Young-Do Nam

Innate immune homeostasis by the homeobox gene caudal and commensal-gut mutualism in Drosophila.

Although commensalism with gut microbiota exists in all metazoans, the host factors that maintain this homeostatic relationship remain largely unknown. We show that the intestinal homeobox gene Caudal regulates the commensal-gut mutualism by repressing nuclear factor kappa B–dependent antimicrobial peptide genes. Inhibition of Caudal expression in flies via RNA interference led to overexpression of antimicrobial peptides, which in turn altered the commensal population within the intestine. In particular, the dominance of one gut microbe, Gluconobacter sp. strain EW707, eventually led to gut cell apoptosis and host mortality. However, restoration of a healthy microbiota community and normal host survival in the Caudal-RNAi flies was achieved by reintroduction of the Caudal gene. These results reveal that a specific genetic deficiency within a host can profoundly influence the gut commensal microbial community and host physiology.

Investigation of archaeal and bacterial diversity in fermented seafood using barcoded pyrosequencing

Little is known about the archaeal diversity of fermented seafood; most of the earlier studies of fermented food have focused on lactic acid bacteria (LAB) in the fermentation process. In this study, the archaeal and bacterial diversity in seven kinds of fermented seafood were culture-independently examined using barcoded pyrosequencing and PCR–denaturing gradient gel electrophoresis (DGGE) methods. The multiplex barcoded pyrosequencing was performed in a single run, with multiple samples tagged uniquely by multiplex identifiers, using different primers for Archaea or Bacteria. Because PCR–DGGE analysis is a conventional molecular ecological approach, this analysis was also performed on the same samples and the results were compared with the results of the barcoded pyrosequencing analysis. A total of 13 372 sequences were retrieved from 15 898 pyrosequencing reads and were analyzed to evaluate the diversity of the archaeal and bacterial populations in seafood. The most predominant types of archaea and bacteria identified in the samples included extremely halophilic archaea related to the family Halobacteriaceae; various uncultured mesophilic Crenarchaeota, including Crenarchaeota Group I.1 (CG I.1a and CG I.1b), Marine Benthic Group B (MBG-B), and Miscellaneous Crenarchaeotic Group (MCG); and LAB affiliated with genus Lactobacillus and Weissella. Interestingly, numerous uncultured mesophilic Crenarchaeota groups were as ubiquitous in the fermented seafood as in terrestrial and aquatic niches; the existence of these Crenarchaeota groups has not been reported in any fermented food. These results indicate that the archaeal populations in the fermented seafood analyzed are diverse and include the halophilic and mesophilic groups, and that barcoded pyrosequencing is a promising and cost-effective method for analyzing microbial diversity compared with conventional approaches.

Insect gut bacterial diversity determined by environmental habitat, diet, developmental stage, and phylogeny of host.

ABSTRACT Insects are the most abundant animals on Earth, and the microbiota within their guts play important roles by engaging in beneficial and pathological interactions with these hosts. In this study, we comprehensively characterized insect-associated gut bacteria of 305 individuals belonging to 218 species in 21 taxonomic orders, using 454 pyrosequencing of 16S rRNA genes. In total, 174,374 sequence reads were obtained, identifying 9,301 bacterial operational taxonomic units (OTUs) at the 3% distance level from all samples, with an average of 84.3 (±97.7) OTUs per sample. The insect gut microbiota were dominated by Proteobacteria (62.1% of the total reads, including 14.1% Wolbachia sequences) and Firmicutes (20.7%). Significant differences were found in the relative abundances of anaerobes in insects and were classified according to the criteria of host environmental habitat, diet, developmental stage, and phylogeny. Gut bacterial diversity was significantly higher in omnivorous insects than in stenophagous (carnivorous and herbivorous) insects. This insect-order-spanning investigation of the gut microbiota provides insights into the relationships between insects and their gut bacterial communities.

