PMID- 33493490
OWN - NLM
STAT- MEDLINE
DCOM- 20210927
LR  - 20220907
IS  - 1872-8359 (Electronic)
IS  - 0167-7012 (Linking)
VI  - 181
DP  - 2021 Feb
TI  - Detection of an alkene monooxygenase in vinyl chloride-oxidizing bacteria with 
      GeneFISH.
PG  - 106147
LID - S0167-7012(21)00015-4 [pii]
LID - 10.1016/j.mimet.2021.106147 [doi]
AB  - Fluorescence in situ hybridization (FISH) can provide information on the 
      morphology, spatial arrangement, and local environment of individual cells 
      enabling the investigation of intact microbial communities. GeneFISH uses 
      polynucleotide probes and enzymatic signal amplification to detect genes that are 
      present in low copy numbers. Previously, this technique has only been applied in 
      a small number of closely related organisms. However, many important functional 
      genes, such as those involved in xenobiotic degradation or pathogenesis, are 
      present in diverse microbial strains. Here, we present a geneFISH method for the 
      detection of the functional gene etnC, which encodes the alpha subunit of an 
      alkene monooxygenase used by aerobic ethene and vinyl chloride oxidizing bacteria 
      (etheneotrophs). The probe concentration was optimized and found to be 100 pg/mul, 
      similar to previous geneFISH reports. Permeabilization was necessary for 
      successful geneFISH labeling of Mycobacteria; sequential treatment with lysozyme 
      and achromopeptidase was the most effective treatment. This method was able to 
      detect etnC in several organisms including Mycobacteria and Nocardioides, 
      demonstrating for the first time that a single geneFISH probe can detect a 
      variety of alleles (>80% sequence similarity) across multiple species. Detection 
      of etnC with geneFISH has practical applications for bioremediation. This method 
      can be readily adapted for other functional genes and has broad applications for 
      investigating microbial communities in natural and engineered systems.
CI  - Copyright (c) 2021 Elsevier B.V. All rights reserved.
FAU - Richards, Patrick M
AU  - Richards PM
AD  - Department of Civil and Environmental Engineering, 4105 Seamans Center, The 
      University of Iowa, Iowa City, IA 52242, USA.
FAU - Mattes, Timothy E
AU  - Mattes TE
AD  - Department of Civil and Environmental Engineering, 4105 Seamans Center, The 
      University of Iowa, Iowa City, IA 52242, USA. Electronic address: 
      tim-mattes@uiowa.edu.
LA  - eng
GR  - P30 ES005605/ES/NIEHS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
DEP - 20210122
PL  - Netherlands
TA  - J Microbiol Methods
JT  - Journal of microbiological methods
JID - 8306883
RN  - EC 1.13.- (Oxygenases)
RN  - EC 1.14.13.- (alkene monooxygenase)
SB  - IM
MH  - Groundwater/*microbiology
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Microbiota
MH  - *Mycobacterium/genetics/isolation & purification
MH  - *Nocardioides/genetics/isolation & purification
MH  - Oxygenases/*genetics
OTO - NOTNLM
OT  - CARD-FISH
OT  - Fluorescence in situ hybridization
OT  - Functional gene
OT  - Plasmid
OT  - etheneotroph, etnC
EDAT- 2021/01/26 06:00
MHDA- 2021/09/28 06:00
CRDT- 2021/01/25 20:10
PHST- 2020/12/08 00:00 [received]
PHST- 2021/01/18 00:00 [revised]
PHST- 2021/01/18 00:00 [accepted]
PHST- 2021/01/26 06:00 [pubmed]
PHST- 2021/09/28 06:00 [medline]
PHST- 2021/01/25 20:10 [entrez]
AID - S0167-7012(21)00015-4 [pii]
AID - 10.1016/j.mimet.2021.106147 [doi]
PST - ppublish
SO  - J Microbiol Methods. 2021 Feb;181:106147. doi: 10.1016/j.mimet.2021.106147. Epub 
      2021 Jan 22.