title: Comparative analysis of polychlorinated biphenyl-dechlorinating communities in enrichment cultures using three different molecular screening techniques.
abstract: The catalysts for many microbially mediated environmental processes such as the dechlorination of polychlorinated biphenyls (PCBs) have been difficult to identify by traditional isolation techniques. Numerous, as yet unsuccessful, attempts have been made to isolate and culture the dechlorinating species. To overcome this limitation, amplified rDNA restriction analysis (ARDRA) of a clone library, denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (TRFLP) were used concurrently to compare their effectiveness for characterizing an enriched microbial community. These methods were applied to enrichment cultures that selectively dechlorinated double-flanked chlorines in the PCB congener 2,3,4,5 chlorinated biphenyl. The methods have different biases, which were apparent from discrepancies in the relative clone frequencies (ARDRA), band intensities (DGGE) or peak heights (TRFLP) from the same enrichment culture. However, each method was effectively qualitative and identified the same organisms: a low G + C Gram-positive eubacterium, an organism most similar to the green non-sulphur bacteria, an Aminobacterium sp. and a Desulfovibrio sp. Overall, in community fingerprinting and preliminary identification, DGGE proved to be the most rapid and effective tool for the monitoring of microorganisms within a highly enriched culture. TRFLP results corroborated DGGE fingerprint analysis; however, identification required the additional step of creating a clone library. ARDRA provided an in-depth analysis of the community and this technique detected slight intraspecies sequence variation in 16S rDNA. These molecular methods are common in environmental microbiology, but rarely are they compared with the same sample site or culture. In general, all three methods detected similar community profiles, but inherent biases resulted in different detection limits for individual OTUs (operational taxonomic units).
authors.affiliation: Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 E. Pratt St., Baltimore, MD 21202, USA.
authors.forename: J E
authors.initials: JE
authors.lastname: Watts
authors.forename: Q
authors.initials: Q
authors.lastname: Wu
authors.forename: S B
authors.initials: SB
authors.lastname: Schreier
authors.forename: H D
authors.initials: HD
authors.lastname: May
authors.forename: K R
authors.initials: KR
authors.lastname: Sowers
mesh_terms: D000667:Ampicillin / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D016588:Anticarcinogenic Agents / Q000494:pharmacology; D001419:Bacteria / Q000145:classification / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D002713:Chlorine / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004275:DNA, Ribosomal / Q000032:analysis / Q000235:genetics; D004586:Electrophoresis / Q000379:methods; D005561:Formates / Q000494:pharmacology; D005650:Fumarates / Q000494:pharmacology; D008969:Molecular Sequence Data; D010406:Penicillins / Q000494:pharmacology; D010802:Phylogeny; D011078:Polychlorinated Biphenyls / Q000378:metabolism*; D012150:Polymorphism, Restriction Fragment Length; D014640:Vancomycin / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D016588:Anticarcinogenic Agents; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D005561:Formates; D005650:Fumarates; D010406:Penicillins; C030544:formic acid; D002713:Chlorine; D014640:Vancomycin; D000667:Ampicillin; C032005:fumaric acid; D011078:Polychlorinated Biphenyls; C078132:2,3,5,6-tetrachlorobiphenyl