St. John's wort promotes adipocyte differentiation and modulates NF-κB activation in 3T3-L1 cells.

St. John's wort (SJW), or Hypericum perforatum, is a perennial herb that has been used in the treatment of depression in several countries. Though its therapeutic effect on depression has been extensively studied, its influence on metabolic syndrome is yet to be fully characterized. Therefore, we investigated the effect of SJW extract on adipocyte differentiation and its anti-inflammatory effects by using 3T3-L1 preadipocytes. Oil Red O staining indicated that SJW promotes adipocyte differentiation, while immunoblots indicated that SJW increases the expression of peroxisome proliferator activated receptor γ (PPARγ), a nuclear receptor regulating adipocyte differentiation, and adiponectin, an anti-inflammatory adipokine. Furthermore, the anti-inflammatory activity of SJW was demonstrated by its inhibition of the activation of nuclear factor-κB (NF-κB), an inflammatory transcription factor. Stimulation of mature 3T3-L1 adipocytes by tumor necrosis factor-α (TNF-α) decreased the expression of the NF-κB inhibitor IκBα, and increased its phosphorylation. Treatment with SJW further decreased the TNF-α-induced perturbation in IκBα expression and phosphorylation, which indicated that SJW mediated the inhibition of NF-κB activation. In addition, SJW decreased the TNF-α-induced increase in the mRNA levels of pro-inflammatory adipokines, interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1). Collectively, our results indicate that SJW treatment could promote adipocyte differentiation probably through its anti-inflammatory activity, which in turn suggests that SJW has the potential to minimize the risk factors of metabolic syndrome.

Electron microscopic analysis of heat-induced leakage of polyphosphate from a phoU mutant of Escherichia coli.

Heat treatment is a simple technique for releasing polyphosphate (polyP) from microbial cells. We investigated the ultrastructural alterations of polyP granules (PPGs) in response to heat treatment at 70 degrees C using a polyP-accumulating phoU mutant of Escherichia coli as a model organism. Electron microscopic and energy-dispersive X-ray analysis suggested concurrent occurrence of PPG degradation and polyP release in the cytoplasm. PolyP is probably released by cytoplasmic extrusion through breaks in the cell envelope.

Effect of cell-surface hydrophobicity on bacterial conversion of water-immiscible chemicals in two-liquid-phase culture systems.

The effect of bacterial cell-surface hydrophobicity on the bioconversion of water-immiscible chemicals in an aqueous-organic (A/O) two-liquid-phase culture system was investigated. Escherichia coli JM109 and Rhodococcus opacus B-4 were used as hydrophilic and hydrophobic whole-cell catalysts, respectively. Hydroxylation reactions of monoaromatics, including toluene (log P(ow)=2.9), ethylbenzene (3.1), n-propylbenzene (3.4), and sec-butylbenzene (3.7), were employed as model conversions. When the todC1C2BA genes encoding Pseudomonas putida toluene dioxygenase were expressed in E. coli JM109, the yield of hydroxylated monoaromatics decreased with increasing substrate hydrophobicity. By contrast, R. opacus transformants, which expressed the todC1C2BA genes, showed high performance in the hydroxylation of monoaromatics, irrespective of substrate hydrophobicity. When the R. opacus transformants were examined for their ability to hydroxylate monoaromatics in an aqueous single-liquid-phase culture, the reaction velocity was markedly lower than that observed in the A/O two-liquid-phase culture. These results suggested that R. opacus B-4 accessed the hydrophobic substrates in the oil phase, thus making it more effective for the bioconversion reactions.

A comparison of various methods to predict bacterial predilection for organic solvents used as reaction media.

Bacterial predilection for organic solvents is important in whole-cell biocatalysis in organic media. Although various methods of measuring bacterial hydrophobicity have been proposed, it is not fully determined whether they are applicable to the assessment of bacterial predilection for organic solvents in whole-cell biocatalytic processes. In this study, bacterial predilection for organic solvents was assessed by bacterial adhesion to hydrocarbon (BATH), contact angle measurement (CAM), hydrophobic interaction chromatography (HIC), and glass adhesion test (GAT). These methods were applied to the cultures of four bacterial species of industrial importance, namely, Rhodococcus opacus B-4, R. erythropolis PR4, Pseudomonas putida T-57, and Escherichia coli JM109, in organic media. Experimental results revealed that CAM assays could be used to predict the dispersibility of bacterial cells in anhydrous organic solvents. However, when bacteria were suspended in aqueous-organic (A/O) two-phase media, the results of BATH assays provided the most reliable assessment of bacterial predilection for organic solvents. This discrepancy noted between CAM and BATH assays was attributed to the effect of electrostatic interaction between bacteria and oil droplets. In A/O two-phase media, the accessibility of a water-immiscible dye, nile red, to the bacterial cell surface, correlated well with BATH assay results.

Expression of Rhodococcus opacus alkB genes in anhydrous organic solvents.

