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Membrane Protein Profiling of Acidovorax Avenae Subsp. Avenae Under Various Growth Conditions

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Membrane Protein Profiling of Acidovorax Avenae Subsp. Avenae Under Various Growth Conditions Arch Microbiol (2015) 197:673–682 DOI 10.1007/s00203-015-1100-9 ORIGINAL PAPER Membrane protein profiling of Acidovorax avenae subsp. avenae under various growth conditions Bin Li1 · Li Wang1 · Muhammad Ibrahim1,3 · Mengyu Ge1 · Yanli Wang2 · Shazia Mannan3 · Muhammad Asif3 · Guochang Sun2 Received: 24 July 2014 / Revised: 1 February 2015 / Accepted: 2 March 2015 / Published online: 13 March 2015 © Springer-Verlag Berlin Heidelberg 2015 Abstract Membrane proteins (MPs) of plant pathogenic transport of small molecules, protein synthesis and secretion bacteria have been reported to be able to regulate many essen- as well as virulence such as NADH, OmpA, secretion pro- tial cellular processes associated with plant disease. The aim teins. Therefore, the result of this study not only suggests that of the current study was to examine and compare the expres- it may be an alternate method to analyze the in vivo expression sion of MPs of the rice bacterial pathogen Acidovorax avenae of proteins by using LE medium to mimic plant conditions, subsp. avenae strain RS-1 under Luria-Bertani (LB) medium, but also reveals that the two sets of differentially expressed M9 medium, in vivo rice plant conditions and leaf extract (LE) MPs, in particular the common MPs between them, might be medium mimicking in vivo plant condition. Proteomic analy- important in energy metabolism, stress response and virulence sis identified 95, 72, 75, and 87 MPs under LB, in vivo, M9 of A. avenae subsp. avenae strain RS-1. and LE conditions, respectively. Among them, six proteins were shared under all tested growth conditions designated as Keywords Acidovorax · Membrane proteins · In vivo · abundant class of proteins. Twenty-six and 21 proteins were Leaf extract · Rice expressed uniquely under in vivo versus LB medium and LE versus M9 medium, respectively, with 17 proteins common among these uniquely induced proteins. Moreover, most of Introduction the shared proteins are mainly related to energy metabolism, Membrane proteins (MPs) are important components of the bacterial membrane, which play an important role in Communicated by Shuang-Jiang Liu. transport of nutrients, cellular processes, energy transduc- Electronic supplementary material The online version of this tion, scaffolding of cell structure and cell-to-cell commu- article (doi:10.1007/s00203-015-1100-9) contains supplementary nications or interactions (Marreddy et al. 2011). Further- material, which is available to authorized users. more, interaction of bacterial plant pathogen with the host is directly associated with the expression of MPs by regu- * Bin Li [email protected] lating the movement of effector proteins from the bacterial cells directly into the eukaryotic plant cells (Ibrahim et al. * Guochang Sun [email protected] 2012; Knief et al. 2011). Indeed, several MPs from Gram- negative bacteria had been found to have the ability to rec- 1 State Key Laboratory of Rice Biology, Institute ognize essential virulence factors and host immune rec- of Biotechnology, Zhejiang University, Hangzhou 310058, ognition target (Schell et al. 2011). Therefore, identifying China abundant and/or novel MPs, and characterizing their func- 2 State Key Laboratory Breeding Base for Zhejiang tion in disease, pathogen physiology and resistance against Sustainable Plant Pest and Disease Control, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China the host are important steps for understanding the genetics and disease intensity of bacterial plant pathogens. 3 Department of Biosciences, COMSATS Institute of Information Technology, Sahiwal Campus, Sahiwal, Membrane proteome has been characterized and ana- Pakistan lyzed in bacteria under various synthetic medium such as 1 3 674 Arch Microbiol (2015) 197:673–682 Luria-Bertani (LB) and M9 medium. However, the expression suspension cultured in LB broth in our previous study of proteins associated with derivative enzymes and transport (Xie et al. 2011) and preserved in 25–35 % sterile glycerol systems and proteins involved in invading the host cells, e.g., (Sangon Biotech, Shanghai, China) at 80 °C. LE broth − type III secretion system (T3SS), are usually expressed during medium was prepared as described by Mehta and Rosato the bacterial infection process in plant (Barnard et al. 2007; (2001). Briefly, about 10.0 g of rice leaves were grinded Mole et al. 2007). Indeed, recently, comprehensive studies of into powder and macerated in 100 mL M9 medium. After proteomics of Methylobacterium extorquens have identified 10 min centrifugation at 5000 RPM, supernatant was more abundant proteins when the commensals colonized plant taken and filtered sequentially using 0.45- and 0.22-µm surface compared to the cells cultured in synthetic medium membranes (Millipore, Bedford, USA) and mixed to M9 (Knief et al. 2011; Gourion et al. 2006). In addition, proteomic broth to final concentration