Taichiro Ishige

Multiple rRNA operons are essential for efficient cell growth and sporulation as well as outgrowth in Bacillus subtilis

The number of copies of rRNA (rrn) operons in a bacterial genome differs greatly among bacterial species. Here we examined the phenotypic effects of variations in the number of copies of rRNA genes in the genome of Bacillus subtilis by analysis of eight mutant strains constructed to carry from two to nine copies of the rrn operon. We found that a decrease in the number of copies from ten to one increased the doubling time, and decreased the sporulation frequency and motility. The maximum levels for transformation activity were similar among the strains, although the competence development was significantly delayed in the strain with a single rrn operon. Normal sporulation only occurred if more than four copies of the rrn operon were present, although ten copies were needed for vegetative growth after germination of the spores. This behaviour was seen even though the intracellular level of ribosomes was similar among strains with four to ten copies of the rrn operon. Furthermore, ten copies of the rrn operon were needed for the highest swarming activity. We also constructed 21 strains that carried all possible combinations of two copies of the rrn operons, and found that these showed a range of growth rates and sporulation frequencies that all fell between those recorded for strains with one or three copies of the rrn operon. The results suggested that the copy number of the rrn operon has a major influence on cellular processes such as growth rate and sporulation frequency.

Comprehensive analysis of transcriptome response to salinity stress in the halophytic turf grass Sporobolus virginicus.

The turf grass Sporobolus virginicus is halophyte and has high salinity tolerance. To investigate the molecular basis of its remarkable tolerance, we performed Illumina high-throughput RNA sequencing on roots and shoots of a S. virginicus genotype under normal and saline conditions. The 130 million short reads were assembled into 444,242 unigenes. A comparative analysis of the transcriptome with rice and Arabidopsis transcriptome revealed six turf grass-specific unigenes encoding transcription factors. Interestingly, all of them showed root specific expression and five of them encode bZIP type transcription factors. Another remarkable transcriptional feature of S. virginicus was activation of specific pathways under salinity stress. Pathway enrichment analysis suggested transcriptional activation of amino acid, pyruvate, and phospholipid metabolism. Up-regulation of several unigenes, previously shown to respond to salt stress in other halophytes was also observed. Gene Ontology enrichment analysis revealed that unigenes assigned as proteins in response to water stress, such as dehydrin and aquaporin, and transporters such as cation, amino acid, and citrate transporters, and H+-ATPase, were up-regulated in both shoots and roots under salinity. A correspondence analysis of the enriched pathways in turf grass cells, but not in rice cells, revealed two groups of unigenes similarly up-regulated in the turf grass in response to salt stress; one of the groups, showing excessive up-regulation under salinity, included unigenes homologos to salinity responsive genes in other halophytes. Thus, the present study identified candidate genes involved in salt tolerance of S. virginicus. This genetic resource should be valuable for understanding the mechanisms underlying high salt tolerance in S. virginicus. This information can also provide insight into salt tolerance in other halophytes.

Strigolactone Regulates Anthocyanin Accumulation, Acid Phosphatases Production and Plant Growth under Low Phosphate Condition in Arabidopsis

Phosphate is an essential macronutrient in plant growth and development; however, the concentration of inorganic phosphate (Pi) in soil is often suboptimal for crop performance. Accordingly, plants have developed physiological strategies to adapt to low Pi availability. Here, we report that typical Pi starvation responses in Arabidopsis are partially dependent on the strigolactone (SL) signaling pathway. SL treatment induced root hair elongation, anthocyanin accumulation, activation of acid phosphatase, and reduced plant weight, which are characteristic responses to phosphate starvation. Furthermore, the expression profile of SL-response genes correlated with the expression of genes induced by Pi starvation. These results suggest a potential overlap between SL signaling and Pi starvation signaling pathways in plants.

Microevolution of Virulence-Related Genes in Helicobacter pylori Familial Infection

Helicobacter pylori, a bacterial pathogen that can infect human stomach causing gastritis, ulcers and cancer, is known to have a high degree of genome/epigenome diversity as the result of mutation and recombination. The bacteria often infect in childhood and persist for the life of the host. One of the reasons of the rapid evolution of H. pylori is that it changes its genome drastically for adaptation to a new host. To investigate microevolution and adaptation of the H. pylori genome, we undertook whole genome sequencing of the same or very similar sequence type in multi-locus sequence typing (MLST) with seven genes in members of the same family consisting of parents and children in Japan. Detection of nucleotide substitutions revealed likely transmission pathways involving children. Nonsynonymous (amino acid changing) mutations were found in virulence-related genes (cag genes, vacA, hcpDX, tnfα, ggt, htrA and the collagenase gene), outer membrane protein (OMP) genes and other cell surface-related protein genes, signal transduction genes and restriction-modification genes. We reconstructed various pathways by which H. pylori can adapt to a new human host, and our results raised the possibility that the mutational changes in virulence-related genes have a role in adaptation to a child host. Changes in restriction-modification genes might remodel the methylome and transcriptome to help adaptation. This study has provided insights into H. pylori transmission and virulence and has implications for basic research as well as clinical practice.

