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 phylog enetic groups when applied to environmental samples containing unknown sequences. We have exploited the restricted physiology of methane-oxi dizing bacteria to assess the specificity and efficiency of probes for this physiological type which target the 165 rRNA or genes involved i n methanotroph physiology. Seawater samples were enriched for methanot rophs by addition of methane and essential nutrients. The changes in c omposition of the bacterial population were monitored by analysis of 1 6S rRNA gene libraries. Methanotroph group-specific probes failed to g ive a signal with samples from these enrichments even though a methano l dehydrogenase structural gene was detected. A 16S rDNA sequence that was abundant only after methane addition was recovered and found to s how a close phylogenetic relationship to Methylomonas. Organisms conta ining this sequence were observed in enrichments by in situ hybridizat ion. The combination of enrichment on methane and screening with the b road specificity methanol dehydrogenase probe allowed detection of nov el methanotrophs that were not detected with the original suite of met hanotroph group-specific probes.