Amplification of uncultured single-stranded DNA viruses from rice paddy soil.

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 phi29 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.

Phylogenetic characterization of two novel commensal bacteria involved with innate immune homeostasis in Drosophila melanogaster.

During a previous study on the molecular interaction between commensal bacteria and host gut immunity, two novel bacterial strains, A911(T) and G707(T), 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 G707(T), a highly pathogenic organism, represents a new species in the genus Gluconobacter, "Gluconobacter morbifer" sp. nov. (type strain G707 = KCTC 22116(T) = JCM 15512(T)). Strain A911(T), 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 22117(T) = JCM 15511(T)).

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.

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.

Characterization of the depth-related changes in the microbial communities in Lake Hovsgol sediment by 16S rRNA gene-based approaches.

The undisturbed sediment of Lake Hovsgol (Mongolia) is scientifically important because it represents a record of the environmental changes that took place between the Holocene (the present age) and Pleistocene (the last ice age; 12,000 14C years before present day). Here, we investigated how the current microbial communities change as the depth increases by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA genes of the microbial communities. The microbial diversity, as estimated by the Shannon index, decreased as the depth increased. In particular, significant changes in archaeal diversity were observed in the middle depth (at 39-42 cm depth of total 60 cm depth) that marks the border between the Holocene and Pleistocene. Phylotype belonging to Beta-and Gamma-Proteobacteria were the predominant bacteria and most of these persisted throughout the depth examined. However, as the depth increased, some bacteria (some genera belonging to Beta-Proteobacteria, Nitrospira, and OP8-9) were not detectable while others (some genera belonging to Alpha-, Beta-, Gamma-Proteobacteria) newly detected by DGGE. Crenarchaea were the predominant archaea and only one phylotype belonging to Euryarchaea was found. Both the archaeal and bacterial profiles revealed by the DGGE band patterns could be grouped into four and three subsets, respectively, subsets that were largely divided by the border between the Holocene and Pleistocene. Thus, the diversity of the current microbial communities in Lake Hovsgol sediments decreases with increasing depth. These changes probably relate to the environmental conditions in the sediments, which were shaped by the paleoclimatic events taking place between the Holocene and Pleistocene.

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 anteiso-C15:0, anteiso-C17:0, iso-C16:0, and iso-C15:0. Analysis of 16S rRNA gene sequences and DNA-DNA relatedness, 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).

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%).

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

A novel bacterium B9(T) was isolated from tidal flat sediment. Its morphology, physiology, biochemical features, and 16S rRNA gene sequence were characterized. Colonies of this strain are yellow and the cells are Gram-negative, rod-shaped, and do not require NaCl for growth. The 16S rRNA gene sequence similarity indicated that strain B9(T) is associated with the genus Lysobacter (< or = 97.2%), Xanthomonas (< or = 96.8%), Pseudomonas (< or = 96.7%), and Luteimonas (< or = 96.0%). However, within the phylogenetic tree, this novel strain shares a branching point with the species Luteimonas composti CC-YY255(T) (96.0%). The DNA-DNA hybridization experiments showed a DNA-DNA homology of 23.0% between strain B9(T) and Luteimonas mephitis B1953/27.1(T). The G+C content of genomic DNA of the type strain is 64.7 mol% (SD, 1.1). The predominant fatty acids are iso-C(11:0), iso-C(15:0), iso-C(16:0), iso-C(17:0), iso-C(17:0) omega9c, and iso-C(11:0) 3-OH. Combined analysis of the 16S rRNA gene sequences, fatty acid profile, and results from physiological and biochemical tests indicated that there is genotypic and phenotypic differentiation of the isolate from other Luteimonas species. For these reasons, strain B9(T) was proposed as a novel species, named Luteimonas aestuarii. The type strain of the new species is B9(T) (= KCTC 22048(T), DSM 19680(T)).

Aliihoeflea aestuarii gen. nov., sp. nov., a novel bacterium isolated from tidal flat sediment.

A novel Gram-negative and rod-shaped bacterium, designated N8(T), was isolated from tidal flat sediment. Phylogenetic analysis based on 16S rRNA gene sequences showed that N8(T) strain is associated with the family Phyllobacteriaceae: two uncultured clones (98.4 and 99.8% 16S rRNA gene sequence similarity) and the genus Mesorhizobium (< or =97.0%). The novel strain formed a separate clade with uncultured clones in the phylogenetic tree based on 16S rRNA gene sequences. Cellular fatty acid profiles predominately comprised C(18:1) omega7c and C(19:0) cyclo omega8c. The major isoprenoid quinone is ubiquinone-10 and genomic DNA G+C content is 53.4 mol%. The polyphasic taxonomic study indicates that the novel strain N8(T) represents a novel species of the new genus in the family Phyllobacteriaceae, named Aliihoeflea aestuarii. The type strain is N8(T) (= KCTC 22052(T)= JCM 15118(T)= DSM 19536(T)).

Quantitative real time PCR assays for the enumeration of Saccharomyces cerevisiae and the Saccharomyces sensu stricto complex in human feces.

