DNA-, rRNA- and mRNA-based stable isotope probing of aerobic methanotrophs in lake sediment.
A stable isotope probing (SIP) approach was used to study aerobic methane-oxidizing bacteria (methanotrophs) in lake sediment. Oligotrophic Lake Stechlin was chosen because it has a permanently oxic sediment surface. 16S rRNA and the pmoA gene, which encodes a subunit of the methane monooxygenase enzyme, were analysed following the incubation of sediment with (13) CH(4) and the separation of (13) C-labelled DNA and RNA from unlabelled nucleic acids. The incubation with (13) CH(4) was performed over a 4-day time-course and the pmoA genes and transcripts became progressively labelled such that approximately 70% of the pmoA genes and 80% of the transcripts were labelled at 96 h. The labelling of pmoA mRNA was quicker than pmoA genes, demonstrating that mRNA-SIP is more sensitive than DNA-SIP; however, the general rate of pmoA transcript labelling was comparable to that of the pmoA genes, indicating that the incorporation of (13) C into ribonucleic acids of methanotrophs was a gradual process. Labelling of Betaproteobacteria was clearly seen in analyses of 16S rRNA by DNA-SIP and not by RNA-SIP, suggesting that cross-feeding of the (13) C was primarily detected by DNA-SIP. In general, we show that the combination of SIP approaches provided valuable information about the activity and growth of the methanotrophic populations and the cross-feeding of methanotroph metabolites by other microorganisms.
Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch Strasse, Marburg, Germany. dumont@mpi-marburg.mpg.de
Marc G
MG
Dumont
Bianca
B
Pommerenke
Peter
P
Casper
Ralf
R
Conrad
D002247:Carbon Isotopes / Q000032:analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D019015:Geologic Sediments / Q000382:microbiology*; D008697:Methane / Q000378:metabolism*; D008772:Methylococcaceae / Q000145:classification* / Q000235:genetics / Q000378:metabolism; D015341:Nucleic Acid Probes / Q000737:chemistry; D010105:Oxygenases / Q000235:genetics; D010802:Phylogeny; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics; D017422:Sequence Analysis, DNA; D013997:Time Factors; D014871:Water Microbiology
D002247:Carbon Isotopes; D004269:DNA, Bacterial; D015341:Nucleic Acid Probes; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D010105:Oxygenases; C021555:methane monooxygenase; D008697:Methane