Comparison of Protein Expression Profiles of Novel Halomonas smyrnensis AAD6^T and Halomonas salina DSMZ 5928^T

Aydan Salman Dilgimen, Kazim Yalcin Arga, Volker A. Erdmann, Brigitte Wittmann-Liebold, Aziz Akin Denizci, Dilek Kazan

Bioengineering Department, Faculty of Engineering, Marmara University (Goztepe Campus), Istanbul, Turkey.
Bioengineering Department, Faculty of Engineering, Marmara University (Goztepe Campus), Istanbul, Turkey;Institute for Chemistry/Biochemistry, Freie University, Berlin, Germany;Present Address: Department of Biochemistry & Molecular Biology, University of Calgary, Calgary, Canada.
Institute for Chemistry/Biochemistry, Freie University, Berlin, Germany.
The Scientific and Technological Research Council of Turkey (TUBITAK), Marmara Research Center (MAM), Genetic Engineering and Biotechnology Institute, Gebze, Turkey.
Wita GmbH, Berlin, Germany.

DOI: 10.4236/ns.2014.69062                

Abstract

In this work, the protein pattern of novel Halomonas smyrnensis AAD6^T was compared to that of Halomonas salina DSMZ5928^T, which is the closest species on the basis of 16S rRNA sequence, to understand how AAD6^T differs from type strains. Using high resolution NEPHEGE technique, the whole cell protein composition patterns of both Halomonas salina DSMZ5928^T and H. smyrnensis AAD6^T were mapped. The expressed proteins of the two microorganisms were mostly located at the acidic side of the gels, at molecular weight values of 60 to 17 kDa, and at isoelectric points 3.8 to 6.0, where they share a significant number of common protein spots. Identification and characterization of protein spots via whole genome sequencing data indicated that these two microorganisms used similar pathways, especially TCA cycle, for their survival; in other words, for their energy requirements. On the other hand, the protein expression differences in AAD6^T and H. salina DSMZ 5928^T showed that they prefer different metabolic pathways for lipid biosynthesis and in adaptation to extreme environments. Thus, we suggested that phylogenetic dissimilarities between these microorganisms could be related to the protein expression differences; in other words, metabolic flux differences in AAD6^T and H. salina DSMZ 5928^T. This is the first study to explain the dissimilarities of phenotypic characters and DNA-DNA hybridization between type strain and novel strain AAD6^T by using protein expression differences.

Keywords

Proteomics, Halomonas salina DSMZ 5928^T, Halomonas smyrnensis AAD6^T, NEPHGE Technique, Genome

