Comparative Genomic Analysis of Lactococcus garvieae Strains Isolated from Different Sources Reveals Candidate Virulence Genes

Lactococcus garvieae is a major pathogen for fish. Two complete (ATCC 49156 and Lg2) and three draft (UNIUD074, 8831, and 21881) genome sequences of L. garvieae have recently been released. We here present the results of a comparative genomic analysis of these fish and human isolates of L. garvieae. The pangenome comprised 1,542 core and 1,378 dispensable genes. The sequenced L. garvieae strains shared most of the possible virulence genes, but the capsule gene cluster was found only in fish-pathogenic strain Lg2. The absence of the capsule gene cluster in other nonpathogenic strains isolated from mastitis and vegetable was also confirmed by PCR. The fish and human isolates of L. garvieae contained the specific two and four adhesin genes, respectively, indicating that these adhesion proteins may be involved in the host specificity differences of L. garvieae. The discoveries revealed by the pangenomic analysis may provide significant insights into the biology of L. garvieae

Intestinal epithelial cell-derived IL-15 determines local maintenance and maturation of intraepithelial lymphocytes in the intestine

Interleukin-15 (IL-15) is a cytokine critical for maintenance of intestinal intraepithelial lymphocytes (IELs), especially CD8αα+ IELs (CD8αα IELs). In the intestine, IL-15 is produced by intestinal epithelial cells (IECs), blood vascular endothelial cells (BECs) and hematopoietic cells. However, the precise role of intestinal IL-15 on IELs is still unknown. To address the question, we generated two kinds of IL-15 conditional knockout (IL-15cKO) mice: villin-Cre (Vil-Cre) and Tie2-Cre IL-15cKO mice. IEC-derived IL-15 was specifically deleted in Vil-Cre IL-15cKO mice, whereas IL-15 produced by BECs and hematopoietic cells is deleted in Tie2-Cre IL-15cKO mice. The cell number and frequency of CD8αα IELs and NK IELs were significantly reduced in Vil-Cre IL-15cKO mice. By contrast, CD8αα IELs were unchanged in Tie2-Cre IL-15cKO mice, indicating that IL-15 produced by BECs and hematopoietic cells is dispensable for CD8αα IELs. Expression of an anti-apoptotic factor, Bcl-2, was decreased, whereas Fas expression was increased in CD8αα IELs of Vil-Cre IL-15cKO mice. Forced expression of Bcl-2 by a Bcl-2 transgene partially restored CD8αα IELs in Vil-Cre IL-15cKO mice, suggesting that some IL-15 signal other than Bcl-2 is required for maintenance of CD8αα IELs. Furthermore, granzyme B production was reduced, whereas PD-1 expression was increased in CD8αα IELs of Vil-Cre IL-15cKO mice. These results collectively suggested that IEC-derived IL-15 is essential for homeostasis of IELs by promoting their survival and functional maturation

Diabetes and obesity are significant risk factors for morning hypertension: From Ibaraki Hypertension Assessment Trial (I-HAT)

AimsAlthough morning hypertension (HT) has been identified as a major cardiovascular risk, susceptible populations remain unknown. This study aimed to clarify the relationship between morning HT and diabetes or obesity in a large-scale population.Main methodsClinic blood pressure (BP) and BP upon awakening were recorded in 2554 outpatients with HT who attended 101 clinics or hospitals for two weeks. Mean clinic and awakening BP > 140/90 and > 135/85 mm Hg, respectively, were considered as HT. The patients were classified according to values for clinic and home BP, into normal BP, white coat HT, masked HT, and sustained HT.Key findingsMorning BP (mm Hg) significantly and progressively elevated in the order of normal glucose tolerance, impaired glucose tolerance and diabetes (134.1 ± 12.2, 135.4 ± 13.1 and 137.5 ± 11.5; p < 0.0001). The incidence of morning HT significantly increased and progressively in the same order (53.4%, 55.6%, 66.4%, p < 0.0001). Morning BP was significantly higher among obese patients with diabetes than among non-obese and non-diabetic patients (138.8 ± 10.5, 133.1 ± 11.9, p < 0.0001). In addition, the incidence of morning HT was significantly higher in obese diabetic patients than in non-obese and non-diabetic patients (73.0% vs. 49.9%, p < 0.0001).SignificanceDiabetic or obese patients frequently have morning HT