Amplification of Uncultured Single-Stranded DNA Viruses from Rice Paddy Soil

ABSTRACT Viruses are known to be the most numerous biological entities in soil; however, little is known about their diversity in this environment. In order to explore the genetic diversity of soil viruses, we isolated viruses by centrifugation and sequential filtration before performing a metagenomic investigation. We adopted multiple-displacement amplification (MDA), an isothermal whole-genome amplification method with φ29 polymerase and random hexamers, to amplify viral DNA and construct clone libraries for metagenome sequencing. By the MDA method, the diversity of both single-stranded DNA (ssDNA) viruses and double-stranded DNA viruses could be investigated at the same time. On the contrary, by eliminating the denaturing step in the MDA reaction, only ssDNA viral diversity could be explored selectively. Irrespective of the denaturing step, more than 60% of the soil metagenome sequences did not show significant hits (E-value criterion, 0.001) with previously reported viral sequences. Those hits that were considered to be significant were also distantly related to known ssDNA viruses (average amino acid similarity, approximately 34%). Phylogenetic analysis showed that replication-related proteins (which were the most frequently detected proteins) related to those of ssDNA viruses obtained from the metagenomic sequences were diverse and novel. Putative circular genome components of ssDNA viruses that are unrelated to known viruses were assembled from the metagenomic sequences. In conclusion, ssDNA viral diversity in soil is more complex than previously thought. Soil is therefore a rich pool of previously unknown ssDNA viruses.

Comparing microarrays and next-generation sequencing technologies for microbial ecology research

Recent advances in molecular biology have resulted in the application of DNA microarrays and next-generation sequencing (NGS) technologies to the field of microbial ecology. This review aims to examine the strengths and weaknesses of each of the methodologies, including depth and ease of analysis, throughput and cost-effectiveness. It also intends to highlight the optimal application of each of the individual technologies toward the study of a particular environment and identify potential synergies between the two main technologies, whereby both sample number and coverage can be maximized. We suggest that the efficient use of microarray and NGS technologies will allow researchers to advance the field of microbial ecology, and importantly, improve our understanding of the role of microorganisms in their various environments.

Development and Evaluation of Genome-Probing Microarrays for Monitoring Lactic Acid Bacteria

ABSTRACT The genome-probing microarray (GPM) was developed for quantitative, high-throughput monitoring of community dynamics in lactic acid bacteria (LAB) fermentation through the deposit of 149 microbial genomes as probes on a glass slide. Compared to oligonucleotide microarrays, the specificity of GPM was remarkably increased to a species-specific level. GPM possesses about 10- to 100-fold higher sensitivity (2.5 ng of genomic DNA) than the currently used 50-mer oligonucleotide microarrays. Since signal variation between the different genomes was very low compared to that of cDNA or oligonucleotide-based microarrays, the capacity of global quantification of microbial genomes could also be observed in GPM hybridization. In order to assess the applicability of GPMs, LAB community dynamics were monitored during the fermentation of kimchi, a traditional Korean food. In this work, approximately 100 diverse LAB species could be quantitatively analyzed as actively involved in kimchi fermentation.

Microbial community analysis of Korean soybean pastes by next-generation sequencing

Soybean pastes, doenjang, have long been consumed as a fortified protein source in Korea. The quality of doenjang is determined by fermentation and Bacillus subtilis is suspected to be the main microorganism responsible. In the current culture-independent analysis, 17,675 bacterial sequences were derived from nine local and two commercial brands of doenjang samples by a barcoded pyrosequencing method targeting the hyper-variable regions V1/V2 of the 16S rRNA gene. In contrast to what has previously been found using plating or conventional molecular biology based methods, doenjang contains a diversity of bacterial species (total 208 species) and each doenjang reflects a region-specific bacterial community. While the Bacillus species was thought to be dominant in soybean pastes, we found that they were in high abundance (58.3-91.6%) only in samples from the central region of Korea, whereas lactic acid bacteria (LAB) (39.8-77.7%) were the dominant bacterial members of other doenjang samples. Compared to local brands of doenjang, commercial brands contain simple microbial communities dominated by Tetragenococcus and Staphylococcus that resemble the microbial communities of Japanese miso; this suggests that artificial inoculation was used for the quality control and standardization of doenjang. In this study, a massive sequencing approach was applied for the first time to analyze the microbial communities of different doenjang samples. Thus, we have determined that massive sequencing is a valid approach for assessing the overall microbial community of Korean fermented soybean pastes.

