Neufeld, Josh D., Chen, Yin, Dumont, Marc G. and Murrell, J. C. (J. Colin). (2008) Marine methylotrophs revealed by stable-isotope probing, multiple displacement amplification and metagenomics. Environmental Microbiology, Vol.10 (No.6). pp. 1526-1535. ISSN 14622912

Full text not available from this repository.

Abstract

The concentrations of one-carbon substrates that fuel methylotrophic microbial communities in the ocean are limited and the specialized guilds of bacteria that use these molecules may exist at low relative abundance. As a result, these organisms are difficult to identify and are often missed with existing cultivation and gene retrieval methods. Here, we demonstrate a novel proof of concept: using environmentally-relevant substrate concentrations in stable-isotope probing (SIP) incubations to yield sufficient DNA for large-insert metagenomic analysis through multiple displacement amplification (MDA). A marine surface-water sample was labelled sufficiently by incubation with near in situ concentrations of methanol. Picogram quantities of labelled C-13-DNA were purified from caesium chloride gradients, amplified with MDA to produce microgram amounts of high-molecular-weight DNA (<= 40 kb) and cloned to produce a fosmid library of > 10 000 clones. Denaturing gradient gel electrophoresis (DGGE) demonstrated minimal bias associated with the MDA step and implicated Methylophaga-like phylotypes with the marine metabolism of methanol. Polymerase chain reaction screening of 1500 clones revealed a methanol dehydrogenase (MDH) containing insert and shotgun sequencing of this insert resulted in the assembly of a 9-kb fragment of DNA encoding a cluster of enzymes involved in MDH biosynthesis, regulation and assembly. This novel combination of methodology enables future structure-function studies of microbial communities to achieve the long-desired goal of identifying active microbial populations using in situ conditions and performing a directed metagenomic analysis for these ecologically relevant microorganisms.

[error in script] [error in script]