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Winker, S. and Woese, C.R. (1991) A Definition of the Domains Archaea, Bacteria and Eucarya in Terms of Small Subunit Ribosomal RNA Characteristics. Systematic and Applied Microbiology, 14, 305-310. http://dx.doi.org/10.1016/S0723-2020(11)80303-6
[2]	Madigan, M.T. and Oren, A. (1999) Thermophilic and Halophilic Extremophiles. Current Opinion in Microbiology, 2, 265-269. http://dx.doi.org/10.1016/S1369-5274(99)80046-0
[3]	Ventosa, A., Nietro, J.J. and Oren, A. (1998) Biology of Moderately Halophilic Aerobic Bacteria. Microbiology and Molecular Biology Reviews, 62, 504-544.
[4]	Poli, A., Nicolaus, B., Denizci, A.A., Yavuzturk, B. and Kazan, D. (2013) Halomonas smyrnensis sp. nov., a Moderately Halophilic, Exopolysaccharide-Producing Bacterium. IJSEM, 63, 10-18.
[5]	Poli, A., Kazak, H., Gürleyendag, B., Tommonaro, G., Pieretti, G., Oner, E.T. and Nicolaus, B. (2009) High Level Synthesis of Levan by a Novel Halomonas Species Growing on Defined Media. Carbohydrate Polymers, 78, 651-657. http://dx.doi.org/10.1016/j.carbpol.2009.05.031
[6]	Sogutcu, E., Emrence, Z., Arikan, M., Cakiris, A., Abaci, N., Oner, E.T., üstek, D. and Arga, K.Y. (2012) Draft Genome Sequence of Halomonas Smyrnensis AAD6T. Journal of Bacteriology, 194, 5690. http://dx.doi.org/10.1128/JB.00559-12
[7]	Molloy, M.P., Herbert, B.R., Walsh, B.J., Tyler, M.I., Traini, M., Sanchez, J.C., Hochstrasser, D.F., Williams, K.L. and Gooley, A.A. (1998) Extraction of Membrane Proteins by Differential Solubilization for Separation Using Two-Dimensional Gel Electrophoresis. Electrophoresis, 19, 837-844. http://dx.doi.org/10.1002/elps.1150190539
[8]	Wittmann-Liebold, B., Graack, H.R. and Pohl, T. (2006) Two-Dimensional Gel Electrophoresis as Tool for Proteomics Studies in Combination with Protein Identification by Mass Spectrometry. Proteomics, 6, 4688-4703. http://dx.doi.org/10.1002/pmic.200500874
[9]	Neuhoff, V., Stamm, R. and Eibl, H. (1985) Clear Background and Highly Sensitive Protein Staining with Coomassie Blue Dyes in Polyacrylamide Gels: A Systematic Analysis. Electrophoresis, 6, 427-448. http://dx.doi.org/10.1002/elps.1150060905
[10]	Switzer, R.C., Merril, C.R. and Shifrin, S. (1979) A Highly Sensitive Silver Stain for Detecting Proteins and Peptides in Polyacrylamide Gels. Analytical Biochemistry, 98, 231-237. http://dx.doi.org/10.1016/0003-2697(79)90732-2
[11]	Bradford, M.M. (1976) A Rapid and Sensitive for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry, 72, 248-254. http://dx.doi.org/10.1016/0003-2697(76)90527-3
[12]	Klose, J. and Kobalz, U. (1995) Two-Dimensional Electrophoresis of Proteins: An Updated Protocol and Implications for a Functional Analysis of the Genome. Electrophoresis, 16, 1034-1059. http://dx.doi.org/10.1002/elps.11501601175
[13]	Jungblut, P. and Seifer, R. (1990) Analysis by High Resolution Two-dimensional Electrophoresis of Differentiation-dependent Alterations in Cytosolic Protein Pattern of HL-60 Leukemic Cells. Journal of Biochemical and Biophysical Methods, 21, 47-58. http://dx.doi.org/10.1016/0165-022X(90)90044-D
[14]	Aziz, R.K., Bartels, D., Best, A.A., DeJongh, M., Disz, T., et al. (2008) The RAST Server: Rapid Annotations using Subsystems Technology. BMC Genomics, 9, 75. http://dx.doi.org/10.1186/1471-2164-9-75
[15]	Kanehisa, M., Goto, S., Sato, Y., Furumichi, M. and Tanabe, M. (2012) KEGG for Integration and Interpretation of Large-Scale Molecular Datasets. Nucleic Acids Research, 40, 109-114. http://dx.doi.org/10.1093/nar/gkr988
[16]	Artimo, P., Jonnalagedda, M., Arnold, K., Baratin, D., Csardi, G., de Castro, E., Duvaud, S., Flegel, V., Fortier, A., Gasteiger, E., Grosdidier, A., Hernandez, C., Ioannidis, V., Kuznetsov, D., Liechti, R., Moretti, S., Mostaguir, K., Redaschi, N., Rossier, G., Xenarios, I. and Stockinger, H. (2012) ExPASy: SIB Bioinformatics Resource Portal. Nucleic Acids Research, 40, 597-603. http://dx.doi.org/10.1093/nar/gks400
[17]	Saier, M.H., Yen, M.R., Noto, K., Tamang, D.G. and Elkan, C. (2009) The Transporter Classification Database (TCDB): Recent Advances. Nucleic Acids Research, 37, 274-278. http://dx.doi.org/10.1093/nar/gkn862
[18]	Dobson, S.J. and Franzmann, P.D. (1996) Unification of the Genera Deleya, Halomonas, and Halovibrio and the Species Paracoccus halodendrificans into a Single Genus, Halomonas, and Placement of the Genus Zymobacter in the Family Halomonadaceae. International Journal of Systematic Bacteriology, 46, 550-558. http://dx.doi.org/10.1099/00207713-46-2-550
[19]	Franzmann, P.D., Wehmeyer, U. and Stackebrandt, E. (1988) Halomonadaceae fam. nov., A New Family of the Class Proteobacteria to Accommodate the Genera Halomonas and Deleya. Systematic and Applied Microbiology, 11, 16-19. http://dx.doi.org/10.1016/S0723-2020(88)80043-2
