Cloning & Sequencing of Genes involved in Sulfur Oxidation in Halothiobacillus neapolitanus

1. Strategies for Cloning & Sequencing of Genes involved in Sulfur Oxidation inHalothiobacillus neapolitanusSuneel A. Chhatre*, Joaquin A. DeLeon, John Latham & Newton P. HilliardDepartment of Chemistry, Eastern New Mexico University, Portales, NM 88130 • Experimental Strategy • Mass production of Halothiobacillus neapolitanus in Bioflo 110 chemostat • Isolation and purification of various enzymes from the harvested cells • Application of degenerate Primers for cloning the key enzymes based on their N-terminus sequence data • Cloning the genes using genomic libraries as template DNA • Sequence analysis to elucidate the molecular mechanisms of sulfur oxidation in Halothiobacillus neapolitanus • Purification of Enzymes • For purification of various enzymes, cells were harvested from reactor and lysed using French press • After centrifugation at 15000 rpm for 1 hr, the cell free extract was loaded on DEAE Sephacel column first and later various fraction were loaded onto different matrix for purification several key enzymes (shown in the flow chart below) • Each fraction was analyzed by spectrophotometry and gel electrophoresis before subjecting to N-termini sequencing Background • Upto 75% of the sulfur in the earth’s crust has been biologically cycled with a good portion of that arising from the microbiological reduction of sulfate to sulfide and corresponding oxidation of reduced forms of sulfur to sulfate. • Despite the widespread interest in the role these microorganisms play in sulfur recycling, there is still much uncertainty regarding the actual metabolic pathways(s) and oxidation reactions. In part this is due to the metabolic diversity amongst sulfur oxidizing microorganisms (SOM). • There appears to be two major modes of oxidation, the ‘S2’ pathways in which a single mole of thiosulfate is sequentially oxidized to two moles of sulfite (well characterized in Paracoccus sp.) and the ‘S4’ pathway in which two moles of thiosulfate are oxidized to form a single mole of tetrathionate which is subsequently degraded and oxidized to sulfate • Halothiobacillus neapolitanus is a chemolithotroph with a different mechanism for sulfur oxidation as it uses ‘S4’ pathway and provides an opportunity to study a single reaction of thiosulfate oxidase • The aim of this study is to clone and overexpress various enzymes involved in sulfur oxidation in Halothiobacillus neapolitanus • Most of the enzymes involved in the sulfur oxidation in Halothiobacillus neapolitanus have been purified successfully in our lab using varying column chromatography methods • Construction of Genomic Libraries • Two libraries were prepared from the genomic DNA of Halothiobacillus neapolitanus • A 3-4 Kb fragment: to look for the individual genes encoding single enzyme involved in sulfur oxidation • A 30-40 Kb fragment library: for the big operons encoding multiple enzymes • 3-4 Kb Library • CloneSmart Blunt Cloning kit was used to construct this library • The genomic DNA was prepared using Qiagen columns with some modifications in the provided protocol and optimized for restriction digestion to yield right size fragments (Fig. 1 and 2) • About 50 µg of genomic DNA was digested with two different blunt cutters, Rsa I and Hinc II, purified by gel extraction and ligated to pSMART Vector (Fig. 3) • A small amount of cells were plated on agar to check the efficiency of library. With both enzymes it was approximately 108, which is more than enough to yield required number of clones (Fig. 4 & 5) • To further confirm the validity of library, ten colonies were picked from the plates and used as template for colony PCR using vector primers. All ten colonies shows 3-4 KB size fragments • In another PCR, 16sRNA primers from Halothiobacillus neapolitanus were designed. The library was used as template to pull out the right size fragment (~500 bp) Lane 1: Molecular Marker Lane 2: End-filled DNA before ligation • Designing Primers for Cloning Relevant Genes • Degenerate primers were designed to “fish out” gene sequences of the key enzymes of sulfur oxidation pathway • First strategy