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Four new yeast species belonging to the genus Apiotrichum and two new yeast species belonging to Cutaneotrichosporon are described for strains isolated from guano samples from bat-inhabited caves in Japan. In 2005, we reported these isolates as Trichosporon species based on sequence analyses of the D1/D2 domain of large subunit (LSU) rRNA genes according to available basidiomycetous yeast classification criteria; however, to date, they have not been officially published as new species with descriptions. Their phylogenetic positions have been reanalysed based on comparison of internal transcribed spacer (ITS) region sequences (including the 5.8S rRNA gene) and the D1/D2 domain of the LSU rRNA gene with those of known species; we confirmed clear separation from previously described species. Physiological and biochemical properties of the isolates also suggest their distinctiveness. Therefore, we describe Apiotrichum akiyoshidainum (holotype JCM 12595T), Apiotrichum chiropterorum (JCM 12594T), Apiotrichum coprophilum (JCM 12596T), Apiotrichum otae (JCM 12593T), Cutaneotrichosporon cavernicola (JCM 12590T) and Cutaneotrichosporon middelhovenii (JCM 12592T) as new species. C. cavernicola showed particularly distinctive morphology including large inflated anomalous cells on the hyphae and germination from the cells, although clear clamp connections on the hyphae were not confirmed. Further study is needed to elucidate the morph of this species.

Four new yeast species belonging to the genus

Analysis of D1/D2 large-subunit (LSU) rRNA gene sequences predicted that 17 yeast isolates, mainly from viscous gels (biofilms) taken from the stone chamber interior of the Kitora tumulus in Nara, Japan, were placed in the Yamadazyma and Zygoascus clades. Polyphasic characterization, including morphological, physiological and chemotaxonomic characteristics, multigene sequence divergence and DNA-DNA hybridization, strongly suggested the assignment of one novel species to each of the clades; these are Yamadazyma kitorensis f.a., sp. nov., with the type strain JCM 31005T (ex-type CBS 14158T=isolate K8617-6-8T), and Zygoascus biomembranicola f.a., sp. nov., with the type strain JCM 31007T (ex-type CBS 14157T=isolate K61208-2-11T). Furthermore, the transfer of five known species of the genus Candida as novel combinations to the genera Yamadazyma and Zygoascus is proposed; these are Yamadazyma olivae f.a., comb. nov. (type strain CBS 11171T=ATCC MYA-4568T), Yamadazyma tumulicola f.a., comb. nov. (type strain JCM 15403T=ex-type CBS 10917T=isolate T6517-9-5T), Yamadazyma takamatsuzukensis f.a., comb. nov. (type strain JCM 15410T=CBS 10916T = isolate T4922-1-1T), Zygoascus polysorbophila f.a., comb. nov. (type strain NRRL Y-27161T=CBS 7317T) and Zygoascus bituminiphila f.a., comb. nov. (type strain CBS 8813T=MUCL 41424T).

During investigation of the biological contamination of the 1300-year-old mural paintings and plaster walls inside the stone chambers of the Takamatsuzuka and Kitora Tumuli (TT and KT) in Asuka-mura, Nara Prefecture, Japan, the identity of 17 bacterial isolates from blackish mouldy spots and viscous gels (biofilms) collected from both tumuli (16 isolates from TT and one from KT) during our 2005–2007 microbiological survey was systematically elucidated. One cluster of the major bacterial isolates was assigned to the genus Stenotrophomonas (class Gammaproteobacteria) by phylogenetic analysis of the 16S rRNA gene sequences. These isolates were divided into two groups A and B. Group A comprised 15 TT isolates that took a phylogenetic position near Stenotrophomonas chelatiphaga LPM-5T. Based on our analysis of the phenotypic (cultural, morphological, physiological and chemotaxonomic) characteristics and genotypic/molecular characteristics (DNA base composition, DNA–DNA relatedness, and 16S rRNA and gyrB gene sequences), the novel species name Stenotrophomonas tumulicola sp. nov. is proposed for the group A isolates with the type strain T5916-2-1bT ( = JCM 30961T = NCIMB 15009T). Group B, which contained only one TT and one KT isolate, was closely related to [Pseudomonas] geniculata, [P.] hibiscicola, [P.] beteli, Stenotrophomonas maltophilia and Stenotrophomonas pavanii. The two isolates were genotypically and phenotypically assignable to S. maltophilia.

Eight strains characterised as Gram-stain-positive, non-spore-forming and non-motile rods were isolated from samples collected from stone chambers of the Takamatsuzuka and Kitora tumuli in Asuka village, Nara Prefecture, Japan. Among them, one strain, T7528-3-6bT, was shown to form a novel lineage within the genus Microbacterium . The most closely phylogenetically related species to T7528-3-6bT was Microbacterium panaciterrae , with 97.8 % sequence similarity. The major isoprenoid quinones of T7528-3-6bT were MK-12, MK-13 and MK-11. The predominant cellular fatty acids for this isolate were anteiso-C15 : 0, anteiso-C17 : 0, iso-C16 : 0 and iso-C15 : 0. The diagnostic diamino acid of the peptidoglycan of this isolate was ornithine. Major polar lipids of the isolate were phosphatidylglycerol, diphosphatidylglycerol and an unknown glycolipid. The G+C content of the genomic DNA of this isolate was 70.1 mol%. On the basis of the results of physiological, biochemical and chemotaxonomic tests and molecular phylogenetic analysis, T7528-3-6bT is considered to represent a novel species of the genus Microbacterium , for which the name M. tumbae sp. nov. has been proposed. The type strain is T7528-3-6bT (=JCM 28836T=NCIMB 15039T). The results of comparisons of both phenotypic and genotypic (16S rRNA gene sequence) characteristics indicated that the remaining seven isolates were very closely related to Microbacterium shaanxiense . Although the sequence similarity between the two was 99.2 %, further detailed multifaceted comparisons are needed to determine their accurate taxonomic assignment.