Item Type:	Journal Article
Subjects:	Q Science > QP Physiology Q Science > QR Microbiology
Divisions:	Faculty of Science > Life Sciences (2010- ) > Biological Sciences ( -2010)
Library of Congress Subject Headings (LCSH):	Methylotrophic microorganisms, Marine microbiology, Stable isotope tracers, Metagenomics, Gene amplification, Methylotrophic bacteria, Polymerase chain reaction
Journal or Publication Title:	Environmental Microbiology
Publisher:	Wiley-Blackwell Publishing Ltd.
ISSN:	14622912
Date:	June 2008
Volume:	Vol.10
Number:	No.6
Number of Pages:	10
Page Range:	pp. 1526-1535
Identification Number:	10.1111/j.1462-2920.2008.01568.x
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Restricted or Subscription Access
Funder:	Natural Environment Research Council (Great Britain) (NERC), Natural Sciences and Engineering Research Council Canada (NSERC)
Grant number:	NE/C001 923/1 (NERC)
References:	Abulencia, C.B., Wyborski, D.L., Garcia, J.A., Podar, M., Chen, W., Chang, S.H., et al. (2006) Environmental wholegenome amplification to access microbial populations in contaminated sediments. Appl Environ Microbiol 72: 3291– 3301. Blanco, L., Bernad, A., Lazaro, J.M., Martin, G., Garmendia, C., and Salas, M. (1989) Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication. J Biol Chem 264: 8935–8940. Carpenter, L.J., Lewis, A.C., Hopkins, J.R., Read, K.A., Longley, I.D., and Gallagher, M.W. (2004) Uptake of methanol to the North Atlantic Ocean surface. Global Biogeochem Cycles 18: GB4027. Dean, F.B., Hosono, S., Fang, L., Wu, X., Faruqi, A.F., Bray- Ward, P., et al. (2002) Comprehensive human genome amplification using multiple displacement amplification. PNAS 99: 5261–5266. Dumont, M., and Murrell, J. (2005) Stable isotope probing – linking microbial identity to function. Nat Rev Microbiol 3: 499–504. Dumont, M.G., Radajewski, S.M., Miguez, C.B., McDonald, I.R., and Murrell, J.C. (2006) Identification of a complete methane monooxygenase operon from soil by combining stable isotope probing and metagenomic analysis. Environ Microbiol 8: 1240–1250. Friedrich, M.W. (2006) Stable-isotope probing of DNA: insights into the function of uncultivated microorganisms from isotopically labeled metagenomes. Curr Opin Biotechnol 17: 59. Galbally, I.E., and Kirstine, W. (2002) The production of methanol by flowering plants and the global cycle of methanol. J Atmos Chem 43: 195–229. Gonzalez, J.M., Portillo, M.C., and Saiz-Jimenez, C. (2005) Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environments. Environ Microbiol 7: 1024–1028. Goodwin, K.D., Varner, R.K., Crill, P.M., and Oremland, R.S. (2001) Consumption of tropospheric levels of methyl bromide by C1 compound-utilizing bacteria and comparison to saturation kinetics. Appl Environ Microbiol 67: 5437– 5443. Heikes, B.G., Chang, W., Pilson, M.E.Q., Swift, E., Singh, H.B., Guenther, A., et al. (2002) Atmospheric methanol budget and ocean implication. Global Biogeochem Cycles 16: 80–81. Hosono, S., Faruqi, A.F., Du Dean, F.B.Y., Sun, Z., Wu, X., et al. (2003) Unbiased whole-genome amplification directly from clinical samples. Genome Res 13: 954–964. Janvier, M., Frehel, C., Grimont, F., and Gasser, F. (1985) Methylophaga marina gen nov., sp. nov. and Methylophaga thalassica sp. nov., marine methylotrophs. Int J Syst Bacteriol 35: 131–139. Kiene, R.P. (1993) Microbial sources and sinks for methylated sulfur compounds in the marine environment. In Microbial Growth on C1 Compounds. Kelly, D.P., and Murrell, J.C. (eds). London, UK: Intercept, pp. 15–33. Lasken, R.S., and Stockwell, T.B. (2007) Mechanism of chimera formation during the multiple displacement amplification reaction. BMC Biotechnol 7: 19. Leckie, S.E., Prescott, C.E., Grayston, S.J., Neufeld, J.D., and Mohn, W.W. (2004) Characterization of humus microbial communities in adjacent forest types that differ in nitrogen availability. Microb Ecol 48: 29–40. Madsen, E.L. (2006) The use of stable isotope probing techniques in bioreactor and field studies on bioremediation. Curr Opin Biotechnol 17: 92–97. Naqvi, S.W.A., Bange, H.W., Gibb, S.W., Goyet, C., Hatton, A.D., and Upstill-Goddard, R.C. (2005) Biogeochemical ocean-atmosphere transfers in the Arabian Sea. Prog Oceanogr 65: 116. Neufeld, J.D., Dumont, M.G., Vohra, J., and Murrell, J.C. (2007a) Methodological considerations for the use of stable isotope probing in microbial ecology. Microb Ecol 53: 