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Applied and Environmental Microbiology, October 2001, p. 4726-4733, Vol. 67, No. 10
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.10.4726-4733.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Family- and Genus-Level 16S rRNA-Targeted Oligonucleotide Probes for Ecological Studies of Methanotrophic Bacteria

Jay Gulledge,1,dagger Azeem Ahmad,1 Paul A. Steudler,2 William J. Pomerantz,1 and Colleen M. Cavanaugh1,*

The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138,1 and The Ecosystems Center, Marine Biological Laboratory, Woods Hole, Massachusetts 025432

Received 9 April 2001/Accepted 17 July 2001

Methanotrophic bacteria play a major role in the global carbon cycle, degrade xenobiotic pollutants, and have the potential for a variety of biotechnological applications. To facilitate ecological studies of these important organisms, we developed a suite of oligonucleotide probes for quantitative analysis of methanotroph-specific 16S rRNA from environmental samples. Two probes target methanotrophs in the family Methylocystaceae (type II methanotrophs) as a group. No oligonucleotide signatures that distinguish between the two genera in this family, Methylocystis and Methylosinus, were identified. Two other probes target, as a single group, a majority of the known methanotrophs belonging to the family Methylococcaceae (type I/X methanotrophs). The remaining probes target members of individual genera of the Methylococcaceae, including Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, and Methylocaldum. One of the family-level probes also covers all methanotrophic endosymbionts of marine mollusks for which 16S rRNA sequences have been published. The two known species of the newly described genus Methylosarcina gen. nov. are covered by a probe that otherwise targets only members of the closely related genus Methylomicrobium. None of the probes covers strains of the newly proposed genera Methylocella and "Methylothermus," which are polyphyletic with respect to the recognized methanotrophic families. Empirically determined midpoint dissociation temperatures were 49 to 57°C for all probes. In dot blot screening against RNA from positive- and negative-control strains, the probes were specific to their intended targets. The broad coverage and high degree of specificity of this new suite of probes will provide more detailed, quantitative information about the community structure of methanotrophs in environmental samples than was previously available.

* Corresponding author. Mailing address: Harvard University, The Biological Laboratories, 16 Divinity Ave., Cambridge, MA 02138. Phone: (617) 496-2177. Fax: (617) 496-6933. E-mail: cavanaug{at}fas.harvard.edu.

dagger Present address: Department of Ecology and Evolutionary Biology, Tulane University, New Orleans, LA 70118.

Applied and Environmental Microbiology, October 2001, p. 4726-4733, Vol. 67, No. 10
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.10.4726-4733.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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