Ten strains of Gram-stain-negative, rod-shaped, non-spore-forming bacteria were isolated from the burial mound soil collected before the dismantling and samples collected during the dismantling work on the Takamatsuzuka Tumulus in Asuka village, Nara Prefecture, Japan in 2007. On the basis of the 16S rRNA gene sequence analysis of the isolates, they were accommodated in the genus Gluconacetobacter (class Alphaproteobacteria ) and can be separated into four groups within the cluster containing the genus Gluconacetobacter . One of the groups demonstrated a phylogenetic position identical to that of Gluconacetobacter asukensis , which was isolated from small holes on plaster walls of the stone chamber interior of Kitora Tumulus in Asuka village, Nara Prefecture, Japan. The remaining three groups consisted of novel lineages within the genus Gluconacetobacter . A total of four isolates were selected from each group and carefully identified using a polyphasic approach. The isolates were characterized on the basis of their possessing Q-10 as the major ubiquinone system and C18 : 1ω7c (58.5–65.2 %) as the predominant fatty acid. A DNA–DNA hybridization test was used to determine that the three lineages represented novel species, for which the names Gluconacetobacter tumulisoli sp. nov., Gluconacetobacter takamatsuzukensis sp. nov. and Gluconacetobacter aggeris sp. nov. are proposed. The type strains are T611xx-1-4aT ( = JCM 19097T = NCIMB 14861T), T61213-20-1aT ( = JCM 19094T = NCIMB 14859T) and T6203-4-1aT ( = JCM 19092T = NCIMB 14860T), respectively.

A Gram-stain-positive, facultatively anaerobic actinomycete, designated strain T6220-5-2bT, was isolated from a sample taken from a mouldy spot on the surface of a mural painting (the white tiger, Byakko) inside the stone chamber of Takamatsuzuka Tumulus in Asuka village, Nara Prefecture, Japan. Based on 16S rRNA gene sequence analysis of the isolate, it was closely related to the genus Promicromonospora , but formed of a novel lineage within the family Promicromonosporaceae . The closest related species to strain T6220-5-2bT was Promicromonospora flava , with which it shared 99.1 % 16S rRNA gene sequence similarity. The isoprenoid quinone systems were menaquinones MK-9(H2), MK-9(H0) and MK-9(H4). The predominant cellular fatty acids for the isolate were anteiso-C15 : 0 and iso-C15 : 0. The peptidoglycan contained glutamic acid, aspartic acid, alanine and lysine, with the last named being the diagnostic diamino acid. The cell-wall acyl type was acetyl. The major polar lipids of the isolate were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositolmannoside, two unknown phospholipids and an unknown phosphoglycolipid. Whole-cell sugars of the isolate were galactose, glucose and ribose. The DNA G+C content of the genomic DNA was 75.2 mol%. Based on the results of phylogenetic, physiological and biochemical analyses and DNA–DNA hybridization experiments, the isolate was considered to represent a novel species of a new genus in the family Promicromonosporaceae , for which the name Krasilnikoviella muralis gen. nov., sp. nov. is proposed. The type strain of Krasilnikoviella muralis is T6220-5-2bT (=JCM 28789T=NCIMB 15040T). The reclassification of Promicromonospora flava as Krasilnikoviella flava comb. nov. is also proposed with the emended description of this species.

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.

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 genusBordetella(classBetaproteobacteria) and could be separated into three groups representing novel lineages within the genusBordetella.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 : 1ω7cand/or C16 : 1ω6c; 10.1–27.0 %) and C17 : 0cyclo (10.8–23.8 %) as the predominant fatty acids. The major hydroxy fatty acids were C12 : 02-OH and C14 : 02-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 namesBordetella muralissp. nov. (type strain T6220-3-2bT = JCM 30931T = NCIMB 15006T),Bordetella tumulicolasp. nov. (type strain T6517-1-4bT = JCM 30935T = NCIMB 15007T) andBordetella tumbaesp. nov. (type strain T6713-1-3bT = JCM 30934T = NCIMB 15008T) are proposed. These results support previous evidence that members of the genusBordetellaexist in the environment and may be ubiquitous in soil and/or water.

During a survey of the mycobiota in the stone chamber of the Takamatsu-zuka tumulus in the village of Asuka, Nara Prefecture, Japan, we isolated 19 yeast strains assigned to the genus Candida from various samples, taken mainly from mouldy spots where the colour of the murals had changed to black, white or another tone, and from viscous gels (biofilms) on plaster walls. The 26S rDNA D1/D2 domain sequence-based phylogeny clearly indicates two groups of isolates. Polyphasic characterization, including morphological, physiological and chemotaxonomic characteristics, and sequence analysis of the 26S rDNA D1/D2 domain and ITS regions suggest that each group is assignable to one of two novel species within the Candida membranifaciens clade. Proposed herein are the names Candida tumulicola sp. nov. (originally T6517-9-5T; holotype JCM 15403T; isotypes CBS 10917T, NBRC 104392T) and Candida takamatsuzukensis sp. nov. (originally T4922-1-1T; holotype JCM 15410T; isotypes CBS 10916T, NBRC 104391T). The 26S rDNA D1/D2 domain sequence divergence indicates that C. tumulicola differs from Candida friedrichii NBRC 10277T, the type strain of the nearest species, in 15 nucleotides (3 %), whereas C. takamatsuzukensis differs from Candida insectorum NBRC 10283T and Pichia mexicana NBRC 10544T, the type strains of the nearest species, in 20 nucleotides (4 %). Both novel species are also clearly distinguishable from the species closest to them by various physiological characteristics.