There have been an increasing number of reports of yeast systemic infection involving Saccharomyces cerevisiae strains. The development of a rapid and reliable diagnostic tool is therefore warranted in order to explore the distribution of S. cerevisiae as an opportunistic pathogen in humans. In this study, we designed and validated five primer sets targeting the 26S rRNA gene of S. cerevisiae and the S. sensu stricto complex using 26 yeast strains. Among them, two sets of primers specifically amplified the 26S rRNA gene and the ITS region of S. cerevisiae strains, and three sets were specific for amplifying the same genes in the S. sensu stricto complex. After determining the optimal conditions of two primer pairs for quantitative real time PCR, human fecal samples were analyzed to examine the distribution of S. cerevisiae and the S. sensu stricto complex. It was possible to detect a single cell of S. cerevisiae in environmental sample. Qualitative PCR revealed that out of eleven fecal samples tested, one sample contained S. cerevisiae and four samples contained the S. sensu stricto complex. Quantitative real time PCR revealed that the target gene copy numbers of S. cerevisiae and the S. sensu stricto complex were 0.84 and 2.44 respectively, in 1 ng of DNA from the bulk fecal community.

Arthrobacter subterraneus sp. nov., isolated from deep subsurface water of the South Coast of Korea.

Strain CH7T, a pale yellow-pigmented bacterium and new isolate from deep subsurface water of the South Coast of Korea, was subjected to a polyphasic taxonomic study. CH7T grew between 5 and 37 degrees C, pH 5.3-10.5, and tolerated up to 13% NaCl. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain CH7T was associated with the genus Arthrobacter and phylogenetically closely related to the type strains Arthrobacter tumbae (99.4%) and Arthrobacter parietis (99.1%). However, DNA-DNA hybridization experiments revealed 2.1% and 12% between strain CH7T and Arthrobacter tumbae and Arthrobacter parietis, respectively. Thus, the phenotypic and phylogenetic differences suggested that CH7T should be placed in the genus Arthrobacter as a novel species, for which the name Arthrobacter subterraneus sp. nov. is proposed. In addition, the type strain for the new species is CH7T (=KCTC 9997T=DSM 17585T).

Comparative genomic analysis of bacteriophage EP23 infecting Shigella sonnei and Escherichia coli.

Bacteriophage EP23 that infects Escherichia coli and Shigella sonnei was isolated and characterized. The bacteriophage morphology was similar to members of the family Siphoviridae. The 44,077 bp genome was fully sequenced using 454 pyrosequencing. Comparative genomic and phylogenetic analyses showed that EP23 was most closely related to phage SO-1, which infects Sodalis glossinidius and phage SSL-2009a, which infects engineered E. coli. Genomic comparison indicated that EP23 and SO-1 were very similar with each other in terms of gene order and amino acid similarity, even though their hosts were separated in the level of genus. EP23 and SSL-2009a displayed high amino acid similarity between their genes, but there was evidence of several recombination events in SSL-2009a. The results of the comparative genomic analyses further the understanding of the evolution and relationship between EP23 and its bacteriophage relatives.

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.

Phenotypic characterization and genomic analysis of the Shigella sonnei bacteriophage SP18.

A novel bacteriophage that infects Shigella sonnei was isolated from the Gap River in Korea, and its phenotypic and genomic characteristics were investigated. The virus, called SP18, showed morphology characteristic of the family Myoviridae, and phylogenetic analysis of major capsid gene (gp23) sequences classified it as a T4-like phage. Based on host spectrum analysis, it is lytic to S. sonnei, but not to Shigella flexneri, Shigella boydii or members of the genera Escherichia and Salmonella. Pyrosequencing of the SP18 bacteriophage genome revealed a 170-kb length sequence. In total, 286 ORFs and 3 tRNA genes were identified, and 259 ORFs showed similarity (BLASTP e-value<0.001) to genes of other bacteriophages. The results from comparative genomic analysis indicated that the enterophage JS98, isolated from human stool, is the closest relative of SP18. Based on phylogenetic analysis of gp23 protein-coding sequences, dot plot comparison and BLASTP analysis of genomes, SP18 and JS98 appear to be closely related to T4-even phages. However, several insertions, deletions, and duplications indicate differences between SP18 and JS98. Comparison of duplicated gp24 genes and the soc gene showed that duplication events are responsible for the differentiation and evolution of T4-like bacteriophages.

Agromyces atrinae sp. nov., isolated from fermented seafood.

A Gram-staining-positive, aerobic, non-motile and rod-shaped bacterium, designated P27(T), was isolated from a traditional fermented seafood. The isolate grew optimally with 0-2.0 % (w/v) NaCl and at pH 6-7 and 30 degrees C. The predominant menaquinones were MK-12 and MK-11. The major cellular fatty acids were anteiso-C(17 : 0), anteiso-C(15 : 0) and iso-C(16 : 0). The major cell-wall sugars were galactose, mannose and rhamnose. The peptidoglycan amino acids of strain P27(T) were 2,4-diaminobutyric acid, alanine, glutamic acid and glycine. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and an unidentified glycolipid. The genomic DNA G+C content of strain P27(T) was 69.0 mol%. Based on its 16S rRNA gene sequence, strain P27(T) showed highest pairwise similarity with Agromyces cerinus subsp. cerinus JCM 9083(T) (97.0 % similarity). Based on phenotypic, genotypic and phylogenetic studies, strain P27(T) represents a novel species in the genus Agromyces, for which the name Agromyces atrinae sp. nov. is proposed. The type strain is P27(T) (=KCTC 19593(T) =JCM 15913(T)).

