TY - JOUR
T1 - Detection in coal tar waste-contaminated groundwater of mRNA transcripts related to naphthalene dioxygenase by fluorescent in situ hybridization with tyramide signal amplification
AU - Bakermans, Corien
AU - Madsen, Eugene L.
N1 - Funding Information:
This work was supported by EPA STAR Fellowship #915386 and NSF Grant #MCB-0084175. Comomonas species, P. putida strain F1, and P. putida mt2 were kindly provided by G. Zylstra, Rutgers University. Burkholderia species were kindly provided by J. Spain, Tyndall Air Force Base, Florida. Clostridium, Bacillus, and Dehalococcoides genomic DNA were kindly provided by J. Weiss, P. Dees, and A. Carroll, respectively, of Cornell University.
PY - 2002
Y1 - 2002
N2 - The ideal ecological metabolic activity assay would be applied to naturally occurring microbial populations immediately fixed in the field, and the assay would focus upon intracellular parameters indicative of a dynamic biogeochemical process. In this study, fluorescent in situ hybridization (FISH) with tyramide signal amplification (TSA) detected intracellular mRNA in bacteria. Detection sensitivity was enhanced by using a Hamamatsu color chilled CCD camera and extended exposure times. Pseudomonas putida NCIB 9816-4, a model naphthalene degrading bacterium, was used to refine the protocol. Probe Ac627BR was developed for detecting naphthalene dioxygenase (nahAc) mRNA transcripts. Only induced cells showed positive hybridization to probe Ac627BR. Results were verified by RNase A or DNase I digestion of samples prior to hybridization. When applied to field-fixed groundwater samples, the naphthalene dioxygenase mRNA probe conferred fluorescence on a subset (∼1%) of the cells present in the contaminated groundwater. This methodology represents progress towards achieving one of the longstanding goals of environmental microbiology: to simultaneously ascertain the identity, activity, and biogeochemical impact of individual microorganisms in situ - in soil, water, or sediment where they dwell.
AB - The ideal ecological metabolic activity assay would be applied to naturally occurring microbial populations immediately fixed in the field, and the assay would focus upon intracellular parameters indicative of a dynamic biogeochemical process. In this study, fluorescent in situ hybridization (FISH) with tyramide signal amplification (TSA) detected intracellular mRNA in bacteria. Detection sensitivity was enhanced by using a Hamamatsu color chilled CCD camera and extended exposure times. Pseudomonas putida NCIB 9816-4, a model naphthalene degrading bacterium, was used to refine the protocol. Probe Ac627BR was developed for detecting naphthalene dioxygenase (nahAc) mRNA transcripts. Only induced cells showed positive hybridization to probe Ac627BR. Results were verified by RNase A or DNase I digestion of samples prior to hybridization. When applied to field-fixed groundwater samples, the naphthalene dioxygenase mRNA probe conferred fluorescence on a subset (∼1%) of the cells present in the contaminated groundwater. This methodology represents progress towards achieving one of the longstanding goals of environmental microbiology: to simultaneously ascertain the identity, activity, and biogeochemical impact of individual microorganisms in situ - in soil, water, or sediment where they dwell.
UR - http://www.scopus.com/inward/record.url?scp=0036220879&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0036220879&partnerID=8YFLogxK
U2 - 10.1016/S0167-7012(02)00015-5
DO - 10.1016/S0167-7012(02)00015-5
M3 - Article
C2 - 11943360
AN - SCOPUS:0036220879
SN - 0167-7012
VL - 50
SP - 75
EP - 84
JO - Journal of Microbiological Methods
JF - Journal of Microbiological Methods
IS - 1
ER -