Complete Genome Sequence and Comparative Analysis of the Fish Pathogen Lactococcus garvieae

Lactococcus garvieae causes fatal haemorrhagic septicaemia in fish such as yellowtail. The comparative analysis of genomes of a virulent strain Lg2 and a non-virulent strain ATCC 49156 of L. garvieae revealed that the two strains shared a high degree of sequence identity, but Lg2 had a 16.5-kb capsule gene cluster that is absent in ATCC 49156. The capsule gene cluster was composed of 15 genes, of which eight genes are highly conserved with those in exopolysaccharide biosynthesis gene cluster often found in Lactococcus lactis strains. Sequence analysis of the capsule gene cluster in the less virulent strain L. garvieae Lg2-S, Lg2-derived strain, showed that two conserved genes were disrupted by a single base pair deletion, respectively. These results strongly suggest that the capsule is crucial for virulence of Lg2. The capsule gene cluster of Lg2 may be a genomic island from several features such as the presence of insertion sequences flanked on both ends, different GC content from the chromosomal average, integration into the locus syntenic to other lactococcal genome sequences, and distribution in human gut microbiomes. The analysis also predicted other potential virulence factors such as haemolysin. The present study provides new insights into understanding of the virulence mechanisms of L. garvieae in fish

An Analysis of the Effect on Human Resource Development by Intrapreneunal Venturing

企業におけるホワイトカラーを取り巻く環境は厳しく,また,求められる技能のレベルも高まり,その育成は企業内に留まらずビジネススクール等の外部機関も活用されている.このような外部機関の利用を社外経験と捉えた場合,社内起業が企業の内部で育成可能な重要な手段として位置づけられるのではないかとの立場に立ち,企業内で社内起業を経験したホワイトカラーと企業本体での業務を経験しているホワイトカラーを対象に,技能及びその習得プロセスの比較を行った。なお,これまでの社内起業に関する先行研究については,社内起業を担うホワイトカラーの育成に焦点を当てたものが少ないため,本研究では,既存の理論に基づく仮説検証型研究ではなく,調査及び分析により仮説的知見を導くという仮説探索型研究を採用した。調査及び分析の結果から,社内起業経験の優位性を主張する仮説的知見とともに,社内起業経験による人材育成モデルを提示した

Multiple-Subunit Genes of the Aromatic-Ring-Hydroxylating Dioxygenase Play an Active Role in Biphenyl and Polychlorinated Biphenyl Degradation in Rhodococcus sp. Strain RHA1

A gram-positive strong polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, can degrade PCBs by cometabolism with biphenyl or ethylbenzene. In RHA1, three sets of aromatic-ring-hydroxylating dioxygenase genes are induced by biphenyl. The large and small subunits of their terminal dioxygenase components are encoded by bphA1 and bphA2, etbA1 and etbA2, and ebdA1 and ebdA2, respectively, and the deduced amino acid sequences of etbA1 and etbA2 are identical to those of ebdA1 and ebdA2, respectively. In this study, we examined the involvement of the respective subunit genes in biphenyl/PCB degradation by RHA1. Reverse transcription-PCR and two-dimensional polyacrylamide gel electrophoresis analyses indicated the induction of RNA and protein products of etbA1 and ebdA1 by biphenyl. Single- and double-disruption mutants of etbA1, ebdA1, and bphA1 were constructed by insertional inactivation. The 4-chlorobiphenyl (4-CB) degradation activities of all the mutants were lower than that of RHA1. The results indicated that all of these genes are involved in biphenyl/PCB degradation. Furthermore, we constructed disruption mutants of ebdA3 and bphA3, encoding ferredoxin, and etbA4, encoding ferredoxin reductase components. The 4-CB degradation activities of these mutants were also lower than that of RHA1, suggesting that all of these genes play a role in biphenyl/PCB degradation. The substrate preferences of etbA1A2/ebdA1A2- and bphA1A2-encoded dioxygenases for PCB congeners were examined using the corresponding mutants. The results indicated that these dioxygenase isozymes have different substrate preferences and that the etbA1A2/ebdA1A2-encoded isozyme is more active on highly chlorinated congeners than the bphA1A2-encoded one

