Characterization of atpD of the Fe-Reducing Thermophilic Bacterium Caloramator celere

1
Characterization of atpD of the Fe-Reducing
Thermophilic Bacterium Caloramator celere

• - Santosh Pudasaini
• Saint Peters College, Jersey City, NJ.
• Dr. Mack Ivey
• Center for Space and Planetary Sciences,
  University of Arkansas, Fayetteville, AR.

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Iron Reduction

• Anaerobic, primitive mode of metabolism
• Chemolithoautotrophic growth
• Evolved very early, thrive under extreme
  conditions

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Scope

• Abundance of Iron and CO2 in Mars.
• A potential metabolic pathway in Martian condition

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Bacterial Iron Reduction

• ATP generated by ATP Synthase
• ATP Synthase
• - transmembrane multiprotein complex
• - sits on bacterial plasma membrane
• - utilizes proton gradient created by the ETC
• - Fe (III) as electron acceptor

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Our Project

• Caloramator celere, a novel Fe (III) reducing
  thermophile
• atpD, a ß-sub unit gene in the atp operon on C.
  celere
• The most highly conserved of the atp operon genes
• Most likely to share sequence similarity with
  previously sequenced orthologs
• Will serve as a probe for isolation of the entire
  atp operon

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Objectives

• Prepare C. celere cultures
• Design primers using MSA
• Extract DNA from the cultures
• Perform PCR to amplify, clone and sequence atpD

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Culture Preparation

• C. celere cultures grown in an anaerobic media.
• Electron acceptor
• 90mM amorphous Fe(III) oxide
• 20mM 9,10-anthraquinone 2,6 disulfonic acid
  (AQDS medium)

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Reduction of Fe (III)
Before reduction
After reduction
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Reduction of AQDS
Before reduction
After reduction
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Multiple Sequence Alignment

• 38 bacterial and Bacillales atpDs gene bank
  of NCBI
• Multiple alignment - vector NTI AlinX tool
• Highly conserved regions- ideal for primer design

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Criteria for Primers

• The oligonucleotides were, in average, about
  20-25 base pairs.
• The GC content was about 50-60 percent.
• The thermodynamic Tm was about 55-60 oC.
• There were no dimers or hairpin loops.
• If present, choose ones with a positive or a
  fairly large dimer dG and loop dG.

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atpD_476_F

• Oligonucleotide (DNA) Length 20
• 5-TTCGGTGGTGCCGGTGTAGG-3
• CONDITIONS
• - Molecular Weight 6298.1
• - GC 65.0
• - Thermodynamic Tm 60.2
• - Hairpin Loops 1 total
• TTCGGTG
• G
• GGATGTGGCCGT
• Stem Length 3 Loop Length 5
• Loop dG -2.3 kcal/mol

• Dimers 3 total
• TTCGGTGGTGCCGGTGTAGG
• GGATGTGGCCGTGGTGGCTT
• Stem Length 3
• Dimer dG -1.7 kcal/mol
• TTCGGTGGTGCCGGTGTAGG
• GGATGTGGCCGTGGTGGCTT
• Stem Length 4
• Dimer dG -4.7 kcal/mol
• TTCGGTGGTGCCGGTGTAGG
• GGATGTGGCCGTGGTGGCTT
• Stem Length 3
• Dimer dG -1.7 kcal/mol

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Primers Design

• 6 Primers designed from MSA
• atpD_476_F 5-TTCGGTGGTGCCGGTGTAGG-3
• atpD_542_F 5-CACGGTGGTATTTCTGTATTCGC-3
• atpD_935_F 5-GCCGATGACTATACTGACCCAGC-3
• atpD_957_R 5-GCTGGGTCAGTATAGTCATCGGC-3
• atpD_1272_R 5-ACCGTAAATTGTTCCGCTACGTGG-3
• atpD_1377_R 5-CCAACTAAACGGAATGCATCTTC-3

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DNA Extraction

• DNA extracted from the cultures grown following
  the protocol - Protocols in Molecular
  Biology-fourth edition.
• Extracted DNA stored in TE buffer overnight.
• Agarose gel electrophoresis performed to check
  for DNA.

Marker
DNA Lanes
? ? ?
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DNA Extraction

• Agarose gel electrophoresis of DNA sample stored
  in isopropanol.
• Gel ran after extracting DNA sample precipitated
  in isopropanol.

Marker
DNA Lanes
?
? ? ?
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DNA Extraction

• Agarose gel electrophoresis of DNA sample freshly
  extracted from culture.
• Agarose gel electrophoresis of cells taken
  directly from culture.

Marker
DNA Lanes
? ? ?
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Polymerase Chain Reaction (PCR)
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Polymerase Chain Reaction (PCR)

• Primers designed from MSA, B. cohnii DNA
• DNA sample extracted from C.celere culture

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Challenges for successful PCR

• Insufficient cell density
• Inefficient cell lysis
• Rapid degradation of the DNA in extracts.

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Future Directions

• Extract DNA samples from cultures, keep them from
  degradation.
• Perform PCR with the Primers designed from MSA.
• Clone the amplified DNA into a plasmid vector and
  perform sequencing.

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Acknowledgements

• Center for Space and Planetary Sciences,
• University of Arkansas, Fayetteville, AR.
• Dr. Mack Ivey
• Jackie Denson
• NASA

Thank You