ANALYSIS OF 16S RIBOSOMAL-RNA AND METHANE MONOOXYGENASE GENE-SEQUENCES REVEALS A NOVEL GROUP OF THERMOTOLERANT AND THERMOPHILIC METHANOTROPHS, METHYLOCALDUM GEN. NOV

Two methanotrophic bacteria with optimum growth temperatures above 40 degrees C were isolated. Thermotolerant strain LK6 was isolated from a gricultural soil, and the moderately thermophilic strain OR2 was isola ted from the effluent of an underground hot spring. When compared to t he described thermophilic methanotrophs Methylococcus capsulatus and M ethylococcus thermophilus, these strains are phenotypically similar to Methylococcus thermophilus. However, their 16S rRNA gene sequences ar e markedly different from the sequence of Methylococcus thermophilus ( similar to 8% divergence) and, together with Methylomonas gracilis, th ey form a distinct, new genus within the gamma-subgroup of the Proteob acteria related to extant Type I methanotrophs. Further phenotypic cha racterisation showed that the isolates possess particulate methane mon ooxygenase (pMMO) but do not contain soluble methane monooxygenase. Th e nucleotide sequence of a gene encoding pMMO (pmoA) was determined fo r both isolates and for Methylomonas gracilis. PmoA sequence compariso ns confirmed the monophyletic nature of this newly recognised group of thermophilic methanotrophs and their relationship to previously descr ibed Type I methanotrophs. We propose that strains OR2 and LK6, togeth er with the misclassified thermophilic strains Methylomonas gracilis V KM-14L(T) and Methylococcus thermophilus IMV-B3122, comprise a new gen us of thermophilic methanotrophs, Methylocaldum gen. nov., containing three new species: Methylocaldum szegediense, Methylocaldum tepidum an d Methylocaldum gracile.