Identification of active methylotroph populations in an acidic forest soil by stable-isotope probing

S. Radajewski, D. S. Reay, S. A. Morris, P. Ineson, D. B. Nedwell, James Ivor Prosser, J. C. Murrell, G. Webster

Research output: Contribution to journalArticle

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Abstract

Stable-isotope probing (SIP) is a culture-independent technique that enables the isolation of DNA from micro-organisms that are actively involved in a specific metabolic process. In this study, SIP was used to characterize the active methylotroph populations in forest soil (pH 3(.)5) microcosms that were exposed to (CH3)-C-13 OH or (CH4)-C-13- Distinct C-13-labelled DNA (13 C-DNA) fractions were resolved from total community DNA by CsCl density-gradient centrifugation. Analysis of 16S rDNA sequences amplified from the 13 C-DNA revealed that bacteria related to the genera Methylocella, Methylocapsa, Methylocystis and Rhodoblastus had assimilated the C-13-labelled substrates, which suggested that moderately acidophilic methylotroph populations were active in the microcosms.

Enrichments targeted towards the active proteobacterial CH3OH utilizers were successful, although none of these bacteria were isolated into pure culture. A parallel analysis of genes encoding the key enzymes methanol dehydrogenase and particulate methane monooxygenase reflected the 16S rDNA analysis, but unexpectedly revealed sequences related to the ammonia monooxygenase of ammonia-oxidizing bacteria (AOB) from the beta-subclass of the Proteobacteria. Analysis of AOB-selective 165 rDNA amplification products identified

Nitrosomonas and Nitrosospira sequences in the C-13-DNA fractions, suggesting certain AOB assimilated a significant proportion of (CO2)-C-13, possibly through a close physical and/or nutritional association with the active methylotrophs. Other sequences retrieved from the C-13-DNA were related to the 165 rDNA sequences of members of the Acidobacterium division, the beta-Proteobacteria and the order Cytophagales, which implicated these bacteria in the assimilation of reduced one-carbon compounds or in the assimilation of the by-products of methylotrophic carbon metabolism. Results from the (CH3OH)-C-13 and (CH4)-C-13 SIP experiments thus provide a rational basis for further investigations 4 into the ecology of methylotroph populations in situ.

Original languageEnglish
Pages (from-to)2331-2342
Number of pages11
JournalMicrobiology
Volume148
Issue numberPt 8
Publication statusPublished - 2002

Keywords

  • methanotrophs
  • community structure
  • culture-independent techniques
  • C-13
  • functional genes
  • 16S RIBOSOMAL-RNA
  • PARTICULATE METHANE MONOOXYGENASE
  • DEHYDROGENASE STRUCTURAL GENE
  • OXIDIZE ATMOSPHERIC METHANE
  • IN-SITU HYBRIDIZATION
  • METHANOTROPHIC COMMUNITIES
  • METHYLOCELLA-PALUSTRIS
  • AMMONIA MONOOXYGENASE
  • MOLECULAR ANALYSES
  • MICROBIAL ECOLOGY

Cite this

Radajewski, S., Reay, D. S., Morris, S. A., Ineson, P., Nedwell, D. B., Prosser, J. I., ... Webster, G. (2002). Identification of active methylotroph populations in an acidic forest soil by stable-isotope probing. Microbiology , 148(Pt 8), 2331-2342.

Identification of active methylotroph populations in an acidic forest soil by stable-isotope probing. / Radajewski, S.; Reay, D. S.; Morris, S. A.; Ineson, P.; Nedwell, D. B.; Prosser, James Ivor; Murrell, J. C.; Webster, G.

In: Microbiology , Vol. 148, No. Pt 8, 2002, p. 2331-2342.

Research output: Contribution to journalArticle

Radajewski, S, Reay, DS, Morris, SA, Ineson, P, Nedwell, DB, Prosser, JI, Murrell, JC & Webster, G 2002, 'Identification of active methylotroph populations in an acidic forest soil by stable-isotope probing', Microbiology , vol. 148, no. Pt 8, pp. 2331-2342.
Radajewski S, Reay DS, Morris SA, Ineson P, Nedwell DB, Prosser JI et al. Identification of active methylotroph populations in an acidic forest soil by stable-isotope probing. Microbiology . 2002;148(Pt 8):2331-2342.
Radajewski, S. ; Reay, D. S. ; Morris, S. A. ; Ineson, P. ; Nedwell, D. B. ; Prosser, James Ivor ; Murrell, J. C. ; Webster, G. / Identification of active methylotroph populations in an acidic forest soil by stable-isotope probing. In: Microbiology . 2002 ; Vol. 148, No. Pt 8. pp. 2331-2342.
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AU - Reay, D. S.

AU - Morris, S. A.

