Introduction Material and Methods Results Diversity of Medicago-nodulationg rhizobial strains from southern Portugal Identification and characterization using Mass Spectrometry Approach Catarina Franco1, Ana Catarina Dourado4, Paula I. Alves1,4, Ana V. Coelho1,3, José F. Marques1,2 Paula Fareleira2, Teresa M.T. Crespo1,4 1. Instituto de Tecnologia Química e Biológica, Oeiras, Portugal; 2. Estação Agronómica Nacional, Oeiras, Portugal; 3. Universidade de Évora, Évora, Portugal; 4. Instituto de Biologia experimental e Tecnológica, Oeiras, Portugal Introduction Current methods for the identification of microorganisms in culture are time-consuming, requiring weeks or even months. With the current instrumentation (MALDI time-of-flight mass spectrometer), cell surface biomolecules (i.e. proteins) provide the most characteristic biomarkers accessible in the analysis of intact organisms. In this methodology the microorganisms cells are introduced intact into the sample holder (MALDI plate) and mixed with a UV-light absorbing matrix which later on, promotes the desorption of biomolecules, allowing differentiation of individual species on basis of the unique mass fingerprints of the biomarker molecules. Approximately 160 strains of the Medicago-nodulating bacteria have been collected from arid environments in southern Portugal and have been MS fingerprinted as also 21 nodulating type strains. The obtained spectra were matched against each other by utilizing extracted peak mass lists and hierarchical clustering. The clustering analysis was used to group the strains by similarity. A comparison between this approach and the classical gene amplification based identification techniques is also discussed. Material and Methods References Isolated rhizobacteria cells Hettick J M; Kashon M L; Simpson J P; Siegel P D; Mazurek G H; Weissman D N, (2004), Anal. Chem., 76: 5769-5776 Fenselau C; Demirev P A, (2001), Mass Spectrometry Reviews, 20: 157-171 Sánchez-Contreras, M., LLoret, J., Martín, M., Villanceros, M. Bonilla, I. & Rivilla, R. (2000), Appl. Environm. Microbiol. 66, 3621-3623. Rhizobacteria identification Mass Spectrometry fingerprinting PCR amplification UV-light absorbing matrix Rhizobacteria intact cells MALDI-TOF Plate Biomarker macromolecule Proteins of intact organisms Don’t need Extraction Separation Purification Biomarker macromolecule DNA Taq DNA extraction Sample stage Laser Detector Ion flight path: Time of Flight Tube m/z Extraction and Amplification (2 primers nod box 4 and muc R) BioNumerics, Applied Maths, Belgium Amplification of nod box 4 and muc R Mass Spectrometry fingerprinting of intact cells Rhizobacteria Fingerprint m/z % Spectra cluster analysis Normalization of DNA ladder patterns Results A Traditional methods Mass Spectrometry approach Sinorhizobium terangae DSMZ 11282 Sinorhizobium meliloti DSMZ ATCC 51124 Sinorhizobium meliloti DSMZ 30135 Sinorhizobium medicae LMG DSMZ 19964 BAsB1-2 BAsE1-1 BAsB1-1 BAsD1-1 BAsE1-2 BAsC1-1 BAsC1-2 BAsA4-4 PTGsI2-1 BAsA4-3 PTGsF1-1 BAsJ2-1 BAsH1-4 Sinorhizobium medicae LMG 19964 SALsB-1 SALsB-3 BAsG1-1 BAsG1-4 PLsTP-3 PLsTP-1 SALsB-6 Sinorhizobium terangae DSMZ 11282 Sinorhizobium meliloti DSMZ 30135 Sinorhizobium meliloti ATCC 51124 Blastobacter denitrificans DSMZ 1113 PTGsB1-1 PLsJD-1 PLsJD-2 Azorhizobium caulinodans DSMZ 5975 BAsI2-1 PTGsG1-4 BAsI2-2 BAsJ2-2 Sinorhizobium saheli DSMZ 11273 Bradyrizobium japonicum DSMZ 30132 Sinorhizobium fredii DSMZ 5851 Mesorhizobium loti DSMZ 2626 PTGsH1-2 BAsD1-2 PTGsI2-3 PTGsI2-4 Burkholderia phymatum DSMZ 17167 BAsG1-3 BAsD1-3 BAsG1-2 PTGsE1-2 BAsH1-3 PTGsI2-2 BAsC1-4 Rhizobium leguminosarum DSMZ 30131 Pearson correlation (Opt:0.34%) [0.0%-100.0%] 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 B A Intensity 20 11 9 8 63 26 37 Intensity 10.00 147 170 168 149 167 152 Sinorhizobium fingerprints using intact cell approach by MALDI-TOF mass spectrometry. These two groups are identified as similiar by the cluster analisys. B Conclusions Acknowledgments Different Sinorhizobium type strains originate different mass spectra. There are different groups of similar nodulating strains collected from arid soils. These data are also consistent with the genetic studies. However, identification of the unknown strains by comparison with the type strains was not yet possible using this approach. This work was financed by Seed Project ITQB/EAN and POCTI/AGG/46371/2002 As would be expected, the majority of the isolates, all obtained with Medicago polymorpha bait plants, group well with Sinorhizobium medicae and Sinorhizobium meliloti type strains. However, some of them group preferentially with other reference strains of Sinorhizobium, Bradyrhizobium, and Mesorhizobium, and also with Burkholderia phymatum and Blastobacter denitrificans, the only clear outlayer being Rhizobium leguminosarum. All the affinities other than those with Sinorhizobium medicae/meliloti deserve further attention. Moreover, some of the isolates group together in a fashion that suggests the influence of environmental stresses. These results also deserve further exploitation and discussion, in conjugation with other analysis for the detection of possible patterns indicative of stress tolerance. Conclusions