Methylomonas rivi sp. nov., Methylomonas rosea sp. nov., Methylomonas aurea sp. nov. and Methylomonas subterranea 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^T, WSC-7^T, SURF-1^T, and SURF-2^T, 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^T and WSC-7^T grew optimally at lower temperatures (25 and 20 °C, respectively) compared to strains SURF-1^T and SURF-2^T (40 °C). Strains WSC-6^T and SURF-2^T were neutrophilic (optimal pH of 7.5 and 7.3, respectively), while strains WSC-7^T and SURF-1^T 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_14 : 0, C_16 : 1  ω8c, C_16 : 1  ω7c, and C_16 : 1  ω5c. The DNA G+C content ranged from 51.5 to 56.0 mol%. Phylogenetic analyses indicated that the strains belonged to the genus Methylomonas, 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 Methylomonas, for which the names Methylomonas rivi (type strain WSC-6^T=ATCC TSD-251^T=DSM 112293^T), Methylomonas rosea (type strain WSC-7^T=ATCC TSD-252^T=DSM 112281^T), Methylomonas aurea (type strain SURF-1^T=ATCC TSD-253^T=DSM 112282^T), and Methylomonas subterranea (type strain SURF-2^T=ATCC TSD-254^T=DSM 112283^T) are proposed.