Elizabethkingia argenteiflava sp. nov., isolated from the pod of soybean, Glycine max.

Soybean pods, separated and enclosed from the outside environment, are considered a suitable place to find new microbes. A Gram-stain-negative, aerobic bacterium, bacterial strain (YB22T) was isolated from the pod of Glycine max (soybean) collected from a rural area in Republic of Korea and characterized by using polyphasic taxonomy. Cells of the strain were rod-shaped (approximately 0.4-0.6 µm wide and 4.0-5.0 µm long), non-flagellated and formed silver-yellow colonies. Cells grew at 25-35 °C (optimum, 28-30 °C), at pH 5.0-9.0 (optimum, pH 7.0) and with 0-2.0% NaCl (optimum, 0 % NaCl). 16S rRNA gene sequencing showed that strain YB22T was phylogenetically closest to the genus Elizabethkingia, and showed highest similarities to Elizabethkingia occulta G4070T (96.7 %), Elizabethkingia meningoseptica ATCC 13253T (96.7 %), Elizabethkingia miricola DSM 14571T (96.6 %), Elizabethkingia bruuniana G0146T (96.5 %), Elizabethkingia ursingii G4122T (96.4 %) and Elizabethkingia anophelis R26T (96.2 %). Average amino acid identity values between strain YB22T and other taxa in the genus Elizabethkingia were all above the threshold range of genus determination. Average nucleotide identity and digital DNA-DNA hybridization values between strain YB22T and other phylogenetic relatives were all found to be below the threshold range for species determination. The respiratory quinone of strain YB22T was menaquinone 6 (MK-6) and the predominant cellular fatty acids were iso-C15 : 0 (47.8 %) and iso-C17 : 0 3-OH (18.5 %). The major polar lipids were phosphatidylethanolamine, four unidentified aminolipids and three unidentified polar lipids. The phylogenetic analysis and physiological and biochemical data showed that strain YB22T should represent a novel species in the genus Elizabethkingia, for which the name Elizabethkingia argenteiflava sp. nov. is proposed. The type strain for this novel species is YB22T (=KCCM 43263T=JCM 32097T).

Overexpression of the putative extracytoplasmic function sigma (σ) factor FujE enhances FK506 production in Streptomyces sp. strain KCCM 11116P.

The role of the putative extracytoplasmic function sigma (σ) factor FujE, which has not been characterized as a member of the FK506 biosynthetic gene cluster, on FK506 production was identified by gene deletion, overexpression, and transcription analysis experiments in Streptomyces sp. strain KCCM 11116P. Inactivation of fujE had no effect on FK506 production, growth, or morphological differentiation. Overexpression of fujE with integrative vectors increased FK506 production by 2.87-fold (24.5 ± 1.4 mg·L(-1)) compared with the wild type (8.5 ± 0.5 mg·L(-1)). Semiquantitative reverse transcription-polymerase chain reaction analysis indicated that the overexpression of fujE stimulates the transcription of the FK506 biosynthetic genes. These results demonstrated that fujE is a new member of the FK506 biosynthetic gene cluster.

Complete genome sequence of Clostridium butyricum DKU-11, isolated from healthy infant feces.

Clostridium butyricum DKU-11, a bacterium has been isolated from the stools of breastfed infants near Cheonan-Asan city, Republic of Korea, and has a genome sequence of 4,630,814 bp. The GC content is 28.7% and a total of 4,137 coding sequences in two contigs.

Anti-Streptococcus mutans and anti-inflammatory effects of ginsenoside Compound K and enzyme-treated red ginseng extract (BTEX-K).