Analysis of yeast and archaeal population dynamics in kimchi using denaturing gradient gel electrophoresis.

Kimchi is a traditional Korean food that is fermented from vegetables such as Chinese cabbage and radish. Many bacteria are involved in kimchi fermentation and lactic acid bacteria are known to perform significant roles. Although kimchi fermentation presents a range of environmental conditions that could support many different archaea and yeasts, their molecular diversity within this process has not been studied. Here, we use PCR-denaturing gradient gel electrophoresis (DGGE) targeting the 16S and 26S rRNA genes, to characterize bacterial, archaeal and yeast dynamics during various types of kimchi fermentation. The DGGE analysis of archaea expressed a change of DGGE banding patterns during kimchi fermentation, however, no significant change was observed in the yeast DGGE banding patterns during kimchi fermentation. No significant difference was indicated in the archaeal DGGE profile among different types of kimchi. In the case of yeasts, the clusters linked to the manufacturing corporation. Haloarchaea such as Halococcus spp., Natronococcus spp., Natrialba spp. and Haloterrigena spp., were detected as the predominant archaea and Lodderomyces spp., Trichosporon spp., Candida spp., Saccharomyces spp., Pichia spp., Sporisorium spp. and Kluyveromyces spp. were the most common yeasts.

Bacterial, archaeal, and eukaryal diversity in the intestines of Korean people

The bacterial, archaeal, and eukaryal diversity in fecal samples from ten Koreans were analyzed and compared by using the PCR-fingerprinting method, denaturing gradient gel electrophoresis (DGGE). The bacteria all belonged to the Firmicutes and Bacteroidetes phyla, which were known to be the dominant bacterial species in the human intestine. Most of the archaeal sequences belonged to the methane-producing archaea but several halophilic archarea-related sequences were also detected unexpectedly. While a small number of eukaryal sequences were also detected upon DGGE analysis, these sequences were related to fungi and stramenopiles (Blastocystis hominis). With regard to the bacterial and archaeal DGGE analysis, all ten samples had one and two prominent bands, respectively, but many individual-specific bands were also observed. However, only five of the ten samples had small eukaryal DGGE bands and none of these bands was observed in all five samples. Unweighted pair group method and arithmetic averages clustering algorithm (UPGMA) clustering analysis revealed that the archaeal and bacterial communities in the ten samples had relatively higher relatedness (the average Dice coefficient values were 68.9 and 59.2% for archaea and bacteria, respectively) but the eukaryal community showed low relatedness (39.6%).

Arthrobacter soli sp. nov., a novel bacterium isolated from wastewater reservoir sediment

A novel Gram-positive bacterium, designated SYB2T, was isolated from wastewater reservoir sediment, and a polyphasic taxonomic study was conducted based on its morphological, physiological, and biochemical features, as well as the analysis of its 16S rRNA gene sequence. During the phylogenetic analysis of the strain SYB2T, results of a 16S rRNA gene sequence analysis placed this bacterium in the genus Arthrobacter within the family Micrococcaceae. SYB2T and Arthrobacter protophormiae ATCC 19271T, the most closely related species, both exhibited a 16S rRNA gene sequence similarity of 98.99%. The genomic DNA G+C content of the novel strain was found to be 62.0 mol%. The predominant fatty acid composition was ante-iso-C15:0, anteiso-C17:0, iso-C16:0, and iso-C15:0. Analysis of 16S rRNA gene sequences and DNA-DNA related-ness, as well as physiological and biochemical tests, showed genotypic and phenotypic differences between strain SYB2T and other Arthrobacter species. The type strain of the novel species was identified as SYB2T (= KCTC 19291T= DSM 19449T).