Characteristic morphology of intracellular microcolonies of Legionella oakridgensis OR-10

Legionella oakridgensis occasionally causes pneumonia in humans. We report here the characteristic morphology of intracellular microcolonies of L. oakridgensis OR-10 in infected epithelial cells. By light microscopy after Gimenez staining, the bacteria showed serpentine-like chain, disk-like conglomerate, and granular forms when they grew intracellularly in Vero cells, HeLa cells, and A549 cells. In a time-lapse study, we observed the progressive change from a serpentine-like chain form to a conglomerate form in Vero cells. Transmission electron microscopy showed that L. oakridgensis OR-10 proliferated both inside membrane structures and in the cytoplasm. Such highly serpentine chain growth has not been reported in any intracellular bacteria. Furthermore, these results imply that L. oakridgensis OR-10 may be proliferating inside the endoplasmic reticulum.     

VopB1 and VopD1 are essential for translocation of type III secretion system 1 effectors of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a pathogenic Vibrio species that causes food-borne acute gastroenteritis, often related to the consumption of raw or undercooked seafood. Vibrio parahaemolyticus has 2 type III secretion systems (T3SS1 and T3SS2). Here, we demonstrate that VP1657 (VopB1) and VP1656 (VopD1), which share sequence similarity with Pseudomonas genes popB (38%) and popD (36%), respectively, are essential for translocation of T3SS1 effectors into host cells. A VP1680CyaA fusion reporter system was constructed to observe effector translocation. Using this reporter assay we showed that the VopB1 and VopD1 deletion strains were unable to translocate VP1680 to host cell but that the secretion of VP1680 into the culture medium was not affected. VopB1 or VopD1 deletion strains did not enhance cytotoxicity and failed to activate mitogen-activated protein kinases and secretion of interleukin-8, which depend on VP1680. Thus, we conclude that VopB1 and VopD1 are essential components of the translocon. To target VopB1 and VopD1 may have therapeutic potential for the treatment or prevention in V.?parahaemolyticus infection.     

Regulation by mitochondrial superoxide and NADPH oxidase of cellular formation of nitrated cyclic GMP: potential implications for ROS signalling

8-Nitro-cGMP (8-nitroguanosine 3',5'-cyclic monophosphate) is a nitrated derivative of cGMP, which can function as a unique electrophilic second messenger involved in regulation of an antioxidant adaptive response in cells. In the present study, we investigated chemical and biochemical regulatory mechanisms involved in 8-nitro-cGMP formation, with particular focus on the roles of ROS (reactive oxygen species). Chemical analyses demonstrated that peroxynitrite-dependent oxidation and myeloperoxidase-dependent oxidation of nitrite in the presence of H2O2 were two major pathways for guanine nucleotide nitration. Among the guanine nucleotides examined, GTP was the most sensitive to peroxynitrite-mediated nitration. Immunocytochemical and tandem mass spectrometric analyses revealed that formation of 8-nitro-cGMP in rat C6 glioma cells stimulated with lipopolysaccharide plus pro-inflammatory cytokines depended on production of both superoxide and H2O2. Using the mitochondria-targeted chemical probe MitoSOX Red, we found that mitochondria-derived superoxide can act as a direct determinant of 8-nitro-cGMP formation. Furthermore, we demonstrated that Nox2 (NADPH oxidase 2)-generated H2O2 regulated mitochondria-derived superoxide production, which suggests the importance of cross-talk between Nox2-dependent H2O2 production and mitochondrial superoxide production. The results of the present study suggest that 8-nitro-cGMP can serve as a unique second messenger that may be implicated in regulating ROS signalling in the presence of NO.     

Potential association of Helicobacter cinaedi with atrial arrhythmias and atherosclerosis

Helicobacter cinaedi has been increasingly recognized as an emerging pathogen. Reports of recurrent bacteremia and isolation of H. cinaedi organisms from a patient with myopericarditis led us to postulate that H. cinaedi is associated with chronic inflammatory cardiovascular diseases such as atrial arrhythmias and atherosclerosis. To assess any association of H. cinaedi with atrial arrhythmias, a retrospective case-control study of patients attending Kumamoto University Hospital from 2005 to 2009 was performed. The arrhythmia status of these patients was determined from their electrocardiography and electrophysiological studies. Multiple logistic regression analysis was used to identify independent risk factors. In a comparison of case patients (n= 132) with control subjects (n= 137), H. cinaedi seropositivity was identified as an independent risk factor for atrial arrhythmia (odds ratio, 5.13; 95% confidence interval, 3.0-8.7; P < 0.001). There were no significant differences, however, between these two groups with respect to anti-H. pylori IgG concentrations, anti-Chlamydophila pneumoniae IgG concentrations, and other studied variables. IgG concentrations against H. cinaedi and H. pylori were inversely correlated, which suggests cross-immunity between these two bacteria. Also, to explore any association of H. cinaedi with atherosclerosis, immunohistochemical analysis of atherosclerotic aortic tissues collected post mortem from nine patients was performed. Immunohistochemistry of atherosclerotic aortic tissues from all nine patients detected H. cinaedi antigens inside CD68(+) macrophages. These findings provide the first evidence, to our knowledge, of a possible association of H. cinaedi with atrial arrhythmias and atherosclerosis.     