OBJECTIVES: Dental caries, or tooth decay, is an oral health issue worldwide. Oral healthcare researchers are considering how to develop safe and effective preventive measures and treatments for dental caries. This study evaluated the potential applications of Compound K and BTEX-K, a Compound K-rich red ginseng extract, for the prevention and treatment of dental caries. Moreover, this study briefly confirmed its inhibitory effect on inflammation, an important factor in dental health. METHODS: The amount of organic acids produced by bacteria in biofilm was determined using in vitro and in vivo assays. The ability of these extracts to promote tooth remineralization and microhardness was evaluated using an in vivo mouse assay. We evaluated their anti-inflammatory potential by inhibiting proinflammatory cytokine expression and lipopolysaccharide-induced nitrous oxide production in cell lines. RESULTS: Compound K (10-20 µg/mL) and BTEX-K (50-100 µg/mL) effectively inhibited the growth of Streptococcus mutans bacteria, demonstrating significant antibacterial properties. They can potentially prevent biofilm formation by reducing lactic acid production in the teeth. These compounds showed a strong ability to promote tooth remineralization and improve the microhardness of acid-producing bacteria. They also possess potent anti-inflammatory properties that downregulate proinflammatory cytokine (interleukin-6, interleukin-1ß, inducible nitric oxide synthase) expression, suppress nuclear factor-kappa B transcription factor activation (∼1.6 times), and reduce nitrous oxide production in lipopolysaccharide-induced RAW264.7 cells. CONCLUSIONS: Compounds K and BTEX-K may provide a novel approach to dental caries prevention as well as inflammation prevention and treatment.

Application of a combined approach involving classical random mutagenesis and metabolic engineering to enhance FK506 production in Streptomyces sp. RM7011.

FK506 production by a mutant strain (Streptomyces sp. RM7011) induced by N-methyl-N'-nitro-N-nitrosoguanidine and ultraviolet mutagenesis was improved by 11.63-fold (94.24 mg/l) compared to that of the wild-type strain. Among three different metabolic pathways involved in the biosynthesis of methylmalonyl-CoA, only expression of propionyl-CoA carboxylase (PCC) pathway led to a 1.75-fold and 2.5-fold increase in FK506 production and the methylmalonyl-CoA pool, respectively, compared to those of the RM7011 strain. Lipase activity of the high FK506 producer mutant increased in direct proportion to the increase in FK506 yield, from low detection level up to 43.1 U/ml (12.6-fold). The level of specific FK506 production and lipase activity was improved by enhancing the supply of lipase inducers. This improvement was approximately 1.88-fold (71.5 mg/g) with the supplementation of 5 mM Tween 80, which is the probable effective stimulator in lipase production, to the R2YE medium. When 5 mM vinyl propionate was added as a precursor for PCC pathway to R2YE medium, the specific production of FK506 increased approximately 1.9-fold (71.61 mg/g) compared to that under the non-supplemented condition. Moreover, in the presence of 5 mM Tween 80, the specific FK506 production was approximately 2.2-fold (157.44 mg/g) higher than that when only vinyl propionate was added to the R2YE medium. In particular, PCC expression in Streptomyces sp. RM7011 (RM7011/pSJ1003) together with vinyl propionate feeding resulted in an increase in the FK506 titer to as much as 1.6-fold (251.9 mg/g) compared with that in RM7011/pSE34 in R2YE medium with 5 mM Tween 80 supplementation, indicating that the vinyl propionate is more catabolized to propionate by stimulated lipase activity on Tween 80, that propionyl-CoA yielded from propionate generates methylmalonyl-CoA, and that the PCC pathway plays a key role in increasing the methylmalonyl-CoA pool for FK506 biosynthesis in RM7011 strain. Overall, these results show that a combined approach involving classical random mutation and metabolic engineering can be applied to supply the limiting factor for FK506 biosynthesis, and vinyl propionate could be successfully used as a precursor of important methylmalonyl-CoA building blocks.

Roles of fkbN in positive regulation and tcs7 in negative regulation of FK506 biosynthesis in Streptomyces sp. strain KCTC 11604BP.

