Identification of Genes Involved in Indole-3-Acetic Acid Biosynthesis by Gluconacetobacter diazotrophicus PAL5 Strain Using Transposon Mutagenesis.

Elisete P Rodrigues,Cleiton De Paula Soares,Patrícia G Galvão,Luc F M Rouws,André L M De Oliveira,Márcia S Vidal,José I Baldani,Eddie L Imada,Jean L Simões-Araújo

doi:10.3389/fmicb.2016.01572

Abstract

Gluconacetobacter diazotrophicus is a beneficial nitrogen-fixing endophyte found in association with sugarcane plants and other important crops. Beneficial effects of G. diazotrophicus on sugarcane growth and productivity have been attributed to biological nitrogen fixation process and production of phytohormones especially indole-3-acetic acid (IAA); however, information about the biosynthesis and function of IAA in G. diazotrophicus is still scarce. Therefore, the aim of this work was to identify genes and pathways involved in IAA biosynthesis in this bacterium. In our study, the screening of two independent Tn5 mutant libraries of PAL5T strain using the Salkowski colorimetric assay revealed two mutants (Gdiaa34 and Gdiaa01), which exhibited 95% less indolic compounds than the parental strain when grown in LGIP medium supplemented with L-tryptophan. HPLC chromatograms of the wild-type strain revealed the presence of IAA and of the biosynthetic intermediates indole-3-pyruvic acid (IPyA) and indole-3-lactate (ILA). In contrast, the HPLC profiles of both mutants showed no IAA but only a large peak of non-metabolized tryptophan and low levels of IPyA and ILA were detected. Molecular characterization revealed that Gdiaa01 and Gdiaa34 mutants had unique Tn5 insertions at different sites within the GDI2456 open read frame, which is predicted to encode a L-amino acid oxidase (LAAO). GDI2456 (lao gene) forms a cluster with GDI2455 and GDI2454 ORFs, which are predicted to encode a cytochrome C and an RidA protein, respectively. RT-qPCR showed that transcript levels of lao. cccA, and ridA genes were reduced in the Gdiaa01 as compared to PAL5T. In addition, rice plants inoculated with Gdiaa01 showed significantly smaller root development (length, surface area, number of forks and tips) than those plants inoculated with PAL5T. In conclusion, our study demonstrated that G. diazotrophicus PAL5T produces IAA via the IPyA pathway in cultures supplemented with tryptophan and provides evidence for the involvement of an L-amino acid oxidase gene cluster in the biosynthesis of IAA. Furthermore, we showed that the mutant strains with reduction in IAA biosynthesis ability, in consequence of the lower transcription levels of genes of the lao cluster, had remarkable effects on development of rice roots.

Highlights

Among the phenotypes altered in the indolic compound synthesis, two mutants exhibiting strong indole-3-acetic acid (IAA)-deficient phenotypes were isolated: mutant Gdiaa34 obtained from the first round of mutagenesis and Gdiaa01 from the second experiment
Gdiaa01 and Gdiaa34 mutants were grown in LGIP medium with L-tryptophan and growth and indolic compounds production was monitored until the stationary phase
The indolic compounds production by Gdiaa01 and Gdiaa34 mutants reached, respectively, about 0.64 and 0.74 μg.mL−1 after 12 h, with maximum levels achieved after 48 h with values up to 3.8 and 3.4 μg.mL−1, respectively, which corresponds to a reduction of approximately 96% as compared to wildtype

Summary

Introduction

Gluconacetobacter diazotrophicus is a nitrogen-fixing endophytic bacterium found colonizing the interior of roots and stems of sugarcane plants (Saccharum officinarum L; Cavalcante and Döbereiner, 1988; Gillis et al, 1989; James et al, 1994) and other crops such as sweet potato, pineapple, coffee, elephant grass, and rice (Jimenez-Salgado et al, 1997; TapiaHernández et al, 2000; Muthukumarasamy et al, 2005; Saravanan et al, 2008; Rouws et al, 2010). G. diazotrophicus has been used as an endophytic model organism to evaluate plant-bacterial interactions with non-legume host (Saravanan et al, 2008). The genome sequence of G. diazotrophicus PAL5T strain was earlier determined providing important insights into metabolism, nitrogen fixation regulation, endophytic relationship, and other processes, including phytohormone production (Bertalan et al, 2009). The function of auxins in the G. diazotrophicus–sugarcane interaction is not well understood, inoculation studies have suggested that bacterial auxin promotes growth of sugarcane in conjunction with biological nitrogen fixation (BNF) (Sevilla et al, 2001; Oliveira et al, 2002; Muñoz-Rojas and Caballero-Mellado, 2003; Lee et al, 2004). Auxin functions as a signaling molecule that participates in gene regulation in some bacteria and as an effector molecule in plant–microbe interactions (Spaepen et al, 2007; Spaepen and Vanderleyden, 2011; Patten et al, 2013)

Objectives

Methods

Results

Conclusion