Poster presented at the Bacterial Electron Transfer Processes and their Regulation Meeting, European Federation of Biotechnology Microbial Physiology Section, 15-18 March 2015, Vimeiro, Portugal

Almeida, M. Gabriela

Moura, Isabel

Moura, José J. G.

Silveira, Célia M.

Sousa, Joana R.

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Joana  R. Sousa1 , Célia M. Silveira 1,2 Isabel Moura 1 , José J. G. Moura1 , M. Gabriela Almeida. 1,3  *email: jrl.sousa@campus.fct.unl.pt   1 UCIBIO@REQUIMTE - Dept. de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Monte da Caparica Portugal                     2 Instituto de Tecnologia Química e Biológica, UNL, Oeiras, Portugal                     3 CiiEM - Instituto Superior de Ciências de Saúde Egas Moniz, Monte Caparica, Portugal.      Introduction Experimental Cells grown in VMN media with: Sulfate (S) or Nitrate (N)	Cell harvesting at the stationary fase (16h)	Cell disruption (French Press)	Centrifugation to remove cell debris	Protein Quantification: - Bradford method; - 2D Quant KIt	2D Electrophoresis: 1) IEF 2) SDS-PAGE	Image Analysis (Melanie 7.0) Mass Spectrometry Protein ID Results Proteomes A B Table 1: Statistical analysis of the total protein extracts from D.d. ATCC 27774 performed with Melanie 7.0 software. Proteins area considered overexpressed if there are two fold variations or more (>2), in relative spot volume.  Statistical Analysis Number of spots Nitrate Sulfate Total 604 519 Overexpressed 22 20 Exclusive to one condition 90 9 Fig.1: Overview of the 2D gel maps of total protein fractions from D.d. ATCC 27774 cells grown for 16h with nitrate (A) and sulfate (B) media. Circles indicate proteins up-regulated in both growths. Proteins (350 µg) were separated by 2D gel electrophoresis (12,5% acrylamide) in the pH gradient 4-7. Gels were stained with colloidal Coomassie blue. [images acquired from Melanie 7.0 software]. Protein Identification Sulfate-reducing bacteria (SRB) are a diverse group of anaerobic microorganisms that obtain their energy from dissimilatory sulfate reduction1. Several studies demonstrated that some SRB species have high respiratory versatility due to the possible use of alternative electron acceptors. A good example is the bacterium Desulfovibrio desulfuricans (Dd) ATCC 27774 which grows in nitrate containing medium with rates and yields ca. 3 times superior to those observed in sulfate containing medium. Yet in the presence of both nutrients, Dd ATCC 27774 cells prefer the thermodynamically less favorable substrate, sulfate.2 So far, little is known about the reasons behind this selection or how the bacterium is able to adapt to different sources of energy.  The main goal of this work is the identification of the metabolic tools involved in the respiratory flexibility of Dd ATCC 27774 cells induced by different electron acceptors (nitrate vs. sulfate).  	Spot Over-expression Protein MW* pI* Putative Function 196 2.6 S Glutaminase 32.8 5.07 Glutaminase activity (glutamine >	glutamate); NH3 excretion 269 2.7 N Glyceraldehyde-3-phosphate dehydrogenase 37.3 6.27 Glucose metabolism 303 2.3 N 2-hydroxyglutaryl dehydratase 47.3 5.05 Glutamate metabolism; NH3 assimilation Table 2: Proteins from the nitrate and sulfate total protein fractions of D. desulfuricans cells identified by MS-TOF-TOF analysis of protein spots from 2D gels. *Theoretical values. N – nitrate, S - sulfate Conclusions and Future Work ü  25% of differential spots;  ü  In the presence of nitrate, a greater number of proteins is produced; this may indicate that the metabolism shifting from sulfate to nitrate reduction has high biosynthetic costs, related with the need to express 90 new proteins.  q  Validation with transcriptomic and metabolomic techniques.  Acknowledgments:  UCBIO@REQUIMTE PEst-C/EQB/LA0006/2013 University of Gent – L-ProBE  	References: 1. Muyzer G et al. (2008) Nat Rev Microbiol 6, 441-54; 2. Marietou A et al. (2009) J Bacteriol 191, 882-9 Respiratory Versatility in Desulfovibrio desulfuricans ATCC 27774 – a proteomic approach 

Respiratory versatility in Desulfovibrio desulfuricans ATCC 27774 – a proteomic approach

http://comum.rcaap.pt/bitstream/10400.26/13904/1/Poster01_ProfGAlmeida_2016.pdf

Respiratory versatility in Desulfovibrio desulfuricans ATCC 27774 – a proteomic approach

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