Novel environmental species isolated from the plaster wall surface of mural paintings in the Takamatsuzuka tumulus: Bordetella muralis sp. nov., Bordetella tumulicola sp. nov. and Bordetella tumbae sp. nov.

Abstract

Ten strains of Gram-stain-negative, non-spore-forming, non-motile coccobacilli were isolated from the plaster wall surface of 1300-year-old mural paintings inside the stone chamber of the Takamatsuzuka tumulus in Asuka village (Asuka-mura), Nara Prefecture, Japan. Based on 16S rRNA gene sequence analysis of the isolates, they belonged to the proteobacterial genus Bordetella (class Betaproteobacteria) and could be separated into three groups representing novel lineages within the genus Bordetella. Three isolates were selected, one from each group, and identified carefully using a polyphasic approach. The isolates were characterized by the presence of Q-8 as their major ubiquinone system and C16 : 0 (30.0–41.8 %), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c; 10.1–27.0 %) and C17 : 0 cyclo (10.8–23.8 %) as the predominant fatty acids. The major hydroxy fatty acids were C12 : 0 2-OH and C14 : 0 2-OH. The DNA G+C content was 59.6–60.0 mol%. DNA–DNA hybridization tests confirmed that the isolates represented three separate novel species, for which the names Bordetella muralis sp. nov. (type strain T6220-3-2bT = JCM 30931T = NCIMB 15006T), Bordetella tumulicola sp. nov. (type strain T6517-1-4bT = JCM 30935T = NCIMB 15007T) and Bordetella tumbae sp. nov. (type strain T6713-1-3bT = JCM 30934T = NCIMB 15008T) are proposed. These results support previous evidence that members of the genus Bordetella exist in the environment and may be ubiquitous in soil and/or water.