The molecular diversity of the methanogenic community in a hypereutrophic freshwater lake determined by PCR-RFLP.
OBJECTIVE
To combine database-held sequence information with a programme of experimental molecular ecology to define the methanogenic community of a hypereutrophic lake by a PCR-restriction fragment length polymorphism (RFLP) analysis.


RESULTS
Methanogen diversity in a hypereutrophic freshwater lake was analysed using 16S rDNA PCR-RFLP. Database-held 16S rRNA gene sequences for 76 diverse methanogens were analysed for specific restriction sites that permitted unequivocal differentiation of methanogens. Restriction digestion and agarose gel electrophoresis of the 16S rDNA from selected methanogen pure cultures generated observed restriction profiles that corroborated the expected patterns. This method was then tested by analysing methanogen diversity in samples obtained over 1 year from sediment and water samples taken from the same sampling site.


CONCLUSIONS
Restriction analysis of the 16S rRNA gene sequences from 157 methanogen clones generated from lakewater and sediment samples showed that over 50% were similar to Methanoculleus spp. Furthermore, a total of 16 RFLP types (1-16) were identified, eight of which contained no cultured representative archaeal 16S rRNA gene sequences.


CONCLUSIONS
This RFLP strategy provides a robust and reliable means to rapidly identify methanogens in the environment.
Whitby
C
C
School of Biological Sciences, University of Liverpool, UK.
Earl
J
J
Lanyon
C
C
Gray
S
S
Robinson
J
J
Meadows
J
J
Edwards
C
C
D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004784:Environmental Monitoring / Q000379:methods; D019605:Euryarchaeota / Q000145:classification / Q000235:genetics*; D005618:Fresh Water / Q000382:microbiology*; D014644:Genetic Variation; D019015:Geologic Sediments; D008697:Methane / Q000378:metabolism*; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D008697:Methane