FK506 is an important 23-member polyketide macrolide with immunosuppressant activity. Its entire biosynthetic gene cluster was previously cloned from Streptomyces sp. strain KCTC 11604BP, and sequence analysis identified three putative regulatory genes, tcs2, tcs7, and fkbN, which encode proteins with high similarity to the AsnC family transcriptional regulators, LysR-type transcriptional regulators, and LAL family transcriptional regulators, respectively. Overexpression and in-frame deletion of tcs2 did not affect the production of FK506 or co-occurring FK520 compared to results for the wild-type strain, suggesting that tcs2 is not involved in their biosynthesis. fkbN overexpression improved the levels of FK506 and FK520 production by approximately 2.0-fold, and a deletion of fkbN caused the complete loss of FK506 and FK520 production. Although the overexpression of tcs7 decreased the levels of FK506 and FK520 production slightly, a deletion of tcs7 caused 1.9-fold and 1.5-fold increases in FK506 and FK520 production, respectively. Finally, fkbN overexpression in the tcs7 deletion strain resulted in a 4.0-fold (21 mg liter(-1)) increase in FK506 production compared to that by the wild-type strain. This suggests that fkbN encodes a positive regulatory protein essential for FK506/FK520 biosynthesis and that the gene product of tcs7 negatively regulates their biosynthesis, demonstrating the potential of exploiting this information for strain improvement. Semiquantitative reverse transcription-PCR (RT-PCR) analyses of the transcription levels of the FK506 biosynthetic genes in the wild-type and mutant strains proved that most of the FK506 biosynthetic genes are regulated by fkbN in a positive manner and negatively by tcs7.

An ABC transporter complex containing S-adenosylmethionine (SAM)-induced ATP-binding protein is involved in antibiotics production and SAM signaling in Streptomyces coelicolor M145.

A sco3956-deletion mutant (ΔSCO3956) of Streptomyces coelicolor was generated to characterize the S-adenosylmethionine (SAM)-induced, ATP-binding cassette transporter (ABC transporter) ATP-binding protein, SCO3956. It produced actinorhodin (ACT) and undecylprodigiosin (RED) decreased by approx. 82 and 64 %, respectively. In addition, the effect of exogenous SAM was lost in the ΔSCO3956. Plasmid-based complementation of sco3956 in ΔSCO3956 restored ACT and RED levels of ΔSCO3956 to wild-type levels (ACT: 20 ± 1.4 mg g(-1) DCW and RED: 5.3 ± 0.6 mg g(-1) DCW) and the exogenous effect significantly increased ACT and RED by approx. 129 and 135 %, respectively, when compared to the exogenous SAM non-treated sco3956 complementation strain. Thus, the ABC transporter ATP-binding protein, SCO3956, plays a critical role in ACT and RED production serving as a transducer of SAM signaling.

NF-κB-mediated anti-inflammatory effects of an organic light-emitting diode (OLED) device in lipopolysaccharide (LPS)-induced in vitro and in vivo inflammation models.

Inflammation is the body's physiological response to harmful agents. However, if not regulated properly, inflammation can become pathological. Macrophages are key players in the inflammatory process, and modulate the immune response. Due to the side effects of anti-inflammatory drugs, non-pharmaceutical therapies for inflammatory diseases must be developed. Photobiomodulation is a non-invasive therapeutic approach to treating certain pathological conditions using light energy. Light-emitting diodes (LEDs) are commonly used as light sources for photobiomodulation treatment, but their clinical applications are limited. Organic LEDs (OLEDs) are thin, lightweight and flexible, enabling consistent and even delivery of light energy to target areas; this makes OLED promising components for therapeutic devices. In the present study, we examined the effects of OLED treatment on inflammation in vitro using a lipopolysaccharide (LPS)-induced macrophage RAW264.7 cell model, and in vivo using a pinna skin mouse model. We found that LPS-induced morphological changes and inflammatory cytokine expression were significantly reduced in RAW264.7 cells subjected to OLED treatment compared to the LPS-induced controls. This work provides evidence for the anti-inflammatory effects of OLEDs, demonstrating their potential to be incorporated into medical devices in the future.

Treatment with Light-Emitting Diodes of Wavelength 863 nm Delays DMBA/TPA-Induced Skin Tumor Formation and Decreases Proinflammatory Cytokine Levels in ICR Mice.

