Novel genes for nitrite reductase and Amorelated proteins indicate a role of uncultivated mesophilic

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Novel genes for nitrite reductase and Amo-related
proteins indicate a role of uncultivated
mesophilic crenarchaeota in nitrogen cycling.
Alexander H. Treusch, Sven Leininger, Arnulf
Kletzin, Stephan C. Schuster, Hans-Peter Klenk
and Christa Schleper
Enivronmental Microbiology, 2005, 7(12)
home.quicknet.nl/.../index_files/Page546.htm
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http//www.mpi-bremen.de/en/Research_Projects_6.ht
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Ammonia oxidation
Crenarchaeota
Nitrosomonas sp.
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Objective

• To identify novel genes encoding for ammonia
  oxidation and nitrite reduction in creanarchaeota
• To determine the expression of novel genes in
  soil ecosystems.
• To understand the ecological impact of
  creanarchaeota in natural environments.

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Experimental Procedure

• Metagenome construction
• A soil sample from calcareous, sandy, grassland
  soil (Rotböll)
• Fosmid library construction
• PCR screen of archaeal 16S rRNA genes

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Gene expression and quantification analysis
Real time Q-PCR
Reverse transcription
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Results
Representation of crenarchaeal fosmid clone 54d9
of 16s rRNA gene showing 43 open reading frames
Copper containing nitrite reductatse gene
Close phylogenetic affiliation to crenarchaeota
amoA amoB genes
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AmoA/PmoA Protein Amino Acid Sequences Aligned
18 A.A. postions are identical White on Black
ORF 37 Amo/PmoB Amino Acid Sequences Aligned 20
A.A. are identical White on black
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Proposed Phylogenetic Tree and Comparison of
fosmid clone 54d9
Fosmid Clone 54d9 compared with sequences from
Sargasso Sea (Venter)
Amo/Pmo Phylogenetic Tree
39 amoA
amoB 37
amoC in sargasso sea crenarchaeota
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RT-PCR of 16S rRNA and amoA transcripts from soil
samples
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Elevation of crenarchaeota amoA mRNA transcritps
in the presence of ammonia
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The NirK gene Nitrite Reductase Gene on Fosmid
54d9
NO3- ? NO2- ? NO ? N2O ? N2
NirS/NirK
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The NirK Phylogenetic Tree
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Summary

• Novel ammonia monooxygenase genes were found in
  crenarchaeal genomes.
• Transcriptional expression of amoA genes was
  observed and increased in soil microcosms.
• Novel nitrite reductase gene was found in
  crenarchaeal genomes.
• This study provides the first evidence of
  crenarchaeotal participation in nitrogen cycling.

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References

• Nicol, Graeme W. Christa Schleper. (2006)
  Ammonia-oxidizing crenarchaeota important
  players in the nitrogen cycle?. Trends in
  Microbiology 145.
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• Venter, J.C., Remington, K., Heidelberg, J.F.,
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