Gluconacetobacter tumulicola sp. nov. and Gluconacetobacter asukensis sp. nov., isolated from the stone chamber interior of the Kitora Tumulus

Abstract

Six Gram-negative, rod-shaped, non-spore-forming bacterial strains were isolated from small holes on plaster walls of the stone chamber interior of the Kitora Tumulus in Asuka village, Nara Prefecture, Japan. These were investigated by means of a polyphasic approach. All the isolates were strictly aerobic and motile by peritrichous flagella. Phylogenetic trees generated based on 16S rRNA gene sequences identified two novel lineages (comprising five isolates and one isolate, respectively) within the genus Gluconacetobacter . The isolates were characterized by having Q-10 as the major ubiquinone system and C18 : 1ω7c (58.7–63.1 % of the total) as the predominant fatty acid. DNA–DNA hybridization experiments endorsed the species rank for the two lineages, for which the names Gluconacetobacter tumulicola sp. nov. (type strain K5929-2-1bT = JCM 17774T = NCIMB 14760T) and Gluconacetobacter asukensis sp. nov. (type strain K8617-1-1bT = JCM 17772T = NCIMB 14759T) are proposed.

Polyphasic insights into the microbiomes of the Takamatsuzuka Tumulus and Kitora Tumulus

“…4): (1) sterile moist cotton swabs bearing the mold samples were rubbed immediately onto moist filter papers in a Petri dish, incubated in a moist chamber at 25∞C, and observed at regular intervals over a period of approximately two months. The newly-appearing colonies were isolated using a sterile needle under a stereomicroscope (Gams et al, 1987;Krug, 2004); (2) swab samples were aseptically transported to the laboratory, where they were smeared directly onto plates containing potato-dextrose agar (for fungi) (PDA; e.g., Kiyuna et al, 2008) and nutrient agar (CM3; e.g., Tazato et al, 2012) and then incubated at 20-25∞C and 30∞C, respectively, for a suitable period under aerobic conditions; and (3) uncovered plates were exposed to air for 10 min and then incubated at 25∞C to isolate airborne microbes. All fungal isolates were cultured on PDA in the dark at 25∞C.…”

“…Based on the sequence data, molecular phylogenetic analyses were made using the standard method. Representative studies that used our methods include Kiyuna et al (2008Kiyuna et al ( , 2011Kiyuna et al ( , 2012Kiyuna et al ( , 2015Kiyuna et al ( , 2017 and An et al (2009) for the major/noteworthy fungal colonizers, Nagatsuka et al (2009Nagatsuka et al ( , 2016Nagatsuka et al ( , 2017 for yeast and yeast-like colonizers, and Tazato et al (2012Tazato et al ( , 2015, Nishijima et al (2013Nishijima et al ( , 2017aNishijima et al ( , 2017b, and Handa et al (2016Handa et al ( , 2017, for the major/noteworthy bacterial colonizers. These original papers have revealed the identities of the TT and KT microbial isolates in question, proposing one novel genus, seventeen novel species, and six novel combinations in total, as listed in Supplementary Materials of this review.…”

“…In September 2005, small holes (approximately 10 mm in diameter; Fig. 1s) caused by acetic acid bacteria (initially identified as members of the genus Gluconacetobacter and subsequently proposed as two novel species, Gluconacetobacter tumulicola and Gluconacetobacter asukensis, by Tazato et al, 2012) became apparent on the plaster wall of the ceiling of KT (Kigawa et al, 2009b(Kigawa et al, , 2012bTazato et al, 2012). In April 2006, the basidiomycetous anamorphic fungus Burgoa sp.…”

“…Until our proposal of two novel species (Tazato et al, 2012) mentioned below, the acetobacteriaceous genus Gluconacetobacter comprised only five species with the type species G. liquefaciens. These species have been isolated from dried fruits, the rhizosphere of coffee plants, sugarcane, and the pink sugar cane mealybug (Komagata et al, 2014).…”

“…These small holes were filled with a black gel or viscous materials, and increased in size with time (Kigawa et al, 2006a). We isolated six acetic-acid-producing bacteria from these black gels and viscous materials (Tazato et al, 2012).…”