Family- and genus-level 16S rRNA-targeted oligonucleotide probes for ecological studies of methanotrophic bacteria

Methanotrophic bacteria play a major role in the global carbon cycle, degra de xenobiotic pollutants, and have the potential for a variety of biotechno logical applications. To facilitate ecological studies of these important o rganisms, we developed a suite of oligonucleotide probes for quantitative a nalysis of methanotroph-specific 16S rRNA from environmental samples. Two p robes target methanotrophs in the family Methylocystaceae (type II methanot rophs) as a group. No oligonucleotide signatures that distinguish between t he two genera in this family, Methylocystis and Methylosinus, were identifi ed. Two other probes target, as a single group, a majority of the known met hanotrophs belonging to the family Methylococcaceae (type I/X methanotrophs ). The remaining probes target members of individual genera of the Methyloc occaceae, including Methylobacter, Methylomonas, Methylomicrobium, Methyloc occus, and Methylocaldum. One of the family-level probes also covers all me thanotrophic endosymbionts of marine mollusks for which 16S rRNA sequences have been published. The two known species of the newly described genus Met hylosarcina gen. nov. are covered by a probe that otherwise targets only me mbers of the closely related genus Methylomicrobium. None of the probes cov ers strains of the newly proposed genera Methylocella and "Methylothermus," which are polyphyletic with respect to the recognized methanotrophic famil ies. Empirically determined midpoint dissociation temperatures were 49 to 5 7 degreesC for all probes. In dot blot screening against RNA from positive- and negative-control strains, the probes were specific to their intended t argets. The broad coverage and high degree of specificity of this new suite of probes will provide more detailed, quantitative information about the c ommunity structure of methanotrophs in environmental samples than was previ ously available.