JOURNAL OF PROTEOMICS 73 (2010) 917– 931 available at www.sciencedirect.com www.elsevier.com/locate/jprot Proteome of Gluconacetobacter diazotrophicus co-cultivated with sugarcane plantlets Marise Fonseca dos Santosa,b,⁎, Vânia Lúcia Muniz de Páduac, Eduardo de Matos Nogueirac,d, Adriana Silva Hemerlyd, Gilberto Barbosa Domontb,⁎ aFederal University of Paraná, Campus Palotina, Brazil bFederal University of Rio de Janeiro, Laboratory of Protein Chemistry/Rio de Janeiro Proteomics Network, Department of Biochemistry, Institute of Chemistry/Brazil cFederal University of the State of Rio de Janeiro, Laboratory of Molecular Biology, Department of Genetics and Molecular Biology, Brazil dFederal University of Rio de Janeiro, Institute of Medical Biochemistry, Brazil ARTICLE INFO ABSTRACT Article history: Gluconacetobacter diazotrophicus is a micro-aerobic bacterium able to fix atmospheric nitrogen Received 16 April 2009 in endophytic mode. A proteomic approach was used to analyze proteins differentially Accepted 8 December 2009 expressed in the presence and absence of sugarcane plantlets. Two-dimensional gel electrophoresis (2-DE) showed 42 spots with altered levels of expression. Analysis of these Keywords: spots by matrix-assisted laser desorption ionization time-of-flight in tandem (MALDI-TOF- Gluconacetobacter diazotrophicus TOF) identified 38 proteins. Differentially expressed proteins were associated with Differential protein expression carbohydrate and energy metabolism, folding, sorting and degradation processes, and Proteome analysis transcription and translation. Among proteins expressed in co-cultivated bacteria, four Endophytic interaction belong to membrane systems; others, like a transcription elongation factor (GreA), a 60 kDa Sugarcane chaperonin (GroEL), and an outer membrane lipoprotein (Omp16) have also been described in other plant–bacteria associations, indicating a common protein expression pattern as a result of symbiosis. A high protein content of 60 kDa chaperonin isoforms was detected as non-differentially expressed proteins of the bacteria proteome. These results allow the assessment of the physiological significance of specific proteins to G. diazotrophicus metabolism and to the pathways involved in bacteria–host endophytic interaction. © 2009 Elsevier B.V. All rights reserved. 1. Introduction benefit diverse plant hosts [5–8]. Furthermore, this bacterium has also been detected in mealy bugs that inhabit sugarcane Gluconacetobacter diazotrophicus is a nitrogen-fixing, aerobic α- fields [4]. Although G. diazotrophicus has been detected in proteobacterium [1] isolated from sugarcane (Saccharum spp.) rizosphere of non-vegetative, propagated plants, its occur- that colonizes the plant's apoplast and xylem [2,3] in an rence in sugarcane rizosphere is less well documented [9]. endophytic manner [4,5]. In addition to sugarcane, G. diazo- The biotechnological relevance of the G. diazotrophicus— trophicus can associate with Cameroon grass (Pennisetum sugarcane symbiosis is due to the economic importance of the purpureum), sweet potato (Ipomoea batatas), pineapple (Ananas crop. Brazil is one of the largest sugar producers and is comosus), and coffee (Coffea arabica), indicating a potential to responsible for 30% of sugarcane culture in the world [10,11]. Abbreviations: BNF, Biological nitrogen fixation. ⁎ Corresponding authors. Domont is to be contacted at Departamento de Bioquimica, Instituto de Química, UFRJ, Brazil. Tel./fax: +55 21 2562 7353. E-mail address: [email protected] (G.B. Domont). 1874-3919/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2009.12.005 918 JOURNAL OF PROTEOMICS 73 (2010) 917– 931 JOURNAL OF PROTEOMICS 73 (2010) 917– 931 919 Brazil also has a sugarcane alcohol program that produces an 2.2. Bacterial strain, maintenance and growth conditions important alternative to fossil fuels if one considers the carbon market incentive and the increasing worry about G. diazotrophicus (PAL 5) was obtained from EMBRAPA–CNPAB environmental preservation [12]. (Rio de Janeiro, Brazil). Bacteria, stored in saturated DYGS Currently, up to 60% of plant nutritional nitrogen comes medium at −80 °C in 20% glycerol [30], were used after two from biological nitrogen fixation (BNF) [9,13–15], requiring successive passages in solid LGI-P medium supplemented fewer nitrogen fertilizers than in the past, thus diminishing with 2 mg% yeast extract at 28 °C. One colony was used to pre- water and atmospheric pollution from farming. G. diazotro- inoculate 5 mL DYGS medium and grown at 28 °C with phicus, which provides BNF for sugarcane, also produces aeration (120 rpm) until an OD of 1.3. Four milliliters of the plant-growth hormones, such as auxins and gibberellins, as pre-inoculum were