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} <i>ω</i>8<i>c</i>, C_{16 : 1} <i>ω</i>7<i>c</i>, and C_{16 : 1} <i>ω</i>5<i>c</i>. The DNA G+C content ranged from 51.5 to 56.0 mol%. Phylogenetic analyses indicated that the strains belonged to the genus <i>Methylomonas</i>, 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 <i>Methylomonas</i>, for which the names <i>Methylomonas rivi</i> (type strain WSC-6^T=ATCC TSD-251^T=DSM 112293^T), <i>Methylomonas rosea</i> (type strain WSC-7^T=ATCC TSD-252^T=DSM 112281^T), <i>Methylomonas aurea</i> (type strain SURF-1^T=ATCC TSD-253^T=DSM 112282^T), and <i>Methylomonas subterranea</i> (type strain SURF-2^T=ATCC TSD-254^T=DSM 112283^T) are proposed.