of 1.0 mg leaves/mL of M9 analysis of cellular outer membrane in bacterial pathogen of broth. rice has shown significant differential expression of proteins under in vivo and in vitro conditions (Ibrahim et al. 2012). Bacterial growth under different conditions Therefore, characterizations of bacterial membrane proteome under the in vivo condition could provide important role in A. avenae subsp. avenae strain RS-1 was cultured on LB understanding the pathogen physiology (Cash 2011). medium, following the incubation at 30 °C for 24 h. For In a previous study, a method for analyzing outer mem- synthetic growth culture medium, single bacterial colony brane proteome of in vivo cultivated bacteria has been was picked and inoculated into 10 mL fresh LB broth, LE developed (Ibrahim et al. 2012; Li et al. 2014). In general, and M9 broth and then incubated at 30 °C. Bacteria were however, due to the difficulty in collecting bacteria from harvested, and the final concentration was adjusted to infected tissues and the high risk of contamination, cellular 1.8–2.0 of OD600nm. The centrifugation was done at 8000 proteome of plant pathogenic bacteria in plants is less char- RPM and 4 °C for 10 min, and bacterial cells were pro- acterized. To solve this problem, some researchers use plant cessed to the steps for extraction of MPs. The MPs of the extracts to mimic the plant environment (Büttner and Bonas in vivo cells were extracted as described in our previous 2010). Proteomic response and changes to plant materials study (Ibrahim et al. 2012). Briefly, 6 days after bacterial have been revealed in previous studies (Knief et al. 2011; inoculation, leaves with visible and prevailing symptoms Andrade et al. 2008; Astua-Monge et al. 2005; Brown et al. were collected and washed with alcohol. The leaves were 2001). However, little is known about the mimic effect of incised to produce very small pieces by using sterile razor plant extracts on the membrane proteome of plant patho- blade. These small pieces of leaves were kept for 30 min genic bacteria Acidovorax avenae subsp. avenae (Xie et al. in contamination-free glass plates containing 20 mL double 2011; Liu et al. 2012; Yang et al. 2014). distilled water to allow leaching bacteria from leaf tissues. In this study, we present the membrane proteome sum- The bacterial suspension and leaf tissues were separated by mary of A. avenae subsp. avenae strain RS-1 under LB, in centrifugation following the wash of bacterial cell pellets vivo rice plant, M9 and leaf extract (LE) culture conditions with PBS and with water. The bacterial cell pellets were using LC–MS/MS and in silico functional characterizations of processed for protein extraction. membrane proteome. Furthermore, comparison of proteomic profiling revealed that the majority of differentially expressed Preparation of MPs MPs under in vivo versus LB medium are common to LE medium versus M9 medium. This result not only indicates that MPs were extracted as described by Jagannadham and it may be an alternate method to analyze the in vivo expression Chowdhury (2012) by using sucrose gradient techniques of proteins by using LE medium to mimic plant conditions, with minor modification. Cells were washed with Tris– but also suggests that the two sets of differentially expressed HCl (30 mM) with pH 8.0 following re-suspension in MPs, in particular the common MPs between them, may be sucrose solution (20 %) made in Tris buffer having 1 mg/ involved in pathogenicity, cell survival, energy metabolism mL lysozyme, DNase (300 μg/mL) and RNase (200 μg/ and stress response of A. avenae subsp. avenae strain RS-1. mL). Cells were lysed using sonication following cen- trifugation for 10 min at 10,000 RPM. The supernatant was taken and loaded onto a double-step sucrose gradi- Materials and methods ent (60 and 70 % sucrose layers) and ultra-centrifuged at 55,000 RPM (Beckman Coulter, USA) for 18 h. After Bacterial strain and LE medium centrifugation, MPs were separated as double bands: The upper band corresponding to the fraction of inner mem- A. avenae subsp. avenae strain RS-1 was isolated from brane, while the lower band to outer membrane. The two diseased rice plants following inoculation with bacterial bands having membrane fractions were collected precisely, 1 3 Arch Microbiol (2015) 197:673–682 675 combined, and subsequently diluted and centrifuged for Genome‑wide in silico functional characterization another round at 55,000 RPM for 1 h. The solubilization of of LC–MS/MS peptides the pellet containing MPs was performed in Triton X-100 (2 %) following the overnight precipitation with 10 % LC–MS/MS-identified proteins were further analyzed for TCA at 20 °C. The resultant pellet was centrifuged for protein subcellular localization using PSORTb (version − 10 min at 14,000 RPM and washed with acetone twice. 3.0.2) and Phobius (Kall et al. 2004; Yu et al. 2010). The Finally, the pellet solubilization for isoelectric focusing parameters selected in PSORTb and Phobius online tools (IEF) was performed using rehydration buffer having 2 % were of normal format with Gram-negative strains, and ASB-14.
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