sp. nov., sp. nov., sp. nov. and sp. nov., type I methane-oxidizing bacteria isolated from a freshwater creek and the deep terrestrial subsurface.

Four methane-oxidizing bacteria, designated as strains WSC-6, WSC-7, SURF-1, and SURF-2, were isolated from Saddle Mountain Creek in southwestern Oklahoma, USA, and the Sanford Underground Research Facility (SURF) in Lead, South Dakota, USA. The strains were Gram-negative, motile, short rods that possessed intracytoplasmic membranes characteristic of type I methanotrophs. All four strains were oxidase-negative and weakly catalase-positive. Colonies ranged from pale pink to orange in colour. Methane and methanol were the only compounds that could serve as carbon and energy sources for growth. Strains WSC-6 and WSC-7 grew optimally at lower temperatures (25 and 20 °C, respectively) compared to strains SURF-1 and SURF-2 (40 °C). Strains WSC-6 and SURF-2 were neutrophilic (optimal pH of 7.5 and 7.3, respectively), while strains WSC-7 and SURF-1 were slightly alkaliphilic, with an optimal pH of 8.8. The strains grew best in media amended with ≤0.5% NaCl. The major cellular fatty acids were C, C  8, C  7, and C  5. The DNA G+C content ranged from 51.5 to 56.0 mol%. Phylogenetic analyses indicated that the strains belonged to the genus , with each exhibiting 98.6-99.6% 16S rRNA gene sequence similarity to closely related strains. Genome-wide estimates of relatedness (84.5-88.4% average nucleotide identity, 85.8-92.4% average amino acid identity and 27.4-35.0% digital DNA-DNA hybridization) fell below established thresholds for species delineation. Based on these combined results, we propose to classify these strains as representing novel species of the genus , for which the names (type strain WSC-6=ATCC TSD-251=DSM 112293), (type strain WSC-7=ATCC TSD-252=DSM 112281), (type strain SURF-1=ATCC TSD-253=DSM 112282), and (type strain SURF-2=ATCC TSD-254=DSM 112283) are proposed.