Spatial distribution of three ARGONAUTEs regulates the anther phasiRNA pathway.

Argonaute protein (AGO) in association with small RNAs is the core machinery of RNA silencing, an essential mechanism for precise development and defense against pathogens in many organisms. Here, we identified two AGOs in rice anthers, AGO1b and AGO1d, that interact with phased small interfering RNAs (phasiRNAs) derived from numerous long non-coding RNAs. Moreover, 3D-immunoimaging and mutant analysis indicated that rice AGO1b and AGO1d cell type-specifically regulate anther development by acting as mobile carriers of these phasiRNAs from the somatic cell layers to the germ cells in anthers. Our study also highlights a new mode of reproductive RNA silencing via the specific nuclear and cytoplasmic localization of three AGOs, AGO1b, AGO1d, and MEL1, in rice pollen mother cells.

3D multiple immunoimaging using whole male organs in rice.

Spatiotemporal regulation of proteins and RNAs is essential for the precise development of reproductive tissues in many organisms. The anther, a prominent part of the male reproductive organ in plants, contains several somatic cell layers named the anther wall and, within it, the germ cells. Here, we successfully developed a simple 3D organ-immunoimaging technique for rice anthers, which distinguishes each individual cell from the four somatic cell layers and germ cells without the need for transformation, embedding, sectioning, or clearing. The 3D immunostaining method is also applicable to the intracellular localization of meiosis-specific proteins in meiocytes, as exemplified by MEL1, a germ cell-specific ARGONAUTE in the cytoplasm, and ZEP1, a pachytene marker on meiotic chromosomes. Our 3D multiple immunostaining method with single-cell and intracellular resolution will contribute to a comprehensive organ-level elucidation of molecular mechanisms and cellular connectivity.

Complete Genome Sequence of Lactococcus cremoris Strain 7-1, a Lactic Acid Bacterium Isolated from a Traditional Mongolian Milk Product Possessing Mucin-Adhesive Ability.

We report the complete genome sequence of Lactococcus cremoris strain 7-1, which was isolated from urum, a traditional Mongolian milk product. Strain 7-1 adhered to porcine gastric mucin in a carbon source-dependent manner. The genome consists of a circular chromosome (2,557,589 bp; GC content, 35.7%) and two circular plasmids.

Complete Genome Sequence of Carotenoid-Producing Enterococcus gilvus CR1, Isolated from Raw Cow's Milk.

Enterococcus gilvus CR1, isolated from raw cow's milk, can produce carotenoids. The complete genome sequence of this strain was determined using the PacBio RS II platform. The assembly was found to contain a circular chromosome, including carotenoid biosynthesis genes, and comprises 2,863,043 bp, with a G+C content of 41.86% and three plasmids.

Complete Genome Sequence of Lactobacillus paracasei EG9, a Strain Accelerating Free Amino Acid Production during Cheese Ripening.

Lactobacillus paracasei EG9 is a strain isolated from well-ripened cheese and accelerates free amino acid production during cheese ripening. Its complete genome sequence was determined using the PacBio RS II platform, revealing a single circular chromosome of 2,927,257 bp, a G+C content of 46.59%, and three plasmids.

Complete Genome Sequence of Lactobacillus plantarum Strain LQ80, Selected for Preparation of Fermented Liquid Feed for Pigs.

Lactobacillus plantarum LQ80 is a strain isolated from liquid feed for pigs. We determined the complete genome sequence of this strain using the PacBio RS II platform. LQ80 contained a single circular chromosome of 3,230,192 bp, with 44.66% G+C content and seven plasmids.

Complete Genome Sequence of Petrimonas sp. Strain IBARAKI, Assembled from the Metagenome Data of a Culture Containing Dehalococcoides spp.

The complete genome sequence of Petrimonas sp. strain IBARAKI in a Dehalococcoides-containing culture was determined using the PacBio RS II platform. The genome is a single circular chromosome of 3,693,233 nucleotides (nt), with a GC content of 44%. This is the first genome sequence of a Petrimonas species.

Complete Genome Sequence of Lactococcus lactis subsp. lactis G50 with Immunostimulating Activity, Isolated from Napier Grass.

Lactococcus lactis subsp. lactis G50 is a strain with immunostimulating activity, isolated from Napier grass (Pennisetum purpureum). We determined the complete genome sequence of this strain using the PacBio RS II platform. The single circular chromosome consists of 2,346,663 bp, with 35.03% G+C content and no plasmids.

Isolation and genomic characterization of a Dehalococcoides strain suggests genomic rearrangement during culture.

