Utility of Environmental Primers Targeting Ancient Enzymes: Methylotroph Detection in Lake Washington

• Published in: Microbial ecology Vol. 48; no. 4; pp. 463 - 472
• Main Authors: Kalyuzhnaya, M. G., Lidstrom, M. E., Chistoserdova, L.
• Format: Journal Article
• Language: English
• Published: New York, NY Springer Science + Business Media, Inc 01.12.2004; Springer; Springer Nature B.V
• Subjects: Bacteria; Bacteria - classification; Bacteria - enzymology; Bacteria - genetics; Bacterial Proteins - classification; Bacterial Proteins - genetics; Bacteriology; Base Sequence; Biological and medical sciences; DNA; DNA Primers; Enzymes; Euryarchaeota - classification; Euryarchaeota - enzymology; Euryarchaeota - genetics; Freshwater; Fundamental and applied biological sciences. Psychology; Genetic Variation; Geologic Sediments - microbiology; Hyphomicrobium; Lake sediments; Lakes; Methane; Methylobacter; Methylobacteriaceae - classification; Methylobacteriaceae - enzymology; Methylobacteriaceae - genetics; Methylobacterium; Methylococcaceae; Methylococcus; Methylocystis; Methylomicrobium; Methylomonas; Methylosinus; Microbiology; Microorganisms; Miscellaneous; Molecular Sequence Data; Phylogenetics; Polymerase chain reaction; Product category rules; Proteobacteria - classification; Proteobacteria - enzymology; Proteobacteria - genetics; Sediments; Washington; Water Microbiology; Xanthobacter; United States; North America; Washington; America; USA, Washington, Washington L; Methylocystis; Targeting; Methylomonas; Enzyme; Methylococcaceae; Method; Phylogeny; Lake sediments; Methylobacter; Methylotrophy; Gene; Melanin concentrating hormone; Bacteria; Environment; Methylosinus; Microorganism; Detection
• ISSN: 0095-3628; 1432-184X
• DOI: 10.1007/s00248-004-0212-6

Methods have been explored for detection of methylotrophs in natural samples, using environmental primers based on genes involved in the tetrahydromethanopterin (H₄MPT)-linked C₁ transfer pathway. The underlying hypotheses were that the H₄MPT-linked pathway is an ancient methylotrophy pathway, based on gene divergence, and that primers targeting more divergent genes will detect a broader variety of methylotrophs compared to the variety uncovered using probes and primers targeting highly conserved genes. Three groups of novel primer sets were developed targeting mch, mtdB, and fae, key genes in the H₄MPT-linked pathway, and these were used to assess the variety of microorganisms possessing these genes in sediments from Lake Washington in Seattle, WA. Environmental clone libraries were constructed for each of the genes and were analyzed by RFLP, and representatives of different RFLP groups were sequenced and subjected to phylogenetic analysis. A combination of all three sets of novel primers allowed detection of the two previously characterized groups of methylotrophs in the site: methanotrophs of the (α- and the γ-proteobacterial groups, belonghg to genera Methylosinus, Methylocystis, Methylomonas, Methylobacter, Methylomicrobium, and Methylococcus. In addition to the genes belonging to known methanotroph populations, novel genes were identified, suggesting existence of previously undetected microbial groups possessing C₁ transfer functions in this site. These included sequences clustering with the well-characterized methylotrophic phyla, Methylobacterium, Hyphomicrobium, and Xanthobacter. In addition, sequences divergent from those known for any groups of methylotrophs or methanogens were obtained, suggesting the presence of a yet unidentified microbial group possessing this H₄MPT-linked C₁ transfer pathway.