PMID- 20406291
OWN - NLM
STAT- MEDLINE
DCOM- 20110315
LR  - 20181201
IS  - 1462-2920 (Electronic)
IS  - 1462-2912 (Linking)
VI  - 12
IP  - 9
DP  - 2010 Sep
TI  - Detection of denitrification genes by in situ rolling circle 
      amplification-fluorescence in situ hybridization to link metabolic potential with 
      identity inside bacterial cells.
PG  - 2508-17
LID - 10.1111/j.1462-2920.2010.02224.x [doi]
AB  - A target-primed in situ rolling circle amplification (in situ RCA) protocol was 
      developed for detection of single-copy genes inside bacterial cells and optimized 
      with Pseudomonas stutzeri, targeting nitrite and nitrous oxide reductase genes 
      (nirS and nosZ). Two padlock probes were designed per gene to target both DNA 
      strands; the target DNA was cut by a restriction endonuclease close to the probe 
      binding sites, which subsequently were made accessible by 5'-3' exonucleolysis. 
      After hybridization, the padlock probe was circularized by ligation and served as 
      template for in situ RCA, primed by the probe target site. Finally, the RCA 
      product inside the cells was detected by standard fluorescence in situ 
      hybridization (FISH). The optimized protocol showed high specificity and 
      signal-to-noise ratio but low detection frequency (up to 15% for single-copy 
      genes and up to 43% for the multi-copy 16S rRNA gene). Nevertheless, multiple 
      genes (nirS and nosZ; nirS and the 16S rRNA gene) could be detected 
      simultaneously in P. stutzeri. Environmental application of in situ RCA-FISH was 
      demonstrated on activated sludge by the differential detection of two types of 
      nirS-defined denitrifiers; one of them was identified as Candidatus 
      Accumulibacter phosphatis by combining in situ RCA-FISH with 16S rRNA-targeted 
      FISH. While not suitable for quantification because of its low detection 
      frequency, in situ RCA-FISH will allow to link metabolic potential with 16S rRNA 
      (gene)-based identification of single microbial cells.
CI  - (c) 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.
FAU - Hoshino, Tatsuhiko
AU  - Hoshino T
AD  - Department of Biological Sciences, Microbiology, Aarhus University, Ny Munkegade 
      114, DK-8000 Aarhus C, Denmark.
FAU - Schramm, Andreas
AU  - Schramm A
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20100416
PL  - England
TA  - Environ Microbiol
JT  - Environmental microbiology
JID - 100883692
RN  - 0 (DNA Primers)
RN  - 0 (DNA, Bacterial)
RN  - 0 (Oligonucleotide Probes)
RN  - 0 (Sewage)
RN  - EC 1.- (Oxidoreductases)
RN  - EC 1.7.2.4 (nitrous oxide reductase)
SB  - IM
MH  - Betaproteobacteria/enzymology/*genetics
MH  - DNA Primers/genetics
MH  - DNA, Bacterial/genetics
MH  - Denitrification/*genetics
MH  - *Genes, Bacterial
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Oligonucleotide Probes
MH  - Oxidoreductases/genetics
MH  - Primed In Situ Labeling
MH  - Pseudomonas stutzeri/enzymology/*genetics
MH  - Sewage/microbiology
EDAT- 2010/04/22 06:00
MHDA- 2011/03/16 06:00
CRDT- 2010/04/22 06:00
PHST- 2010/04/22 06:00 [entrez]
PHST- 2010/04/22 06:00 [pubmed]
PHST- 2011/03/16 06:00 [medline]
AID - EMI2224 [pii]
AID - 10.1111/j.1462-2920.2010.02224.x [doi]
PST - ppublish
SO  - Environ Microbiol. 2010 Sep;12(9):2508-17. doi: 10.1111/j.1462-2920.2010.02224.x. 
      Epub 2010 Apr 16.