paula Ferreira
authors.forename: Jean Luiz
authors.initials: JL
authors.lastname: Simões Araújo
authors.forename: Luc Felicianus Marie
authors.initials: LF
authors.lastname: Rouws
authors.forename: José Ivo
authors.initials: JI
authors.lastname: Baldani
authors.forename: Marcia Soares
authors.initials: MS
authors.lastname: Vidal
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001692:Biological Transport / Q000235:genetics; D003470:Culture Media / Q000737:chemistry; D005816:Genetic Complementation Test; D041821:Gluconacetobacter / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics*; D016254:Mutagenesis, Insertional; D009154:Mutation; D009591:Nitrogenase / Q000235:genetics; D010641:Phenotype; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D017262:Siderophores / Q000032:analysis / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D008565:Membrane Proteins; D017262:Siderophores; C057616:tonB protein, Bacteria; D007501:Iron; D009591:Nitrogenase