Characterization of the Gallate Dioxygenase Gene: Three Distinct Ring Cleavage Dioxygenases Are Involved in Syringate Degradation by Sphingomonas paucimobilis SYK-6

Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate (PCA) and 3-O-methylgallate (3MGA) in reactions with the tetrahydrofolate-dependent O-demethylases LigM and DesA, respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, whereas 3MGA is metabolized via three distinct pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and 3MGA O-demethylase (LigM) are involved. In the 3MGA O-demethylation pathway, LigM converts 3MGA to gallate, and the resulting gallate appears to be degraded by a dioxygenase other than LigAB or DesZ. Here, we isolated the gallate dioxygenase gene, desB, which encodes a 418-amino-acid protein with a molecular mass of 46,843 Da. The amino acid sequences of the N-terminal region (residues 1 to 285) and the C-terminal region (residues 286 to 418) of DesB exhibited ca. 40% and 27% identity with the sequences of the PCA 4,5-dioxygenase β and α subunits, respectively. DesB produced in Escherichia coli was purified and was estimated to be a homodimer (86 kDa). DesB specifically attacked gallate to generate 4-oxalomesaconate as the reaction product. The K(m) for gallate and the V(max) were determined to be 66.9 ± 9.3 μM and 42.7 ± 2.4 U/mg, respectively. On the basis of the analysis of various SYK-6 mutants lacking the genes involved in syringate degradation, we concluded that (i) all of the three-ring cleavage dioxygenases are involved in syringate catabolism, (ii) the pathway involving LigM and DesB plays an especially important role in the growth of SYK-6 on syringate, and (iii) DesB and LigAB are involved in gallate degradation

2-Hydroxypenta-2,4-dienoate Metabolic Pathway Genes in a Strong Polychlorinated Biphenyl Degrader, Rhodococcus sp. Strain RHA1

A gram-positive polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, metabolizes biphenyl through the 2-hydroxypenta-2,4-dienoate (HPD) and benzoate metabolic pathways. The HPD metabolic pathway genes, the HPD hydratase (bphE1), 4-hydroxy-2-oxovalerate aldolase (bphF1), and acetaldehyde dehydrogenase (acylating) (bphG) genes, were cloned from RHA1. The deduced amino acid sequences of bphGF1E1 have 30 to 58% identity with those of the HPD metabolic pathway genes of gram-negative bacteria. The order of these genes, bphG-bphF1-bphE1, differs from that of the HPD metabolic pathway genes, bphE-bphG-bphF, in gram-negative degraders of PCB, phenol, and toluene. Reverse transcription-PCR experiments indicated that the bphGF1E1 genes are inducibly cotranscribed in cells grown on biphenyl and ethylbenzene. Primer extension analysis revealed that the transcriptional initiation site exists within the bphR gene located adjacent to and upstream of bphG, which is deduced to code a transcriptional regulator. The respective enzyme activities of bphGF1E1 gene products were detected in Rhodococcus erythropolis IAM1399 carrying a bphGF1E1 plasmid. The insertional inactivation of the bphE1, bphF1, and bphG genes resulted in the loss of the corresponding enzyme activities and diminished growth on both biphenyl and ethylbenzene. Severe growth interference was observed during growth on biphenyl. The growth defects were partially restored by the introduction of plasmids containing the respective intact genes. These results indicated that the cloned bphGF1E1 genes are not only responsible for the primary metabolism of HPD during growth on both biphenyl and ethylbenzene but are also involved in preventing the accumulation of unexpected toxic metabolites, which interfere with the growth of RHA1