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Applied and Environmental Microbiology, November 2005, p. 6885-6899, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.6885-6899.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Bacterial Populations Active in Metabolism of C1 Compounds in the Sediment of Lake Washington, a Freshwater Lake

Olivier Nercessian,1,{dagger} Emma Noyes,2,{ddagger} Marina G. Kalyuzhnaya,1 Mary E. Lidstrom,1,3 and Ludmila Chistoserdova1*

Department of Chemical Engineering,1 Department of Microbiology, University of Washington, Seattle, Washington 98195,3 Omak High School, Omak, Washington 988412

Received 14 April 2005/ Accepted 13 June 2005

Active members of the bacterial community in the sediment of Lake Washington, with special emphasis on C1 utilizers, were identified by employing two complementary culture-independent approaches: reverse transcription of environmental mRNA and 16S rRNA combined with PCR (RT-PCR) and stable-isotope probing (SIP) of DNA with the 13C-labeled C1 substrates methanol, methylamine, formaldehyde, and formate. Analysis of RT-PCR-amplified fragments of 16S rRNA-encoding genes revealed that gammaproteobacterial methanotrophs belonging to Methylobacter and Methylomonas dominate the active methylotroph population, while only one other known methylotrophic lineage, Methylophilaceae, was detected via this approach. Analysis of RT-PCR-amplified functional genes, pmoA and fae, allowed detection of alphaproteobacterial (Methylosinus) and gammaproteobacterial (Methylobacter, Methylomonas, and Methylomicrobium) methanotrophs, methylotrophs of the genus Methylobacterium, and yet-unidentified proteobacteria. SIP experiments allowed detection of a broad variety of groups actively metabolizing C1 compounds. Comparisons between 16S rRNA gene pools amplified from [13C]DNA and from [12C]DNA revealed that the proportion of Methylophilus-related sequences increased in the presence of [13C]methanol, [13C]methylamine, and [13C]formaldehyde; Novosphingobium-related sequences were enriched in the presence of [13C]methanol; Gemmatimonadaceae-related sequences were enriched in the presence of [13C]formaldehyde and [13C]formate; and Xanthomonadaceae-related sequences were enriched in the presence of [13C]formate. Analysis of fae genes amplified from [13C]DNAs isolated from different microcosms revealed specific shifts in populations in response to a specific C1 compound: Methylosinus sequences dominated the [13C]methanol microcosm pool, and beta- and gammaproteobacterial sequences dominated the [13C]methylamine microcosm pool. The [13C]formaldehyde microcosm was dominated by betaproteobacterial sequences and by sequences of a nonaffiliated group, while the [13C]formate microcosm was dominated by alpha- and betaproteobacterial sequences. Overall, these data point toward the presence of a diverse population of active methylotrophs in Lake Washington sediments and toward the existence of yet-uncultivated organisms.

* Corresponding author. Mailing address: 231 Wilcox Hall, Box 352125, University of Washington, Seattle, WA 98195. Phone: (206) 543-6683. Fax: (206) 616-5721. E-mail: milachis{at}u.washington.edu.

{dagger} Present address: CEA/Cadarache, DSV/DEVM/LEMiR, Bat 161, 13108 St-Paul lez Durance, France.

{ddagger} Present address: Department of Microbiology, University of Washington, Seattle, WA 98195.

Applied and Environmental Microbiology, November 2005, p. 6885-6899, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.6885-6899.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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