Application of quantitative real-time PCR for enumeration of total bacterial, archaeal, and yeast populations in kimchi.

Kimchi is a Korean traditional fermented food made of brined vegetables, with a variety of spices. Various microorganisms are associated with the kimchi fermentation process. This study was undertaken in order to apply quantitative real-time PCR targeting the 16S and 26S rRNA genes for the investigation of dynamics of bacterial, archaeal, and yeast communities during fermentation of various types of kimchi. Although the total bacterial and archaeal rRNA gene copy numbers increased during kimchi fermentation, the number of yeasts was not significantly altered. In 1 ng of bulk DNA, the mean number of rRNA gene copies for all strains of bacteria was 5.45 x 10(6) which was 360 and 50 times greater than those for archaea and yeast, respectively. The total gene copy number for each group of microorganisms differed among the different types of kimchi, although the relative ratios among them were similar. The common dominance of bacteria in the whole microbial communities of various types of kimchi suggests that bacteria play a principal role in the kimchi fermentation process.

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.

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

A novel bacterium (strain K4(T)) belonging to the genus Sphingomonas was isolated from tidal flat sediment in Korea. Its morphology, physiology, biochemical features and 16S rRNA gene sequence were characterized. Colonies of this strain are yellow in colour and the cells are rod-shaped, exhibiting negative Gram staining. The strain grows at 0-5 % (w/v) NaCl and 20-35 degrees C, with optimal growth occurring at 0 % (w/v) NaCl and 30 degrees C. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain K4(T) is associated with the genus Sphingomonas. Within the phylogenetic tree, this novel strain shares a branching point with Sphingomonas asaccharolytica Y-345(T), with which it shares 97.3 % 16S rRNA gene sequence similarity. The polyamine pattern predominantly contains the Sphingomonas-specific triamine sym-homospermidine. Combined analysis of 16S rRNA gene sequences, DNA-DNA relatedness, physiological and biochemical test results identified genotypic and phenotypic differences between strain K4(T) and other Sphingomonas species. On the basis of these differentiating features, it is concluded that strain K4(T) (=KCTC 22050(T)=DSM 19475(T)) represents a novel species of the genus Sphingomonas, for which the name Sphingomonas aestuarii sp. nov. is proposed.

Haloterrigena jeotgali sp. nov., an extremely halophilic archaeon from salt-fermented food.

A novel red-pigmented halophilic archaeon, strain A29T, was isolated from shrimp jeotgal, a traditional salt-fermented food from Korea. This strain grows in the ranges 10-30% (w/v) NaCl, 17-50 degrees C and pH 6.5-8.5, with optimal growth occurring at 15-20% NaCl, 37-45 degrees C and pH 7.0-7.5. The isolate is Gram-negative and non-motile. Phylogenetic analysis, based on 16S rRNA gene sequences, showed that strain A29T is associated with the genus Haloterrigena and closely related to the species Haloterrigena thermotolerans (99.0% similarity). However, DNA-DNA hybridization experiments revealed that the level of hybridization between strain A29T and related strains of Haloterrigena is less than 70%. The polar lipid fraction consists of phosphatidylglyerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me) and mannose-2,6-disulfate(1-2)-glucose glycerol diether (S2-DGD). The G+C content of genomic DNA of the type strain is 62.3 mol%. On the basis of this polyphasic taxonomic study, strain A29T should be placed in the genus Haloterrigena as a novel species, for which the name Haloterrigena jeotgali sp. nov. is proposed. The type strain of the new species is A29T (=KCTC 4020T=DSM 18794T=JCM 14585T=CECT 7218T).

Alishewanella aestuarii sp. nov., isolated from tidal flat sediment, and emended description of the genus Alishewanella.

A Gram-negative strain, B11(T), was isolated from tidal flat sediment in Yeosu, Republic of Korea. Strain B11(T) did not require NaCl for growth and grew between 18 and 44 degrees C. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain B11(T) was associated with the genus Alishewanella and was closely related to the type strain of Alishewanella fetalis (98.3 % similarity). Within the phylogenetic tree, the novel isolate shared a branching point with A. fetalis. Analysis of 16S rRNA gene sequences and DNA-DNA relatedness, as well as physiological and biochemical tests, indicated genotypic and phenotypic differences between strain B11(T) and the type strain of A. fetalis. Thus, strain B11(T) is proposed as a representative of a novel species, Alishewanella aestuarii sp. nov.; the type strain is B11(T) (=KCTC 22051(T) =DSM 19476(T)).