title: Detection in coal tar waste-contaminated groundwater of mRNA transcripts related to naphthalene dioxygenase by fluorescent in situ hybridization with tyramide signal amplification.
abstract: 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 (approximately 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.
authors.affiliation: Department of Microbiology, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Corien
authors.initials: C
authors.lastname: Bakermans
authors.forename: Eugene L
authors.initials: EL
authors.lastname: Madsen
mesh_terms: D001419:Bacteria / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification*; D003033:Coal Tar / Q000032:analysis*; D049308:Dioxygenases; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D009097:Multienzyme Complexes / Q000235:genetics*; D010105:Oxygenases / Q000235:genetics*; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014439:Tyramine / Q000031:analogs & derivatives; D014871:Water Microbiology; D014874:Water Pollutants, Chemical / Q000032:analysis*; D014881:Water Supply
chemical_list: D009097:Multienzyme Complexes; D012329:RNA, Bacterial; D012333:RNA, Messenger; D014874:Water Pollutants, Chemical; D003033:Coal Tar; D010105:Oxygenases; D049308:Dioxygenases; C038703:naphthalene dioxygenase; D014439:Tyramine