We have developed and characterized a bacterial consortium that reductively dechlorinates trichloroethene to ethene. Quantitative PCR analysis for the 16S rRNA and reductive dehalogenase genes showed that the consortium is highly enriched with Dehalococcoides spp. that have two vinyl chloride reductive dehalogenase genes, bvcA and vcrA, and a trichloroethene reductive dehalogenase gene, tceA. The metagenome analysis of the consortium by the next generation sequencer SOLiD 3 Plus suggests that a Dehalococcoides sp. that is highly homologous to D. mccartyi 195 and equipped with vcrA and tceA exists in the consortium. We isolated this Dehalococcoides sp. and designated it as D. mccartyi UCH-ATV1. As the growth of D. mccartyi UCH-ATV1 is too slow under isolated conditions, we constructed a consortium by mixing D. mccartyi UCH-ATV1 with several other bacteria and performed metagenomic sequencing using the single molecule DNA sequencer PacBio RS II. We successfully determined the complete genome sequence of D. mccartyi UCH-ATV1. The strain is equipped with vcrA and tceA, but lacks bvcA. Comparison with tag sequences of SOLiD 3 Plus from the original consortium shows a few differences between the sequences. This suggests that a genome rearrangement of Dehalococcoides sp. occurred during culture.

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area.

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II's sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.

First Complete Genome Sequence of the Skin-Improving Lactobacillus curvatus Strain FBA2, Isolated from Fermented Vegetables, Determined by PacBio Single-Molecule Real-Time Technology.

The first complete genome sequence of Lactobacillus curvatus was determined by PacBio RS II. The single circular chromosome (1,848,756 bp, G+C content of 42.1%) of L. curvatus FBA2, isolated from fermented vegetables, contained low G+C regions (26.9% minimum) and 43 sets of >1,000-bp identical sequence pairs. No plasmids were detected.

Gene expression analysis of disabled and re-induced isoprene emission by the tropical tree Ficus septica before and after cold ambient temperature exposure.

Isoprene is the most abundant type of nonmethane, biogenic volatile organic compound in the atmosphere, and it is produced mainly by terrestrial plants. The tropical tree species Ficus septica Burm. F. (Rosales: Moraceae) has been shown to cease isoprene emissions when exposed to temperatures of 12 °C or lower and to re-induce isoprene synthesis upon subsequent exposure to temperatures of 30 °C or higher for 24 h. To elucidate the regulation of genes underlying the disabling and then induction of isoprene emission during acclimatization to ambient temperature, we conducted gene expression analyses of F. septica plants under changing temperature using quantitative real-time polymerase chain reaction and western blotting. Transcription levels were analyzed for 17 genes that are involved in metabolic pathways potentially associated with isoprene biosynthesis, including isoprene synthase (ispS). The protein levels of ispS were also measured. Changes in transcription and protein levels of the ispS gene, but not in the other assessed genes, showed identical temporal patterns to isoprene emission capacity under the changing temperature regime. The ispS protein levels strongly and positively correlated with isoprene emission capacity (R(2) = 0.92). These results suggest that transcriptional regulation of ispS gave rise to the temporal variation in isoprene emission capacity in response to changing temperature.

Heterogeneous composition of key metabolic gene clusters in a vent mussel symbiont population.

Chemosynthetic symbiosis is one of the successful systems for adapting to a wide range of habitats including extreme environments, and the metabolic capabilities of symbionts enable host organisms to expand their habitat ranges. However, our understanding of the adaptive strategies that enable symbiotic organisms to expand their habitats is still fragmentary. Here, we report that a single-ribotype endosymbiont population in an individual of the host vent mussel, Bathymodiolus septemdierum has heterogeneous genomes with regard to the composition of key metabolic gene clusters for hydrogen oxidation and nitrate reduction. The host individual harbours heterogeneous symbiont subpopulations that either possess or lack the gene clusters encoding hydrogenase or nitrate reductase. The proportions of the different symbiont subpopulations in a host appeared to vary with the environment or with the host's development. Furthermore, the symbiont subpopulations were distributed in patches to form a mosaic pattern in the gill. Genomic heterogeneity in an endosymbiont population may enable differential utilization of diverse substrates and confer metabolic flexibility. Our findings open a new chapter in our understanding of how symbiotic organisms alter their metabolic capabilities and expand their range of habitats.

First Complete Genome Sequence of Clostridium sporogenes DSM 795T, a Nontoxigenic Surrogate for Clostridium botulinum, Determined Using PacBio Single-Molecule Real-Time Technology.

The first complete genome sequence of Clostridium sporogenes DSM 795(T), a nontoxigenic surrogate for Clostridium botulinum, was determined in a single contig using the PacBio single-molecule real-time technology. The genome (4,142,990 bp; G+C content, 27.98%) included 86 sets of >1,000-bp identical sequence pairs and 380 tandem repeats.

First Complete Genome Sequence of Pseudomonas aeruginosa (Schroeter 1872) Migula 1900 (DSM 50071T), Determined Using PacBio Single-Molecule Real-Time Technology.