Production of Aromatic Compounds by Metabolically Engineered Escherichia coli with an Expanded Shikimate Pathway

Escherichia coli was metabolically engineered by expanding the shikimate pathway to generate strains capable of producing six kinds of aromatic compounds, phenyllactic acid, 4-hydroxyphenyllactic acid, phenylacetic acid, 4-hydroxyphenylacetic acid, 2-phenylethanol, and 2-(4-hydroxyphenyl)ethanol, which are used in several fields of industries including pharmaceutical, agrochemical, antibiotic, flavor industries, etc. To generate strains that produce phenyllactic acid and 4-hydroxyphenyllactic acid, the lactate dehydrogenase gene (ldhA) from Cupriavidus necator was introduced into the chromosomes of phenylalanine and tyrosine overproducers, respectively. Both the phenylpyruvate decarboxylase gene (ipdC) from Azospirillum brasilense and the phenylacetaldehyde dehydrogenase gene (feaB) from E. coli were introduced into the chromosomes of phenylalanine and tyrosine overproducers to generate phenylacetic acid and 4-hydroxyphenylacetic acid producers, respectively, whereas ipdC and the alcohol dehydrogenase gene (adhC) from Lactobacillus brevis were introduced to generate 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, respectively. Expression of the respective introduced genes was controlled by the T7 promoter. While generating the 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, we found that produced phenylacetaldehyde and 4-hydroxyphenylacetaldehyde were automatically reduced to 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol by endogenous aldehyde reductases in E. coli encoded by the yqhD, yjgB, and yahK genes. Cointroduction and cooverexpression of each gene with ipdC in the phenylalanine and tyrosine overproducers enhanced the production of 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol from glucose. Introduction of the yahK gene yielded the most efficient production of both aromatic alcohols. During the production of 2-phenylethanol, 2-(4-hydroxyphenyl)ethanol, phenylacetic acid, and 4-hydroxyphenylacetic acid, accumulation of some by-products were observed. Deletion of feaB, pheA, and/or tyrA genes from the chromosomes of the constructed strains resulted in increased desired aromatic compounds with decreased by-products. Finally, each of the six constructed strains was able to successfully produce a different aromatic compound as a major product. We show here that six aromatic compounds are able to be produced from renewable resources without supplementing with expensive precursors.     

Detection and characterization of a thermophilic biotin biosynthetic enzyme, 7-keto-8-aminopelargonic acid synthase, from various thermophiles

By detailed BLAST searches of the genome database of various thermophiles, five ORFs with similarity to the bioF gene, which encodes 7-keto-8-aminopelargonic acid synthase (BioF) involved in biotin biosynthesis, of Escherichia coli were found: AqbioF, CltbioF, GkbioF, SytbioF, and TsebioF, from Aquifex aeolicus VF5, Clostridium thermocellum ATCC27405, Geobacillus kaustophilus JCM12893, Symbiobacterium thermophilum IAM14863, and Thermosynechococcus elongatus BP-1 respectively. The five purified recombinant bioF gene products, which were overexpressed in E. coli, had the enzyme activity of BioF. The optimum temperature range and thermostability of five BioFs, AqBioF, CltBioF, GkBioF, SytBioF, and TseBioF, were higher than those of E. coli BioF. In particular, AqBioF was found to show the highest thermostability of the α-oxoamine synthase family enzymes reported to date. Substrate specificity experiments revealed that SytBioF was also able to catalyze the reaction of 2-amino-3-ketobutyrate CoA ligase, a member of the α-oxoamine synthase family, and that it used acetyl-CoA and glycine as substrates, like the TTHA1582 protein of Thermus thermophilus. The other purified BioFs, AqBioF and GkBioF, did not show any activity with acyl-CoAs and amino acids other than pimeloyl-CoA and L-alanine as substrates.     

Identification of the actinorhodin monomer and its related compound from a deletion mutant of the actVA-ORF4 gene of Streptomyces coelicolor A3(2)

An oxygenated derivative of dihydrokalafungin (DHK) was isolated from a deletion mutant of the actVA-ORF4 gene involved in the biosynthesis of a dimeric benzoisochromanequinone (BIQ) antibiotic, actinorhodin (ACT), in Streptomyces coelicolor A3(2). Spectroscopic analysis elucidated its structure as 8-hydroxy-DHK, corresponding to the monomeric unit of ACT. Further metabolite analysis identified its related compound, clearly derived from the reduction of 8-hydroxy-DHK. The structures of these metabolites indicate the essential role of ActVA-ORF4 in ACT biosynthesis, specifically in dimerization of a BIQ intermediate via C-C bond formation.     

Bimodal effect of retinoic acid on melanocyte differentiation identified by time-dependent analysis

Retinoic acid (RA) is considered to control melanocytes; however, its precise mechanism remains unclear because of a bimodal effect, which promotes or inhibits melanin synthesis depending on the cell type, culture condition of melanocytes and skin conditions. In this study, we examined the effects of RA throughout each stage of differentiation of melanocytes using a mouse embryonic stem cell culture system to induce melanocytes. The results showed that RA has significantly different effects depending on the stage of differentiation of melanocytes. More specifically, RA promoted differentiation in earlier stages, wherein embryonic stem cells became melanoblasts via neural crest cells, and inhibited differentiation in later stages, wherein melanoblasts became melanocytes. It was revealed for the first time that melanocytes show markedly different reactions to RA depending on the stage of differentiation.