Three actinobacterial strains, 27D-LEPIT, 1B-Mac and 36A-HELLB, were isolated from small standing and running freshwater habitats located in Salzburg, Austria. Phylogenetic reconstructions based on 16S rRNA gene sequences and genome based on concatenated amino acid sequences of 119 single-copy genes referred the three strains within the family Microbacteriaceae to the genus Rhodoluna. The strains showed 100 % 16S rRNA gene sequence similarities to the previously described Candidatus Rhodoluna limnophila. Cells were very small, approximately 0.5×0.3 µm, and showed a red pigmentation in liquid nutrient broth–soyotone–yeast extract medium as well as on agar plates. The strains assimilated a broad variety of substrates; however, the patterns differed a great deal between the three investigated strains. For strain 27D-LEPIT, the major fatty acids were iso-C14 : 0 and anteiso-C15 : 0; the identified polar lipids were phosphatidylglycerol and diphosphatidylglycerol; the major respiratory quinone was MK-11; and analysis of the peptidoglycan structure indicated the presence of type B2β (typeB10). The genomic DNA of the three strains was very small (1.4 Mbp) and had a DNA G+C content of 54 mol%. The investigated traits suggested that strains 36A-HELLB (=DSM 107805=JCM 32927), 1B-Mac (=DSM 107802=JCM 32925) and 27D-LEPIT (=JCM 32926T =DSM 107804T) represent a new species for which the name Rhodolunalimnophila sp. nov. is proposed, with strain 27D-LEPIT as the type strain.
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