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1. Characterization of the Sulfur Oxidation Pathways in HalothiobacillusneapolitanusANTON B. ILIUK* and NEWTON P. HILLIARDDepartment of Physical Sciences, Eastern New Mexico University, Portales, NM-88130 • Abstract: Certain specialized microbes are capable of using Spectroscopy and pH:Halothiobacillus neapolitanus grown • redox reactions with inorganic sulfur compounds in the energy on different media was subjected to UV/Vis spectrometer every producing pathways of their metabolism. These reactions can be 8 hours to examine the cytochrome peaks in the region of • very useful in bioleaching, biomining, etc.; they were introduced 300-750 nm (Figure 6 and Figure 7). Moreover, the acidity • for relief in catastrophic environmental problems (acid mine drainage level of the media was observed, as an indication of the • and deterioration of concretes). Complete oxidation of sulfur results bacteria’s growth (figures 2, 3, 4, 5). • in the formation of sulfuric acid and a subsequent drop in pH in the • local environment. One such microbe, Halothiobacillus neapolitanus, Results and discussion: From the four selected media • is capable of using thiosulfate as its sole energy source. Pathways Halothiobacillus neapolitanus has shown growth only when • for the complete oxidation of sulfur, which appear in the literature, subcultured with thiosulfate (common medium for the bacteria) • indicate numerous intermediate forms of inorganic sulfur compounds and tetrathionate (compound of interest); however, no growth • in the proposed pathway for complete oxidation of sulfur. In order to was observed when sulfite (compound of interest) or sulfate • fully understand the proposed pathway, proposed intermediates and (negative control) were used. This poses a question if the • their relationship to one another, this project will analyze the effects bacteria differentiates between thiosulfate and tetrathionate, • of growth on several of the sulfur compounds proposed as and if yes, what are the differences. The absorbances of • intermediates in the oxidation pathways. Compounds chosen include Figure 2: Avg. pH drop of thiosulfate (first cycle) Figure 3: Avg. pH drop of tetrathionate (first of these two media with the growing bacterium are quite • sulfite, sulfate, and tetrathionate. Growth rates and maximum cell compared to avg. pH drop of sulfate and sulfite cycle) compared to avg. pH drop of sulfate & sulfite similar (Figures 6 and 7), as well as the rates of growth • yield will be measured for growth on each compound. Acidity, pH, according to the change in acidity levels with time • will be monitored to determine if sulfuric acid is being produced. (Figures 2, 3, 4, and 5).These phenomena could possibly • Sulfite and tetrathionate are the two compounds used for experimental due to closeness of tetrathionate to thiosulfate in sulfur • growth, while sulfate is used as a negative control since, according oxidation reactions scheme. Thus, differential proteomics • to the sulfur pathways scheme, no further reduction can occur. The will be used for the advancement of the experiment. • primary results showed: because of the lack of acid production • (pH did not change with time), Halothiobacillus neapolitanus can not Conclusions: This experiment has shown possibilities of • reduce sulfite. It can be also noticed that the bacteria is as likely to growing Halothiobacillus neapolitanus using various sulfur • use tetrathionate as thiosulfate for the energy production, since only compounds (tetrathionate particularly) as shown on the • 24-32 hours is needed for the media pH to drop to 3.5-3.8 using either Sulfur Oxidation Pathways scheme (figure 1). We’ve come • one of these two compounds. Substrate induced expression of proteins to believe, that tetrathionate is the only observed by us • will be monitored using 2-dimensional polyacrylamide gel electrophoresis compound that is able to achieve similar effects on the • (2D-PAGE). All parameters will be compared to reference culture grown bacteria as thiosulfate, and it might be able to replace • using thiosulfate as energy source. thiosulfate if needed. • Sulfur Oxidation Pathways: • Figure 1Acknowledgements: This experiment would not be possible to complete without the financial assistance of NIH NCRR grant Number RR-16480. I also would like to thank Dr. Newton HilliardFigure 4: Avg. pH drop of Figure 5: Avg. pH drop of for his support and guidance. tthiosulfate (first cycle) compared to thiosulfate a tetrathionate (first cycle) compared to (second cycle) tetrathionate a (second cycle) References: Hempfling, W. P. and Vishniac, (1967). W. Yield Coefficients of Halothiobacillus neapolitanus in Continuous Culture. • Materials and Methods:Halothiobacillus neapolitanusJournal of Bacteriology. 874-878. • bacteria was grown on ATCC 23461 medium of Vishniac and Parker, C. D. and Prisk, J. (1953). The Oxidation of • Satner using the following ingredients: for 1L. Of medium – 10 g. Inorganic Compounds of Sulfur by Various Sulfur Bacteria. • of thiosulfate, 4 g. of KH2PO4, 4 g. of K2HPO4, 0.39 g. of MgSO4, Journal of General Microbiology. 8:344. • 0.4 g. of NH4Cl. When the bacteria appeared grown, it was Roy, A. B. and Trudifner, P. A. (1970). The Biochemistry • centrifuged in order to obtain the cell pellets. Furthermore, the of Inorganic Compounds of Sulfur. University Press, Cambridge: 1-40. • pellets were resuspended in the similar media as described above, • however, instead of 10 g/L of thiosulfate, we introduced four flasks • with 10 g/L of tetrathionate, sulfate, sulfite, and thiosulfate • accordingly. Three experiments was performed with each the four • sulfur compounds simultaneously for statistical purposes. The • measurements of pH and absorbance were taken from each media • every 8 hours. Additionally, The method of differential expression • in proteomics using 2-D polyacrylamide gel electrophoresis will be • used to observe the differences in protein expression depending on • various media and time of growth. Figure 6: Avg. absorbance of the Figure 7: Avg. absorbance of the bacteria in thiosulfate medium (time = 24 hrs) bacteria in tetrathionate medium (time = 24 hrs)