[20]	Lee, J.C., Jeon, C.O., Lim, J.M., Lee, S.M., Lee, J.M., Song, S.M., Park, D.J., Li, W.J. and Kim, C.J. (2005) Halomonas taeanensis sp. nov., a Novel Moderately Halophilic Bacterium Isolated from a Solar Saltern in Korea. IJSEM, 55, 2027-2032.
[21]	Cho, C.W., Lee, S.H., Choi, J., Park, S.J., Ha, D.J., Kim, H.J. and Kim, C.W. (2003) Improvement of the Two Dimensional Gel Electrophoresis Analyses for the Proteome Study of Halobacterium salinarum. Proteomics, 3, 23252329. http://dx.doi.org/10.1002/pmic.200300525
[22]	Shukla, H.D. (2006) Proteomic Analyses of Acidic Chaperons and Stress Proteins in Extreme Halophile Halobacterium NRC-I: A Comparative Proteomic Aproach to Steady Heat Shock Response. Proteome Science, 4, 6-14. http://dx.doi.org/10.1186/1477-5956-4-6
[23]	Saum, S.H., Pfeiffer, F., Palm, P., Rampp, M., Schuster, S.C., Müller, V. and Oesterhelt, D. (2012) Chloride and Organic Osmolytes: A Hybrid Strategy to Cope with Elevated Salinities by the Moderately Halophilic, Chloride-Dependent Bacterium Halobacillus halophilus. Environmental Microbiology, 15, 1619-1633. http://dx.doi.org/10.1111/j.1462-2920.2012.02770.x
[24]	Kuntz, I.D. (1971) Hydration of Macromolecules: III. Hydration of Polypeptides. Journal of the American Chemical Society, 93, 514-516. http://dx.doi.org/10.1021/ja00731a036
[25]	Elock, A.G. and McCommon, J.A. (1998) Electrostatic Contributions to the Stability of Halophilic Proteins. Journal of Molecular Biology, 280, 731-748. http://dx.doi.org/10.1006/jmbi.1998.1904
[26]	Isarankura-Na-Ayudhya, P., Isarankura-Na-Ayudhya, C., Yainoy, S., Thippakorn, C., Singhagamol, W., Polprachum, W., Roytrakul, S. and Prachayasittikul, V. (2010) Proteomic Alterations of Escherichia coli by Paraquat. EXCLI Journal, 9, 108-118.
[27]	Starai, V.J. and Escalante-Semerena, J.C. (2004) Acetyl-Coenzyme A Synthetase (AMP forming). Cellular and Molecular Life Sciences, 61, 2020-2030. http://dx.doi.org/10.1007/s00018-004-3448-x
[28]	Weidenhaupt, M., Rossi, P., Beck, C., Fischer, H.M. and Hennecke, H. (1996) Bradyrhizobium japonicum Possesses Two Discrete Sets of Electron Transfer Flavoprotein Genes: fixA, fixB and etfS, etfL. Archives of Microbiology, 165, 169-178.
[29]	Roberts, D.L., Frerman, F.E. and Kim, J.J. (1996) Three-Dimensional Structure of Human Electron Transfer Flavoprotein to 2.1-A Resolution. Proceedings of the National Academy of Sciences, 93, 14355-14360. http://dx.doi.org/10.1073/pnas.93.25.14355
[30]	Roberts, D.L., Salazar, D., Fulmer, J.P., Frerman, F.E. and Kim, J.J. (1999) Crystal Structure of Paracoccus denitrificans Electron Transfer Flavoprotein: Structural and Electrostatic Analysis of a Conserved Flavin Binding Domain. Biochemistry, 38, 1977-1989. http://dx.doi.org/10.1021/bi9820917
[31]	Leys, D., Basran, J., Talfournier, F., Sutcliffe M.J. and Scrutton, N.S. (2003) Extensive Conformational Sampling in a Ternary Electron Transfer Complex. Nature Structural Biology, 10, 219-25. http://dx.doi.org/10.1038/nsb894
[32]	Canovas, D., Vargas, C., Csonka, L.N., Ventosa, A. and Nieto, J.J. (1998) Synthesis of Glycine Betaine from Exogenous Choline in the Moderately Halophilic Bacterium Halomonas elongate. Applied and Environmental Microbiology, 64, 4095-4097.
[33]	Canovas, D., Vargas, C., Calderon, M.I., Ventosa, A. and Nieto, J.J. (3043) Characterization of the Genes for the Biosynthesis of the Compatible Solute Ectoine in the Moderately Halophilic Bacterium Halomonas elongata DSM. Systematic and Applied Microbiology, 21, 487-497. http://dx.doi.org/10.1016/S0723-2020(98)80060-X
[34]	Schouler, C., Clier, F., Lerayer, A.L., Ehrlich, S.D. and Chopin, M.C. (1998) A Type IC Restriction-Modification System in Lactococcus lactis. Journal of Bacteriology, 180, 407-411.
[35]	Flower, A. and McHenry, C. (1991) Transcriptional Organization of the Escherichia coli dnaX Gene. Journal of Molecular Biology, 220, 649-658. http://dx.doi.org/10.1016/0022-2836(91)90107-H
[36]	Bell, K.S., Sebaihia, M., Pritchard, L., Holden, M.T., Hyman, L.J., Holeva, M.C., Thomson, N.R., Bentley, S.D., Churcher, L.J., Mungall, K., Atkin, R., Bason, N., Brooks, K., Chillingworth, T., Clark, K., Doggett, J., Fraser, A., Hance, Z., Hauser, H., Jagels, K., Moule, S., Norbertczak, H., Ormond, D., Price, C., Quail, M.A., Sanders, M., Walker, D., Whitehead, S., Salmond, G.P., Birch, P.R., Parkhill, J. and Toth, I.K. (2004) Genome Sequence of the Enterobacterial Phytopathogen Erwinia carotovora subsp. Atroseptica and Characterization of Virulence Factors. Proceedings of the National Academy of Sciences, 101, 11105-11110. http://dx.doi.org/10.1073/pnas.0402424101