Detection of novel marine methanotrophs using phylogenetic and functional gene probes after methane enrichment.
A major limitation of rRNA-targeted group-specific probes is that they may cross-react with organisms of other physiological, or even phylogenetic groups when applied to environmental samples containing unknown sequences. We have exploited the restricted physiology of methane-oxidizing bacteria to assess the specificity and efficiency of probes for this physiological type which target the 16S rRNA or genes involved in methanotroph physiology. Seawater samples were enriched for methanotrophs by addition of methane and essential nutrients. The changes in composition of the bacterial population were monitored by analysis of 16S rRNA gene libraries. Methanotroph group-specific probes failed to give a signal with samples from these enrichments even though a methanol dehydrogenase structural gene was detected. A 16S rDNA sequence that was abundant only after methane addition was recovered and found to show a close phylogenetic relationship to Methylomonas. Organisms containing this sequence were observed in enrichments by in situ hybridization. The combination of enrichment on methane and screening with the broad specificity methanol dehydrogenase probe allowed detection of novel methanotrophs that were not detected with the original suite of methanotroph group-specific probes.
Holmes
Andrew J
AJ
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
Owens
Nick J P
NJP
Department of Marine Sciences and Coastal Management, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, UK.
Murrell
J Colin
JC
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
D000429:Alcohol Oxidoreductases / Q000235:genetics; D001483:Base Sequence; D004275:DNA, Ribosomal / Q000235:genetics*; D017753:Ecosystem; D004783:Environmental Microbiology; D015723:Gene Library; D017404:In Situ Hybridization, Fluorescence; D008697:Methane / Q000378:metabolism*; D008772:Methylococcaceae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D010105:Oxygenases / Q000235:genetics; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics*
D004275:DNA, Ribosomal; D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor); D010105:Oxygenases; C021555:methane monooxygenase; D008697:Methane