435–442. Neufeld, J.D., Schäfer, H., Cox, M.J., Boden, R., McDonald, I.R., and Murrell, J.C. (2007b) Stable-isotope probing implicates Methylophaga spp. and novel Gammaproteobacteria in marine methanol and methylamine metabolism. ISME J 1: 480–491. Neufeld, J.D., Wagner, M., and Murrell, J.C. (2007c) Who eats what, where and when? Isotope-labelling experiments are coming of age. ISME J 1: 103–110. Neufeld, J.D., Vohra, J., Dumont, M.G., Lueders, T., Manefield, M., Friedrich, M.W., and Murrell, J.C. (2007d) DNA stable-isotope probing. Nat Protoc 2: 860–866. Radajewski, S., Ineson, P., Parekh, N., and Murrell, J.C. (2000) Stable-isotope probing as a tool in microbial ecology. Nature 403: 646–649. Rappé, M.S., and Giovannoni, S.J. (2003) The uncultured microbial majority. Annu Rev Microbiol 57: 369–394. Ricke, P., Kube, M., Nakagawa, S., Erkel, C., Reinhardt, R., and Liesack, W. (2005) First genome data from uncultured upland soil cluster alpha methanotrophs provide further evidence for a close phylogenetic relationship to Methylocapsa acidiphila B2 and for high-affinity methanotrophy involving particulate methane monooxygenase. Appl Environ Microbiol 71: 7472–7482. Rusch, D.B., Halpern, A.L., Sutton, G., Heidelberg, K.B., Williamson, S., Yooseph, S., et al. (2007) The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical Pacific. PLoS Biol 5: e77. Sambrook, J., and Maniatis, T. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory Press. Schäfer, H. (2007) Isolation of Methylophaga spp. from marine dimethylsulfide-degrading enrichment cultures and identification of polypeptides induced during growth on dimethylsulfide. Appl Environ Microbiol 73: 2580–2591. Schloss, P.D., and Handelsman, J. (2004) Status of the microbial census. Microbiol Mol Biol Rev 68: 686–691. Singh, H.B., Tabazadeh, A., Evans, M.J., Field, B.D., Jacob, D.J., Sachse, G., et al. (2003) Oxygenated volatile organic chemicals in the oceans: inferences and implications based on atmospheric observations and air-sea exchange models. Geophys Res Lett 30: 1862. Stein, J.L., Marsh, T.L., Wu, K.Y., Shizuya, H., and DeLong, E.F. (1996) Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon. J Bacteriol 178: 591–599. Tringe, S.G., von Mering, C., Kobayashi, A., Salamov, A.A., Chen, K., Chang, H.W., et al. (2005) Comparative metagenomics of microbial communities. Science 308: 554–557. Tyson, G.W., Chapman, J., Hugenholtz, P., Allen, E.E., Ram, R.J., Richardson, P.M., et al. (2004) Community structure and metabolism through reconstruction of microbial genomes from the environment. Nature 428: 37–43. Venter, J.C., Remington, K., Heidelberg, J.F., Halpern, A.L., Rusch, D., Eisen, J.A., et al. (2004) Environmental genome shotgun sequencing of the Sargasso Sea. Science 304: 66–74. Vergin, K.L., Urbach, E., Stein, J.L., DeLong, E.F., Lanoil, B.D., and Giovannoni, S.J. (1998) Screening of a fosmid library of marine environmental genomic DNA fragments reveals four clones related to members of the order Planctomycetales. Appl Environ Microbiol 64: 3075–3078. Vila-Costa, M., del Valle, D.A., Gonzalez, J.M., Slezak, D., Kiene, R.P., Sanchez, O., and Simo, R. (2006) Phylogenetic identification and metabolism of marine dimethylsulfide-consuming bacteria. Environ Microbiol 8: 2189–2200. Wang, Q., Garrity, G.M., Tiedje, J.M., and Cole, J.R. (2007) A naïve Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73: 5261–5267. Williams, J., Holzinger, R., Gros, V., Xu, X., Atlas, E., and Wallace, D.W.R. (2004) Measurements of organic species in air and seawater from the tropical Atlantic. Geophys Res Lett 31: L23S06. Wu, L., Liu, X., Schadt, C.W., and Zhou, J. (2006) Microarraybased analysis of subnanogram quantities of microbial community DNAs by using whole-community genome amplification. Appl Environ Microbiol 72: 4931–4941. Yokouchi, H., Fukuoka, Y., Mukoyama, D., Calugay, R., Takeyama, H., and Matsunaga, T. (2006) Wholemetagenome amplification of a microbial community asso- ciated with scleractinian coral by multiple displacement amplification using f29 polymerase. Environ Microbiol 8: 1155–1163. Zhang, K., Martiny, A.C., Reppas, N.B., Barry, K.W., Malek, J., Chisholm, S.W., and Church, G.M. (2006) Sequencing genomes from single cells by polymerase cloning. Nat Biotechnol 24: 680–686.
URI:	http://wrap.warwick.ac.uk/id/eprint/30078

Data sourced from Thomson Reuters' Web of Knowledge

Request changes to a record

Actions (login required)

View Item