AU - Ineson, P.

AU - Nedwell, D. B.

AU - Prosser, James Ivor

AU - Murrell, J. C.

AU - Webster, G.

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N2 - Stable-isotope probing (SIP) is a culture-independent technique that enables the isolation of DNA from micro-organisms that are actively involved in a specific metabolic process. In this study, SIP was used to characterize the active methylotroph populations in forest soil (pH 3(.)5) microcosms that were exposed to (CH3)-C-13 OH or (CH4)-C-13- Distinct C-13-labelled DNA (13 C-DNA) fractions were resolved from total community DNA by CsCl density-gradient centrifugation. Analysis of 16S rDNA sequences amplified from the 13 C-DNA revealed that bacteria related to the genera Methylocella, Methylocapsa, Methylocystis and Rhodoblastus had assimilated the C-13-labelled substrates, which suggested that moderately acidophilic methylotroph populations were active in the microcosms.Enrichments targeted towards the active proteobacterial CH3OH utilizers were successful, although none of these bacteria were isolated into pure culture. A parallel analysis of genes encoding the key enzymes methanol dehydrogenase and particulate methane monooxygenase reflected the 16S rDNA analysis, but unexpectedly revealed sequences related to the ammonia monooxygenase of ammonia-oxidizing bacteria (AOB) from the beta-subclass of the Proteobacteria. Analysis of AOB-selective 165 rDNA amplification products identifiedNitrosomonas and Nitrosospira sequences in the C-13-DNA fractions, suggesting certain AOB assimilated a significant proportion of (CO2)-C-13, possibly through a close physical and/or nutritional association with the active methylotrophs. Other sequences retrieved from the C-13-DNA were related to the 165 rDNA sequences of members of the Acidobacterium division, the beta-Proteobacteria and the order Cytophagales, which implicated these bacteria in the assimilation of reduced one-carbon compounds or in the assimilation of the by-products of methylotrophic carbon metabolism. Results from the (CH3OH)-C-13 and (CH4)-C-13 SIP experiments thus provide a rational basis for further investigations 4 into the ecology of methylotroph populations in situ.

AB - Stable-isotope probing (SIP) is a culture-independent technique that enables the isolation of DNA from micro-organisms that are actively involved in a specific metabolic process. In this study, SIP was used to characterize the active methylotroph populations in forest soil (pH 3(.)5) microcosms that were exposed to (CH3)-C-13 OH or (CH4)-C-13- Distinct C-13-labelled DNA (13 C-DNA) fractions were resolved from total community DNA by CsCl density-gradient centrifugation. Analysis of 16S rDNA sequences amplified from the 13 C-DNA revealed that bacteria related to the genera Methylocella, Methylocapsa, Methylocystis and Rhodoblastus had assimilated the C-13-labelled substrates, which suggested that moderately acidophilic methylotroph populations were active in the microcosms.Enrichments targeted towards the active proteobacterial CH3OH utilizers were successful, although none of these bacteria were isolated into pure culture. A parallel analysis of genes encoding the key enzymes methanol dehydrogenase and particulate methane monooxygenase reflected the 16S rDNA analysis, but unexpectedly revealed sequences related to the ammonia monooxygenase of ammonia-oxidizing bacteria (AOB) from the beta-subclass of the Proteobacteria. Analysis of AOB-selective 165 rDNA amplification products identifiedNitrosomonas and Nitrosospira sequences in the C-13-DNA fractions, suggesting certain AOB assimilated a significant proportion of (CO2)-C-13, possibly through a close physical and/or nutritional association with the active methylotrophs. Other sequences retrieved from the C-13-DNA were related to the 165 rDNA sequences of members of the Acidobacterium division, the beta-Proteobacteria and the order Cytophagales, which implicated these bacteria in the assimilation of reduced one-carbon compounds or in the assimilation of the by-products of methylotrophic carbon metabolism. Results from the (CH3OH)-C-13 and (CH4)-C-13 SIP experiments thus provide a rational basis for further investigations 4 into the ecology of methylotroph populations in situ.

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KW - community structure

KW - culture-independent techniques

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KW - functional genes

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KW - PARTICULATE METHANE MONOOXYGENASE

KW - DEHYDROGENASE STRUCTURAL GENE

KW - OXIDIZE ATMOSPHERIC METHANE

KW - IN-SITU HYBRIDIZATION

KW - METHANOTROPHIC COMMUNITIES

KW - METHYLOCELLA-PALUSTRIS

KW - AMMONIA MONOOXYGENASE

KW - MOLECULAR ANALYSES

KW - MICROBIAL ECOLOGY

M3 - Article

VL - 148

SP - 2331

EP - 2342

JO - Microbiology

JF - Microbiology

SN - 1350-0872

IS - Pt 8

ER -