Key Enzymes

1. Construction of Genomic Libraries and Cloning of Genes involved in Sulfur Oxidation in Halothiobacillus neapolitanusS. Chhatre*, B. Goldbaum, S. Panchalingala & N. Hilliard, Department of Chemistry, Eastern New Mexico University, Portales, NM 88130 • Experimental Strategy • Construction of genomic libraries of Halothiobacillus neapolitanus • Application of degenerate Primers for cloning the key enzymes in the sulfur oxidation pathway • Sequence analysis to elucidate the molecular mechanisms of sulfur oxidation in Halothiobacillus neapolitanus • Site-directed and insertional mutagenesis to knock out specific enzymes 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 • 30-40 Kb Library • For this library, CopyControl Fosmid Library Kit was used • Genomic DNA was sheared by repeated pipetting before end filling (to make blunt ended 5’-phosphorylated DNA) • The end-repaired DNA was size selected by low melting point (LMP) agarose gel electrophoresis • 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 Overview of library construction (Epicentre Lit#171) The size selected fragments of DNA are run on a gel before ligating with fosmid to make sure the DNA is not lost in the gel extraction process Map of fosmid used to make the library • 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