Two strains of Gram-negative, aerobic, non-pigmented, non-motile, rod-shaped bacteria were isolated from beechwood blocks during decay by the white-rot fungus Hypholoma fasciculare and were designated strains BW863(T) and BW872. They are capable of methylotrophic growth and assimilate carbon via the ribulose-bisphosphate pathway. In addition to methanol, the novel isolates utilized ethanol, pyruvate and malate. Strains BW863(T) and BW872 are obligately acidophilic, mesophilic organisms capable of growth at pH 3.1-6.5 (with an optimum at pH 4.5-5.0) and at 4-30 degrees C. Phospholipid fatty acid profiles of these bacteria contain unusually large amounts (about 90 %) of C(18 : 1)omega7c, thereby resembling the profiles of Methylobacterium strains. The predominant quinone is Q-10. The DNA G+C content of the novel isolates is 61.8-62.8 mol%. On the basis of 16S rRNA gene sequence similarity, strains BW863(T) and BW872 are most closely related to the acidophilic methanotroph Methylocapsa acidiphila B2(T) (96.5-97 %). Comparative sequence analysis of mxaF, the gene encoding the large subunit of methanol dehydrogenase, placed the MxaF sequences of the two novel strains in a cluster that is distinct from all previously described MxaF sequences of cultivated methylotrophs. The identity between the MxaF sequences of the acidophilic isolates and those from known alpha-, beta- and gammaproteobacterial methylotrophs was respectively 69-75, 61-63 and 64-67 %. The data therefore suggest that strains BW863(T) and BW872 represent a novel genus and species of methylotrophic bacteria, for which the name Methylovirgula ligni gen. nov., sp. nov. is proposed. Strain BW863(T) (=DSM 19998(T) =NCIMB 14408(T)) is the type strain of Methylovirgula ligni.