Yuka Nagatsuka

Citeromyces siamensis sp. nov., a novel halotolerant yeast isolated in Thailand

Two halotolerant yeast strains, H130(T) and H149, were isolated from dry salted squid and fermented soybeans, respectively, in Thailand. Both isolates grew by multilateral budding, produced asci that had one roughened spherical ascospore and contained ubiquinone Q-8. These characteristics were shared by Citeromyces matritensis, the only species of the genus Citeromyces. Strains H130(T) and H149 were differentiated from C matritensis by their ability to assimilate L-sorbose and L-lysine and to grow at 37 degrees C. The novel isolates were more tolerant to higher concentrations of cations (3 M NaCl or 0.8 M LiCI) and to higher osmotic pressure (60% glucose) than C. matritensis. A phylogenetic analysis of 18S rRNA gene sequence data indicated that the two novel isolates represented a sister species to C. matritensis. Furthermore, DNA-DNA hybridization data indicated that the isolates were clearly distinct from the type strain of C. matritensis (IFO 0954(T). Based on the above characteristics, strains H130(T) and H149 are proposed to represent a novel species within the genus Citeromyces, Citeromyces siamensis; the type strain is H130(T) (= IFO 11052(T) = JCM 11522(T) = TISTR 5777(T) = CBS 9153(T)).

Stenotrophomonas tumulicola sp. nov., a major contaminant of the stone chamber interior in the Takamatsuzuka Tumulus.

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.

Candida tumulicola sp. nov. and Candida takamatsuzukensis sp. nov., novel yeast species assignable to the Candida membranifaciens clade, isolated from the stone chamber of the Takamatsu- zuka tumulus

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.

Yamadazyma kitorensis f.a., sp. nov. and Zygoascus biomembranicola f.a., sp. nov., novel yeasts from the stone chamber interior of the Kitora tumulus, and five novel combinations in Yamadazyma and Zygoascus for species of Candida

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).

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

Microbial outbreaks and related biodeterioration problems have affected the 1300-year-old multicolor (polychrome) mural paintings of the special historic sites Takamatsuzuka Tumulus (TT) and Kitora Tumulus (KT). Those of TT are designated as a national treasure. The microbiomes of these tumuli, both located in Asuka village, Nara, Japan, are critically reviewed as the central subject of this report. Using culture-dependent methods (conventional isolation and cultivation), we conducted polyphasic studies of the these microbial communities and identified the major microbial colonizers (Fusarium spp., Trichoderma spp., Penicillium spp., dark Acremonium spp., novel Candida yeast spp., Bacillus spp., Ochrobactrum spp., Stenotrophomonas tumulicola, and a few actinobacterial genera) and noteworthy microbial members (Kendrickiella phycomyces, Cephalotrichum verrucisporum (≡Doratomyces verrucisporus), Sagenomella striatispora, Sagenomella griseoviridis, two novel Cladophialophora spp., Burgoa anomala, one novel species Prototheca tumulicola, five novel Gluconacetobacter spp., three novel Bordetella spp., and one novel genus and species Krasilnikoviella muralis) involved in the biodeterioration of mural paintings, plaster walls, and stone chamber interiors. In addition, we generated microbial community data from TT and KT samples using culture-independent methods (molecular biological methods, including PCR-DGGE, clone libraries, and pyrosequence analysis). These data are comprehensively presented, in contrast to those derived from culture-dependent methods. Furthermore, the microbial communities detected using both methods are analytically compared, and, as a result, the complementary roles of these methods and approaches are highlighted. In related contexts, knowledge of similar biodeterioration problems affecting other prehistoric cave paintings, mainly at Lascaux in France and Altamira in Spain, are referred to and commented upon. Based on substrate preferences (or ecological grouping) and mapping (plotting detection sites of isolates), we speculate on the possible origins and invasion routes whereby the major microbial colonizers invaded the TT stone chamber interior. Finally, concluding remarks, lessons, and future perspectives based on our microbiological surveys of these ancient tumuli, and similar treasures outside of Japan, are briefly presented. A list of the microbial taxa that have been identified and fully or briefly described by us as known and novel taxa for TT and KT isolates since 2008 is presented in Supplementary Materials.