is to PCR amplify the genes using forward primer based on the N-terminal sequence of C554 and the reverse primer would be from the the vector • The second strategy is to design primer on the basis of consensus sequence generated from the divergent set of C554. For this various C554 sequences were pulled out from data bases, fed into DS view Pro 6.0 and aligned using block maker • Several primers have been designed based on these approaches for the amplification of C554, tetrathionate hydrolase and thiosulfate oxidase and experiments are underway for cloning of these key enzymes Key Enzymes • This picture shows is the diagrammatic representation of proposed sulfur oxidation pathways. It is clear from the series of reactions outlined that any comprehensive scheme of sulfur oxidation may consist of multiple pathways that overlap and interconnect to varying degrees • Some of these reactions and enzymes involved have been well characterized in other organisms • Based on the structures and sequences available in databanks, an approach is being made to decipher molecular mechanism of sulfur oxidation in Halothiobacillus neapolitanus in this study Fig. 3 Vector Map (pSMART-AMP from Lucigen Corp) Fig. 2 Optimization of restriction digestion with Hin III Fig.1 Optimization of restriction digestion with RsaI gi|115244|sp|P259 ( 11) YDASCASCHGMQAQGQ 100 gi|27358154|gb|AA ( 34) SEKQCDACHGANGVSG 84 gi|37926569|pdb|1 ( 460) KVAVCGACHGVDGNSP 97 gi|46140339|ref|Z ( 154) YEKECLECHGKTGEGN 88 gi|53804702|ref|Y ( 144) LIPPCSACHGAHGQGW 92 gi|56708937|ref|Y ( 29) YAESCAGCHGETGQGN 77 gi|66797508|ref|Z ( 51) SAGSCQNCHGANGNST 83 Fig. 4 Hinc II clones on TB+Amp Plates Fig. 5 Rsa I clones on TB+Amp Plates PDB 1H31 Iverson et al. 2001 Crystal structure of SoxAX protein from Rhodovulum sulfidophilum. The SoxAX is a heterodimeric c-type cytochrome involved in thiosulfate oxidation. The SoxA of Paracoccus has 25% identity with thiosulfate oxidase of Halothiobacillus The sequence alignment with c554 from various organisms. The highlighted residues are conserved and make a consensus sequence in all c type cytochromes. Halothiobacillus neapolitanus contains ~ 20 c type cytochromes and this alignment can be used to design a common primer set to pull out them from the library Crystal structure of c554 from Nitrosomonas europea. C554 is a tetra-heme cytochrome with conserved heme-packing motifs that are present in other heme-containing proteins Crystal structure of sulfur oxygenase reductase from Acidianus ambivalens which catalyzes an oxygen-dependent disproportionation of elemental sulfur. Twenty four monomers form a large hollow sphere enclosing a positively charged nanocompartment Acknowledgements This work was supported by NIH NCRR grant number R-16480. The contents of this poster are solely the responsibility of the authors and do not necessarily represent the official view of NIH We thank A. Iliuk, B. Goldbaum, Srikanth P. and Joauqin for experimental assistance. Urich et al. 2006 • References • Compton T. 1990. Degenerate primers for DNA amplification. pp. 39-45 in: PCR Protocols (Innis, Gelfand, Sninsky and White, eds.); Academic Press, New York. • Trudinger, P.A. 1961. Thiosulfate oxidation and cytochromes in Thiobacillus X. J.of Biochemistry. 78:673-680. • Trudinger P.A., Meyer T.E., Bartsch R.G. and Kamen M.D. 1985. The major soluble cytochromes of the obligately aerobic sulfur bacterium, Thiobacillus neapolitanus Arch Microbiol.; 141(4): 273-8. • Kelly, D.P. 1968. Biochemistry of Oxidation of inorganic sulphur compounds .Australian J. of science. 31:165-173. • Ambler R.P., Meyer T. E., Trudinger P.A., and Kamen M. D. 1985. The amino acid sequence of the cytochrome c-554(547) from chemolithotropic bacterium Thiobacillus neapoliatnus,Biochem. J. 227, 1009-1013. • Urich, T., Gomes, C., 2006, X-ray structure of a sulfur cycle metalloenzyme, Science, 311, 996-1002 • Iverson, T., Arciero, D., Rees, D., 2001, High resolution structures of the oxidized and reduced states of cytochrome c554 from Nitrosomonas europea J. Biol. Inorg.. Chem. 6, 390-397 • Epicentre Literature # 171 CopyControl Fosmid Kit • CloneSMART blunt Cloning Kit Manual, Lucigen Corp