Strategies for Cloning

1
Strategies for Cloning Sequencing of Genes
involved in Sulfur Oxidation in Halothiobacillus
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 earths 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)

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
gi115244spP259 ( 11) YDASCASCHGMQAQGQ
100 gi27358154gbAA ( 34) SEKQCDACHGANGVSG
84 gi37926569pdb1 ( 460) KVAVCGACHGVDGNSP
97 gi46140339refZ ( 154) YEKECLECHGKTGEGN
88 gi53804702refY ( 144) LIPPCSACHGAHGQGW
92 gi56708937refY ( 29) YAESCAGCHGETGQGN
77 gi66797508refZ ( 51) SAGSCQNCHGANGNST 83
Fig. 5 Rsa I clones on TBAmp Plates
Fig. 4 Hinc II clones on TBAmp 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
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• Trudinger P.A., Meyer T.E., Bartsch R.G. and
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  Thiobacillus neapolitanus Arch Microbiol.
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• Kelly, D.P. 1968. Biochemistry of Oxidation of
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  science. 31165-173.
• Ambler R.P., Meyer T. E., Trudinger P.A., and
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• 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.
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• Epicentre Literature 171 CopyControl Fosmid Kit
• CloneSMART blunt Cloning Kit Manual, Lucigen Corp