Rhodococcus opacus B-4 is a benzene-tolerant bacterium which was isolated from a gasoline-contaminated soil sample. We previously demonstrated that this organism was able to survive and exhibit biocatalytic activity in anhydrous organic solvents for at least 5 d. In the present study, we cloned the alkB1 and alkB2 genes encoding alkane hydroxylases from R. opacus B-4. Heterologous expression of the alkB1 and alkB2 genes in Escherichia coli JM109 showed that they encode functional alkane hydroxylases with a substrate range of C(5)-C(16). Promoters of the alkB1 and alkB2 genes, designated P(alkB1) and P(alkB2), respectively, were examined for activity in anhydrous bis (2-ethylhexyl) phthalate (BEHP) containing C(5)-C(16)n-alkanes. Two recombinant plasmids, pP(alkB1)EGFP and pP(alkB2)EGFP, were constructed by inserting the egfp gene downstream of P(alkB1) and P(alkB2), respectively and transformed into R. opacus B-4. Resting cells of R. opacus B-4 (pP(alkB1)EGFP) showed greater levels of EGFP fluorescence in anhydrous BEHP than in 0.85% NaCl, when C(8)-C(16)n-alkanes were supplied as an inducer. Furthermore, n-alkane inducibility of P(alkB1) activity in anhydrous BEHP was noticeably different from that in 0.85% NaCl. This paper presents the first evidence that bacteria can express their genes in essentially anhydrous organic solvents.

Stabilization of water-in-oil emulsion by Rhodococcus opacus B-4 and its application to biotransformation.

Rhodococcus opacus B-4, which has recently been isolated as an organic solvent-tolerant bacterium, stabilized water-in-oil (w/o) emulsions by inhibition of droplet coalescence when the cells were dispersed in 90% (v/v) organic solvents. Confocal microscopy revealed that many bacterial cells assembled at the interface between oil and water droplets, though free cells were also detectable at the inside of water droplets. Bacterial cells in the w/o emulsion were capable of utilizing both a water-soluble (glucose) and an oil-soluble substrate (oleic acid) as an energy source. Availability of the w/o emulsion as an immobilized cell system in organic solvents was demonstrated using production of indigo from indole and production of o-cresol from toluene as model conversions. When glucose and oleic acid were simultaneously supplied as energy sources, the w/o emulsion culture of R. opacus B-4 produced indigo and o-cresol at levels of 0.217 and 2.12 mg ml(-1), respectively, by 12 h.

Utilization of hydrophobic bacterium Rhodococcus opacus B-4 as whole-cell catalyst in anhydrous organic solvents.

Rhodococcus opacus strain B-4, which has recently been isolated as an organic solvent-tolerant bacterium, has a high hydrophobicity and exhibits a high affinity for hydrocarbons. This bacterium was able to survive for at least 5 days in organic solvents, including n-tetradecane, oleyl alcohol, and bis(2-ethylhexyl) phthalate (BEHP), which contained water less than 1% (w/v). The biocatalytic ability of R. opacus B-4 was demonstrated in the essentially nonaqueous BEHP using indigo production from indole as a model conversion. By the catabolism of oleic acid for NADH regeneration, indigo production increased up to 71.6 microg ml(-1) by 24 h.

Protein O-mannosyltransferase A of Aspergillus awamori is involved in O-mannosylation of glucoamylase I.

Industrially important extracellular enzymes from filamentous fungi are often O-mannosylated. The structure and function of the pmtA (AapmtA) gene encoding the protein O-D-mannosyltransferase of Aspergillus awamori were characterized. The AapmtA disruptant, designated AaPMTA, was constructed by homologous recombination. The strain AaPMTA exhibited fragile cell morphology with respect to hyphal extension, as well as swollen hyphae formation and conidia formation in potato dextrose medium. Moreover, the AapmtA disruptant showed increased sensitivity to high temperature and Congo red. Thus, the AaPmtA protein is involved in the formation of the normal cell wall. The strain AaPMTA could grow well in liquid synthetic medium and secrete glucoamylase I (GAI-AaPMTA) to a similar extent to the wild-type strain (GAI-WT). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the GAIs revealed that approximately 33 mannose moieties of GAI were absent in strain AaPMTA. This result indicates that the AaPmtA protein is responsible for the transfer of mannose to GAI. Structural analysis of the O-linked oligosaccharides of GAI also demonstrated that the AapmtA disruption resulted in a reduction of the amounts of O-linked oligosaccharides, such as D-mannose and alpha-1,2-mannotriose, in GAI-AaPMTA. However, the amount of alpha-1,2-mannobiose was comparable between GAI-WT and GAI-AaPMTA. The result suggests the presence of a compensatory mechanism in the synthetic pathway of O-mannosylation in A. awamori.

Molecular characterization of protein O-mannosyltransferase and its involvement in cell-wall synthesis in Aspergillus nidulans.

Protein O-glycosylation is essential for protein modification and plays important roles in eukaryotic cells. O-Mannosylation of proteins occurs in the filamentous fungus Aspergillus. The structure and function of the pmtA gene, encoding protein O-d-mannosyltransferase, which is responsible for the initial O-mannosylation reaction in Aspergillus nidulans, was characterized. Disruption of the pmtA gene resulted in the reduction of in vitro protein O-d-mannosyltransferase activity to 6 % of that of the wild-type strain and led to underglycosylation of an extracellular glucoamylase. The pmtA disruptant exhibited abnormal cell morphology and alteration in carbohydrate composition, particularly reduction in the skeletal polysaccharides in the cell wall. The results indicate that PmtA is required for the formation of a normal cell wall in A. nidulans.

Thr/Ser-rich domain of Aspergillus glucoamylase is essential for secretion.

The recombinant Aspergillus awamori strain carrying the mutant glucoamylase-encoding gene in which the entire Thr/Ser-rich Gp-I domain was deleted abolished secretion of mutant glucoamylase. The transcription of the Bip-encoding bipA was low in the wild type (wt) strain, but elevated in the recombinant strain under the condition of glaA expression. The results indicate that the Gp-I domain is vital for glucoamylase secretion.