The first complete genome sequence of the type strain Pseudomonas aeruginosa (Schroeter 1872) Migula 1900 (DSM 50071(T)) was determined in a single contig by PacBio RS II. The genome (6,317,050 bp, G+C content of 66.52%) contained 10 sets of >1,000-bp identical sequence pairs and 183 tandem repeats.

Complete Genome Sequences of Low-Passage Virulent and High-Passage Avirulent Variants of Pathogenic Leptospira interrogans Serovar Manilae Strain UP-MMC-NIID, Originally Isolated from a Patient with Severe Leptospirosis, Determined Using PacBio Single-Molecule Real-Time Technology.

Here, we report the complete genome sequences of low-passage virulent and high-passage avirulent variants of pathogenic Leptospira interrogans serovar Manilae strain UP-MMC-NIID, a major causative agent of leptospirosis. While there were no major differences between the genome sequences, the levels of base modifications were higher in the avirulent variant.

First Complete Genome Sequences of Staphylococcus aureus subsp. aureus Rosenbach 1884 (DSM 20231T), Determined by PacBio Single-Molecule Real-Time Technology.

The first complete genome sequences of Staphylococcus aureus subsp. aureus Rosenbach 1884 strain DSM 20231(T), the type strain of the bacterium causing staphylococcal disease, were determined using PacBio RS II. The sequences represent the chromosome (2,755,072 bp long; G+C content, 32.86%) and a plasmid (27,490 bp long; G+C content, 30.69%).

Genome sequence determination and metagenomic characterization of a Dehalococcoides mixed culture grown on cis-1,2-dichloroethene.

A Dehalococcoides-containing bacterial consortium that performed dechlorination of 0.20 mM cis-1,2-dichloroethene to ethene in 14 days was obtained from the sediment mud of the lotus field. To obtain detailed information of the consortium, the metagenome was analyzed using the short-read next-generation sequencer SOLiD 3. Matching the obtained sequence tags with the reference genome sequences indicated that the Dehalococcoides sp. in the consortium was highly homologous to Dehalococcoides mccartyi CBDB1 and BAV1. Sequence comparison with the reference sequence constructed from 16S rRNA gene sequences in a public database showed the presence of Sedimentibacter, Sulfurospirillum, Clostridium, Desulfovibrio, Parabacteroides, Alistipes, Eubacterium, Peptostreptococcus and Proteocatella in addition to Dehalococcoides sp. After further enrichment, the members of the consortium were narrowed down to almost three species. Finally, the full-length circular genome sequence of the Dehalococcoides sp. in the consortium, D. mccartyi IBARAKI, was determined by analyzing the metagenome with the single-molecule DNA sequencer PacBio RS. The accuracy of the sequence was confirmed by matching it to the tag sequences obtained by SOLiD 3. The genome is 1,451,062 nt and the number of CDS is 1566, which includes 3 rRNA genes and 47 tRNA genes. There exist twenty-eight RDase genes that are accompanied by the genes for anchor proteins. The genome exhibits significant sequence identity with other Dehalococcoides spp. throughout the genome, but there exists significant difference in the distribution RDase genes. The combination of a short-read next-generation DNA sequencer and a long-read single-molecule DNA sequencer gives detailed information of a bacterial consortium.

Complete annotated genome sequence of Mycobacterium tuberculosis (Zopf) Lehmann and Neumann (ATCC35812) (Kurono).

We report the completely annotated genome sequence of Mycobacterium tuberculosis (Zopf) Lehmann and Neumann (ATCC35812) (Kurono), which is a used for virulence and/or immunization studies. The complete genome sequence of M. tuberculosis Kurono was determined with a length of 4,415,078 bp and a G+C content of 65.60%. The chromosome was shown to contain a total of 4,340 protein-coding genes, 53 tRNA genes, one transfer messenger RNA for all amino acids, and 1 rrn operon. Lineage analysis based on large sequence polymorphisms indicated that M. tuberculosis Kurono belongs to the Euro-American lineage (lineage 4). Phylogenetic analysis using whole genome sequences of M. tuberculosis Kurono in addition to 22 M. tuberculosis complex strains indicated that H37Rv is the closest relative of Kurono based on the results of phylogenetic analysis. These findings provide a basis for research using M. tuberculosis Kurono, especially in animal models.

First Complete Genome Sequence of Salmonella enterica subsp. enterica Serovar Typhimurium Strain ATCC 13311 (NCTC 74), a Reference Strain of Multidrug Resistance, as Achieved by Use of PacBio Single-Molecule Real-Time Technology.

We report the first complete genomic sequence of Salmonella enterica subsp. enterica serovar Typhimurium strain ATCC 13311, the leading food-borne pathogen and a reference strain used in drug resistance studies. De novo assembly with PacBio sequencing completed its chromosome and one plasmid. They will accelerate the investigation into multidrug resistance in Salmonella Typhimurium.