Ho-Won Chang

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.

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.

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).

Statistical superiority of genome-probing microarrays as genomic DNA-DNA hybridization in revealing the bacterial phylogenetic relationship compared to conventional methods.

The genomic DNA-DNA hybridization (DDH) method has been widely used as a practical method for the determination of phylogenetic relationships between closely related biological strains. Traditional DDH methods have serious limitations including low reproducibility, a high background and a time-consuming procedure. The DDH method using a genome-probing microarray (GPM) has been recently developed to complement conventional methods and could be used to overcome the limitations that are typically encountered. It is necessary to compare the GPM-based DDH method to the conventional methods before using the GPM for the estimation of genomic similarities since all of the previous scientific data have been entirely dependent on conventional DDH methods. In order to address this issue we compared the DDH values obtained using the GPM, microplate and nylon membrane methods to multi-locus sequence typing (MLST) data for 9 Salmonella genomes and an Escherichia coli type strain. The results showed that the genome similarity values and the degrees of standard deviation obtained using the GPM method were lower than those obtained with the microplate and nylon membrane methods. The dendrogram from the cluster analysis of GPM DDH values was consistent with the phylogenetic tree obtained from the multi-locus sequence typing (MLST) data but was not similar to those obtained using the microplate and nylon membrane methods. Although the signal intensity had to be maximal when the targets were hybridized to their own probe, the methods using membranes and microplates frequently produced higher signals in the heterologous hybridizations than those obtained in the homologous hybridizations. Only the GPM method produced the highest signal intensity in homologous hybridizations. These results show that the GPM method can be used to obtain results that are more accurate than those generated by the other methods tested.

Metatranscriptome analysis of lactic acid bacteria during kimchi fermentation with genome-probing microarrays.

We constructed genome probing microarrays (GPM) that are specific to 39 lactic acid bacteria (LAB) in an effort to monitor microbial diversity and biological activity during the fermentation of kimchi, a traditional Korean vegetable product known to contain various health-promoting and immunity-boosting factors. Metagenomes and metatranscriptomes extracted from periodically sampled kimchi soup were labeled, hybridized and comparatively analyzed using GPMs. Each metatranscriptome was prepared by subtracting 16S rRNA and 23S rRNA from the total RNA, and selectively synthesizing mRNA-specific cDNAs from the rRNA-subtracted samples. Metagenomic analysis revealed 23 LAB related to kimchi fermentation [defined as bacteria with more than a 1% average relative composition (ARC)]. Metatranscriptome analysis revealed that, with the exception of two microorganisms, all LAB probed in the microarray contributed to kimchi fermentation. Moreover, the relative compositions of the major LAB remained unchanged (there was less than a 1.5% difference between the maximum and minimum values) in our metagenome analysis, while our metatranscriptome analysis revealed significant differences in the relative compositions of major LAB during fermentation (relative compositions changed by 2.4% to 9.5%). These data indicate that microorganisms that are less abundant in the flora (those with less than a 5% ARC in the metagenomic analysis) also participated in kimchi fermentation with relatively high activities.

Phylogenetic Characterization of Two Novel Commensal Bacteria Involved with Innate Immune Homeostasis in Drosophila melanogaster

ABSTRACT During a previous study on the molecular interaction between commensal bacteria and host gut immunity, two novel bacterial strains, A911T and G707T, were isolated from the gut of Drosophila melanogaster. In this study, these strains were characterized in a polyphasic taxonomic study using phenotypic, genetic, and chemotaxonomic analyses. We show that the strains represent novel species in the family Acetobacteraceae. Strain G707T, a highly pathogenic organism, represents a new species in the genus Gluconobacter, “Gluconobacter morbifer” sp. nov. (type strain G707 = KCTC 22116T = JCM 15512T). Strain A911T, dominantly present in the normal Drosphila gut community, represents a novel genus and species, designated “Commensalibacter intestini” gen. nov., sp. nov. (type strain A911 = KCTC 22117T = JCM 15511T).

Paracoccus aestuarii sp. nov., isolated from tidal flat sediment

A Gram-negative micro-organism, designated strain B7(T), was isolated from tidal flat sediment and subjected to a polyphasic taxonomic study involving morphological, physiological, biochemical and 16S rRNA gene sequence analyses. A phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain B7(T) belonged to the genus Paracoccus and was closely related phylogenetically to Paracoccus marcusii MH1(T) (97.5 % sequence similarity), Paracoccus marinus KKL-A5(T) (97.5 %), Paracoccus haeundaensis BC74171(T) (97.3 %), Paracoccus carotinifaciens E-396(T) (97.3 %), Paracoccus homiensis DD-R11(T) (97.2 %), Paracoccus seriniphilus MBT-A4(T) (96.9 %) and other type strains of the genus Paracoccus (95.2-96.7 %). The G+C content of the genomic DNA and the major isoprenoid quinone of the type strain were 62.0 mol% and ubiquinone-10, respectively. The major fatty acid components were C(18 : 1)omega7c (68.9 %) and C(18 : 0) (18.1 %); this profile, with C(18 : 1)omega7c as the predominant fatty acid, was characteristic of members of the genus Paracoccus. The 16S rRNA gene sequence analysis, DNA-DNA hybridization studies and physiological and biochemical tests identified genotypic and phenotypic differences between strain B7(T) and recognized Paracoccus species. On the basis of these data, therefore, strain B7(T) represents a novel species of the genus Paracoccus, for which the name Paracoccus aestuarii sp. nov. is proposed. The type strain is B7(T) (=KCTC 22049(T)=DSM 19484(T)=JCM 15119(T)).

Joostella marina gen. nov., sp. nov., a novel member of the family Flavobacteriaceae isolated from the East Sea

A Gram-negative, non-spore-forming, non-motile, yellow-pigmented, strictly aerobic bacterial strain, designated En5(T), was isolated from the East Sea of Korea and was subjected to a polyphasic taxonomy study. Strain En5(T) grew optimally at 30 degrees C, in the presence of 1-3 % (w/v) NaCl and at pH 5.3-7.6. The major respiratory lipoquinone was MK-6 and the major fatty acids were iso-C(15 : 0), iso-C(17 : 0) 3-OH and iso-C(17 : 1)omega9c. The DNA G+C content of strain En5(T) was 30.1 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain En5(T) formed a distinct evolutionary lineage within the family Flavobacteriaceae and shared 93 % sequence similarity with the type strains of both Galbibacter mesophilus and Zhouia amylolytica. On the basis of its phenotypic and phylogenetic properties, strain En5(T) is suggested to represent a novel species of a new genus in the family Flavobacteriaceae, for which the name Joostella marina gen. nov., sp. nov. is proposed. The type strain is En5(T) (=KCTC 12518(T)=DSM 19592(T)=CGMCC 1.6973(T)).