Phylogenetic and population genetic analysis of Salmonella enterica subsp. enterica serovar Infantis strains isolated in Japan using whole genome sequence data

Salmonella enterica subsp. enterica serovar Infantis has been reported to be carried asymptomatically in chickens and isolated from some human patients with diarrhea. The aim of this study was to investigate the phylogeny of S. Infantis strains isolated in Japan from chicken meat, chicken egg shells, environmental samples from a grading and packing center for chicken eggs (GP center), diarrhea patients, and asymptomatic carriers based on whole genome sequence data. The S. Infantis strains were in five clusters in a phylogenetic tree reconstructed by the maximum likelihood method. The five clusters were confirmed by neighbor-net and Bayesian cluster analyses. Two of the five clusters formed a group containing all of the strains isolated from chicken meat and some of the strains isolated from diarrhea patients and asymptomatic carriers. The median-joining network reconstructed in this study showed that strains in one of these two clusters diverged from one node with similar relatively short branches, suggesting clonal dissemination of these strains. The other three clusters formed a group containing all of the strains isolated from chicken egg shells and the GP center, and the remaining strains from diarrhea patients and asymptomatic carriers. Interestingly, strains isolated from patients did not cluster in only one group, indicating that none of the S. Infantis strains in this study had significantly higher human pathogenicity. The population genetic analyses in this study showed the separation of the five clusters into two groups was concordant with the sources where the strains in the clusters were isolated. These results suggested that evolutionary groups with higher hierarchy than the clusters identified in this study may be present, although such groups could not be determined by phylogenetic, neighbor-net, and Bayesian analyses in this study. Determination of higher level S. Infantis evolutionary groups should be investigated using other types of genetic markers.

A novel subpopulation of Salmonella enterica serovar Infantis strains isolated from broiler chicken organs other than the gastrointestinal tract.

Salmonella enterica subsp. enterica serovar Infantis strains were isolated from broiler chickens from six farms in Japan and the pathogenicity associated with the recently reported 280kbp mega plasmid was examined by possession of the plasmid and histopathology of tissues from these chickens. S. Infantis strains were isolated from 10 of 24 chickens. Phylogenetic, network and Bayesian cluster analyses were used to determine whether these strains were in the previously defined Clusters 1-5. Phylogenetic analysis classified the strains isolated in this study in two groups (Groups A and B). Both groups contained strains from gastrointestional contents, but only Group A also contained strains from spleen, liver, and lymphoid tissues. Histopathology showed suppurative splenitis in a spleen from which Group A strains were isolated. Although network and Bayesian cluster analyses were unable to differentiate Group A and B strains from the previously defined Clusters 1-5, population genetic analysis indicated that Group A was a different population from Cluster 5, indicating that Group A would be a subpopulation of Cluster 5. The irp2 gene, which is in the mega plasmid carried by a pathogenic S. Infantis strain recently isolated in Israel, was found in both Groups A and B strains and in the previously reported Clusters 4 and 5 strains. These results suggested that Group A would be a novel subpopulation of the previously defined Cluster 5, and presence of the mega plasmid may not be related whether S. Infantis strains can infect certain organs.

Comparative Transcriptome Analysis of the Less-Dormant Taiwanese Pear and the Dormant Japanese Pear during Winter Season

The flower bud transcriptome in the less dormant Taiwanese pear ‘Hengshanli’ and high-chilling requiring Japanese pear strain TH3 subjected to the same chilling exposure time were analyzed during winter using next-generation sequencing. In buds sampled on January 10th and on February 7th in 2014, 6,978 and 7,096 genes, respectively, were significantly differentially expressed in the TH3 and ‘Hengshanli’ libraries. A comparative GO analysis revealed that oxidation-reduction process (biological process) and ATP binding (molecular function), were overrepresented during the ecodormancy period (EP) when compared to the endodormancy deepest period (DP), indicating that ATP synthesis was activated during the transition between these dormancy stages. Among the 11 differently expressed genes (DEGs) annotated as probable dehydrins or LEA protein-related genes, 9 DEGs showed higher transcript levels in the DP than in the EP. In order to focus on transcription factors induced by low temperature or drought, 7 differently expressed genes (DEGs) annotated as probable ICE1 or DREB proteins were analyzed by real-time PCR. Expression levels of 3 genes were higher in TH3 than in ‘Hengshanli’ on all sampling days. Their expression increased during the endodormancy deepest period (DP) and then decreased before endodormancy breaking in TH3 buds. Taken together, these results suggest that these genes annotated as ICE1, DREB and ERF are involved in endodormancy maintenance and in the transition from endodormancy to ecodormancy.

