The Journal of General and Applied Microbiology Vol. 63(2017) No. 2

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.

In the present study, high throughput 16S rRNA gene sequencing was used to investigate soil invaded by the aggressive weed Ageratina adenophora to determine its effect on the species composition, distribution, and biodiversity of the bacterial communities. Soil samples from 12 micro-sites containing a monoculture of A. adenophora plants, mixtures of A. adenophora and different native plant species, and native species alone were studied. We found that the invasion of this weed resulted in a selection of bacteria belonging to phyla Acidobacteria and Verrucomicrobia and the lack of bacteria belonging to phyla Actinobacteria and Planctomycetes, but did not affect significantly the percentage abundances of members of other phyla. A similar bacterial population selection was also observed at genus or subgroup levels. The NO₃^–-N level was an important factor affecting soil bacterial communities and contributed to the dominance of A. adenophora. However, the numbers of total bacterial species, and the diversity and structure of soil bacterial microbiome did not (P > 0.05) change significantly following invasion by this weed.

The rumen microbiome plays a vital role in ruminant nutrition and health, and its community is affected by environmental factors. However, little is known about the rumen bacterial community of ruminants living in the special ecological environment of the Qinghai-Tibetan Plateau (QTP) of China. The objectives of this study were to investigate the rumen bacterial community of the typical plateau sheep (Tibetan sheep, TS, and Gansu alpine fine-wool sheep, GS) grazing on the QTP, using 16S rRNA gene sequence analysis, and to evaluate the relationship between the rumen bacterial community and the QTP environment. A total of 116 sequences (201 clones) were examined and divided into 53 operational taxonomic units (OTUs) in the TS library and 46 OTUs in the GS library. Phylogenetic analysis showed that the sequences that belonged to the Firmicutes were the most predominant bacteria in both TS and GS libraries, representing 79.4% and 62.8% of the total clones, respectively. The remaining sequences belonged to Bacteroidetes, Proteobacteria, Actinobacteria, or were unclassified bacteria. Sequence analysis revealed that the TS and GS rumens harbored many novel sequences associated with uncultured bacteria that accounted for 63.6% and 46.8% of the total clones, respectively. Comparison of the composition and diversity of the TS and GS rumen bacteria revealed few overlapping known bacteria between the two breeds, and a higher diversity in TS. The rumen bacteria of the plateau sheep showed higher percentages of bacteria that belonged to Firmicutes and novel species compared with the low-elevation sheep. The unique bacterial community in the plateau sheep rumens is perhaps one of the major reasons that they can adapt to the harsh plateau environment. These results can help identify the rumen bacterial community of the ruminants in the QTP, and provide bacteria resources and basic data to improve ruminant productivity.

Cyanobacteria are photosynthetic prokaryotes that perform oxygenic photosynthesis by extracting electrons from water, with the generation of oxygen as a byproduct. Cyanobacteria use oxygen not only for respiration to produce energy in the dark but also for biosynthesis of various metabolites, such as heme and chlorophyll. Oxygen levels dynamically fluctuate in the field environments, from hyperoxic at daytime to almost anaerobic at night. Thus, adaptation to anaerobiosis should be important for cyanobacteria to survive in low-oxygen and anaerobic environments. However, little is known about the molecular mechanisms of cyanobacterial anaerobiosis because cyanobacteria have been regarded as aerobic organisms. As a first step to elucidate cyanobacterial adaptation mechanisms to low-oxygen environments, we isolated five mutants, T-1–T-5, exhibiting growth defects under microoxic conditions. The mutants were obtained from a transposon-tagged mutant library of the cyanobacterium Synechocystis sp. PCC 6803, which was produced by in vitro transposon tagging of cyanobacterial genomic DNA. Southern blot analysis indicated that a kanamycin resistance gene was inserted in the genome as a single copy. We identified the chromosomal transposon-tagged locus in T-5. Two open reading frames (sll0577 and sll0578) were partially deleted by the insertion of the kanamycin resistance gene in T-5. A reverse transcription polymerase chain reaction suggested that these co-transcribed genes are constitutively expressed under both aerobic and microoxic conditions. Then, we isolated two mutants in which one of the two genes was individually disrupted. Only the mutants partially lacking an intact sll0578 gene showed growth defects under microoxic conditions, whereas it grew normally under aerobic conditions. sll0578 is annotated as purK encoding N⁵-carboxy-aminoimidazole ribonucleotide synthetase involved in purine metabolism. This result implies the unexpected physiological importance of PurK under low-oxygen environments.

Autophagy is a conserved cellular degradation process in eukaryotes, in which cytoplasmic components and organelles are digested in vacuoles/lysosomes. Recently, autophagic degradation of nuclear materials, termed “nucleophagy”, has been reported. In the multinucleate filamentous fungus Aspergillus oryzae, a whole nucleus is degraded by nucleophagy after prolonged culture. While developing an H2B-EGFP processing assay for the evaluation of nucleophagy in A. oryzae, we found that nucleophagy is efficiently induced by carbon or nitrogen depletion. Microscopic observations in a carbon depletion condition clearly demonstrated that autophagosomes selectively sequester a particular nucleus, despite the presence of multiple nuclei in the same cell. Furthermore, AoNsp1, the A. oryzae homolog of the yeast nucleoporin Nsp1p, mainly localized at the nuclear periphery, but its localization was restricted to the opposite side of the autophagosome being formed around a nucleus. In contrast, the perinuclear ER visualized with the calnexin AoClxA was not morphologically affected by nucleophagy. The findings of nucleophagy-inducing conditions enabled us to characterize the morphological process of autophagic degradation of a whole nucleus in multinucleate cells.

The aim of this study was to select aerobic spore-formers for animal feed based on their in vitro probiotic potential, including their enzyme-producing ability and safety assessment. Seven isolates out of 187 spore-forming bacteria were selected for their ability to produce cellulase (89.21–1668.32 U/ml), xylanase (1399.68–4351.10 U/ml), and phytase (2.72–28.70 U/ml). Among seven isolates, five had activities towards a broad range of p-nitrophenyl esters with acyl chain lengths from C2 to C12. The probiotic properties of all selected isolates varied with respect to their acid and bile salt tolerance under simulated gastrointestinal tract (GIT) conditions, and their adherence ability to human intestinal cell lines (Caco-2 and HT-29). The safety assessment revealed that the isolate CM40 was not cytotoxic to Caco-2 and HT-29, did not exhibit hemolytic activity, carried no enterotoxin or emetic toxin genes, and was susceptible to ten antibiotics, including six key antibiotics (chloramphenicol, erythromycin, gentamicin, tetracycline, streptomycin, and kanamycin) as recommended by the European Food Safety Authority (EFSA). Co-incubation of isolate CM40 with enteric bacteria (Salmonella Typhi, Salmonella Enteritidis 1781, and Escherichia coli) demonstrated that CM40 significantly decreased the number of pathogens (about 30–48%) adhering to Caco-2 and HT-29 (P < 0.05). Analysis of gene encoding 16S rRNA, gyrase A (gyrA) and the cheA histidine kinase revealed that CM40 belongs to Bacillus subtilis. On the basis of probiotic properties and basic safety aspects, the B. subtilis strain CM40 was found to possess desirable in vitro probiotic properties, and may be a potential candidate for supplementation of animal feed.