transferred to 200 mL of DYGS medium at indicated by in vitro experiments [16–18].Moreover,a 28 °C with the same aeration and, after approximately 22 h, possible biological defense role has been ascribed to G. two milliliters of bacteria at 1.3 OD was aseptically transferred diazotrophicus, due to its antagonistic activity against to the co-culture medium. Xanthomonas albilineans and Colletotrichum falcatum through, respectively, the production of bacteriocin [19,20] and likely 2.3. Plant growth and bacteria co-culture through its ability to ferment sugars and lower the pH medium to under 3.0 [21]. Sugarcane genotype SP70-1143, a high BNF variety, was used Anatomical and physiological changes in sugarcanes of throughout this work. Sugarcane plantlets free of micro- several genotypes were observed during association with organisms were obtained from sterile meristem culture and diazotrophic bacteria such as G. diazotrophicus [22]. A previous micropropagated according to the method of Hendre [31]. survey of sugarcane genes involved in symbiosis showed that Plantlets were cultured in rooting and shooting medium, a they are not only involved in general stress responses due to modified Murashige and Skoog (MS) liquid medium [32], for association with microorganisms, but rather are part of an 50–60 days, then were transferred to 30 mL flasks containing intricate system of plant/microbe signaling; several physio- MS liquid medium diluted 10-fold without hormones and logical processes are actively engaged in this particular type sucrose (MS/10 medium). The plantlets rested for seven days of plant–bacteria interaction [23]. Recently, sugarcane recep- until sterile transfer to a new set of MS/10 vials containing G. tors were described to participate directly in signaling diazotrophicus cultures inoculated two days before (item 2.2). pathways related to the establishment of plant–endophytic Approximately 40 MS/10 vials inoculated with bacteria were bacteria interaction [23–26]. It was suggested that G. diazo- prepared and maintained at 28 °C with an irradiance of 60- − − trophicus is able to contribute to plant development even mmol photons m 2 s 1 for 12 h per day. Seven days after while supporting extreme conditions of apoplastic fluid [27], plant relocation, the bacteria were collected from the liquid and that its biological activity might be regulated by organic medium by centrifugation at 12,000 g for 10 min at 4 °C. The or inorganic nitrogen compounds during its interaction with harvested cells were stored at −80 °C. Controls, that is, the plant [28]. bacteria grown in the absence of plantlets, were prepared To better understand this host–bacteria interaction, G. in a similar manner. Four biological replicate experiments diazotrophicus proteins were studied using differential protein were performed. expression analysis after in vitro culture in the absence and presence of a high BNF sugarcane host. The work was based on 2.4. Sample preparation a bottom–up proteomics approach [29], using statistical tools to evaluate the relative protein expression in 2-DE gels and Cells from each biological replicate were washed twice with − − − MALDI-TOF-TOF MS for protein identification. 10 mmol L 1 Tris, 50 mmol L 1 NaCl, 3 mmol L 1 KCl, − − 5mmolL 1 EDTA, and 1 mmol L 1 PMSF, pH 8.8 [33].Protein extractions were conducted as by Lery et al. [34] with some 2. Materials and methods modification. Pelleted cells were suspended in lysis buffer, − [8.1 mol L 1 urea, 4% (w/v) CHAPS, 1% (w/v) DTT, 2% (v/v) − 2.1. Materials Pharmalyte, 3−10, 8 mmol L 1 PMSF, 0.5% (v/v) Triton X100] − using a 1:2.5 ratio (wet weight, cells mL 1, lysis solution). Cells Reagents and 2-DE units were from GE Health Care (Uppsala, were disrupted by 20 cycles of −170° to 20 °C thermal shock, Sweden); PMSF, CHCA and bicinchoninic acid solution were sonication, and vortexing. Lysis solution was added to a 1:3.5 from Sigma (St. Louis, MO). Sequencing grade-modified final ratio, and the mixture was homogenized and centrifuged trypsin was from Promega (Madison, WI). MilliQ water was at 57,000 g for 2.5 h at 5 °C. Total protein was determined using used throughout out (Millipore, MA). All other solvents and the bicinchoninic acid solution assay [35]. Extracts were stored reagents were of analytical grade. at −80 °C until analysis. Fig. 1 – 2-DE maps of proteins from Gluconacetobacter diazotrophicus. (a) biological replicates: (A) to (D) — control bacteria; (E) to (H) — bacteria co-cultivated with sugarcane plantlets. Eight spots, representing 30% of the total protein volume, are highlighted: HSP70 (spot 388), GroEL (366, 382 and 386), GroES (107) TF (377), CSP-E (86) and 50S ribosomal protein L7/L12 (119); (b) non-differentially expressed identified proteins.