Basic characterization of avian β-defensin genes in the Japanese quail, Coturnix japonica.

In this study, we identified a cluster of 14 avian β-defensins (AvBD; approximately 66 kbp) in the Japanese quail, Coturnix japonica. Except for AvBD12 (CjAvBD12) and -13, the CjAvBDs coding sequences exhibited greater than 78.0% similarity to the respective orthologous chicken AvBD genes (GgAvBD). The putative amino acid sequence encoded by each CjAvBD contained six cysteine residues and the GXC (X1-2) motif considered essential for the β-defensin family. Each CjAvBDs also formed a sub-group with the respective orthologous genes of various bird species in a phylogenetic tree analysis. Synteny between the CjAvBD cluster and GgAvBD cluster was confirmed. The CjAvBD cluster was mapped on the long-arm end of chromosome 3 by linkage analysis based on single nucleotide polymorphisms (SNPs) of CjAvBD1 and CjAvBD12 (approximately 46 kbp), as well as GgAvBD cluster. We also confirmed that CjAvBD1, -4, -5, -9, and -10 are transcribed in 20 tissues, including immune and digestive tissues. However, our experimental data indicated that the CjAvBD cluster lacks the AvBD3 and -7 loci, whereas the CjAvBD101α, -101β, and -101θ loci arose from gene duplication of the AvBD6 orthologous locus in the CjAvBD cluster after differentiation between Coturnix - Gallus.

Single-Nucleotide Polymorphisms in the Whole-Genome Sequence Data of Shiga Toxin-Producing Escherichia coli O157:H7/H- Strains by Cultivation

Nine Shiga toxin-producing Escherichia coli O157:H7/H- (O157) strains were serially cultured three times on LB agar plates. After each sub-culture, five colonies were picked for DNA isolation and whole genome sequence (WGS) analysis. After exclusion of possible recombination-related SNPs, 11, 9, and 34 single-nucleotide polymorphisms (SNPs) were detected in genes in the backbone, O-island, and mobile elements gene categories. This suggested that those SNPs due to cultivation could influence the threshold value set for molecular epidemiological studies of O157. Significant differences were observed by the Kruskal–Wallis test (P < 0.01) when the number of the SNPs in a strain was compared to that in other strains. This indicated that a specific number of strains could be used for setting the threshold value in molecular epidemiological studies. Due to cultivation, the SNPs were also detected in genes in a few core genome or core gene sets, suggesting that those SNPs could affect studies of phylogeny as well as molecular epidemiology. To improve the accuracy of phylogenetic and molecular epidemiological studies, genes in which the SNPs have arisen due to cultivation should be excluded from WGS data.

Evaluating the Phylogenetic Status of the Extinct Japanese Otter on the Basis of Mitochondrial Genome Analysis

The Japanese otter lived throughout four main Japanese islands, but it has not been observed in the wild since 1979 and was declared extinct in 2012. Although recent taxonomic and molecular phylogenetic studies suggest that it should be treated as an independent species, International Union for Conservation of Nature Red List considers it as subspecies of Lutra lutra. Therefore, the taxonomic status of this species needs to be resolved. Here we determined the complete mitochondrial genome of two Japanese otters caught in Kanagawa and Kochi prefectures and five Eurasian otters (L. lutra). We reconstructed a molecular phylogenetic tree to estimate the phylogenetic position of the Japanese otter in Lutrinae using the Japanese otters and the other 11 Lutrinae species on the basis of ND5 (692 bp) and cytochrome b (1,140 bp) sequences. We observed that the two Japanese otters had close relationships with Eurasian otters, forming a monophyletic group (100% bootstrap probability). To elucidate detailed phylogenetic relationships among the Japanese and Eurasian otters, we reconstructed a maximum likelihood tree according to mitochondrial genome sequences (14,740 bp). The Japanese otter (JO1) collected in Kanagawa was deeply nested in the Eurasian otter clade, whereas the Japanese otter (JO2) collected in Kochi formed a distinct independent lineage in the Lutra clade. The estimated molecular divergences time for the ancestral lineages of the Japanese otters was 0.10 Ma (95%: 0.06–0.16 Ma) and 1.27 Ma (95%: 0.98–1.59 Ma) for JO1 and JO2 lineages, respectively. Thus, JO1 was identified as a member of L. lutra; JO2 represented the old Japanese otter lineage, which may be a distinct new species or subspecies of Lutra. We suggest that the ancestral population of the JO2 lineage migrated to Japan via the land bridge that existed between western Japanese islands and Asian continent at 1.27 Ma.