The popularity of light/energy devices for cosmetic purposes (e.g., skin care) is increasing. However, the effects and underlying mechanisms remain poorly understood. Commencing in the 1960s, various studies have evaluated the beneficial effects of a light source on cells and tissues. The techniques evaluated include low-level light (laser) therapy and photobiomodulation (PBM). Most studies on PBM used red light sources, but, recently, many studies have employed near-infrared light sources including those of wavelength 800 nm. Here, we used a light-emitting diode (LED) array with a wavelength of 863 nm to treat DMBA/TPA-induced mouse skin tumors; treatment with the array delayed tumor development and reduced the levels of systemic inflammatory cytokines. These results suggest that light therapy could be beneficial. However, the effects were small. Further studies on different skin tumors using an optimized LED setup are required. Combination therapies (conventional methods and an LED array) may be useful.

Biosynthesis of the allylmalonyl-CoA extender unit for the FK506 polyketide synthase proceeds through a dedicated polyketide synthase and facilitates the mutasynthesis of analogues.

The allyl moiety of the immunosuppressive agent FK506 is structurally unique among polyketides and critical for its potent biological activity. Here, we detail the biosynthetic pathway to allylmalonyl-coenzyme A (CoA), from which the FK506 allyl group is derived, based on a comprehensive chemical, biochemical, and genetic interrogation of three FK506 gene clusters. A discrete polyketide synthase (PKS) with noncanonical domain architecture presumably in coordination with the fatty acid synthase pathway of the host catalyzes a multistep enzymatic reaction to allylmalonyl-CoA via trans-2-pentenyl-acyl carrier protein. Characterization of this discrete pathway facilitated the engineered biosynthesis of novel allyl group-modified FK506 analogues, 36-fluoro-FK520 and 36-methyl-FK506, the latter of which exhibits improved neurite outgrowth activity. This unique feature of FK506 biosynthesis, in which a dedicated PKS provides an atypical extender unit for the main modular PKS, illuminates a new strategy for the combinatorial biosynthesis of designer macrolide scaffolds as well as FK506 analogues.

Treatment with LEDs at a wavelength of 642†nm enhances skin tumor proliferation in a mouse model.

Photobiomodulation (PBM) is attracting increased attention in the fields of dermatology and cosmetics. PBM with a variety of light parameters has been used widely in skin care, but can cause certain types of unwanted cells to proliferate in the skin; this can lead to skin tumors, such as papillomas and cancers. We constructed a mouse model of human skin tumors using DMBA as an initiator and TPA as a promoter, and confirmed that LEDs with a wavelength of 642 nm (red light) increased tumor size, epidermal thickness, and systemic proinflammatory cytokine levels. These results indicated that skin tumor cell proliferation may result from the use of 642 nm LEDs, suggesting the need for regulation of skin care based on LED light therapy.

470 nm LED Irradiation Inhibits the Invasiveness of CD133-positive Human Colorectal Cancer Stem Cells by Suppressing the Cyclooxygenase-2/prostaglandin E2 Pathway.

BACKGROUND/AIM: Recurrence and metastasis of cancer caused by cancer stem cells (CSCs) is a challenge to overcome. Low level laser therapy is a new treatment strategy to suppress their invasiveness. We have assessed the inhibitory effects of 470 nm blue LED on the invasiveness of them to determine the molecular mechanisms of anti-invasiveness. MATERIALS AND METHODS: The effects of blue LEDs on their viability, proliferation and invasion were analyzed using MTT and transwell methods. In addition, the anti-invasiveness effect of blue LED on them was evaluated by zymography, semi-quantitative RT-PCR and western blot analysis. RESULTS: Irradiation with blue LED at 3 J/cm2 resulted in inhibition of their viability, proliferation and invasiveness. Their matrix metalloproteinase 2 (MMP-2) and MMP-9 activities were reduced by blue LED irradiation. Semi-quantitative RT-PCR also showed similar results. In addition, western blotting analyses showed that cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) synthesis were significantly inhibited by LED irradiation in CD133+ colorectal CSCs. CONCLUSION: Down-regulation of the COX-2/PGE2 signaling pathway by blue LED irradiation led to reduce expression of MMP-2 and MMP-9, inhibiting the invasiveness of CD133+ colorectal CSC.

Liquid chromatography-mass spectrometry characterization of FK506 biosynthetic intermediates in Streptomyces clavuligerus KCTC 10561BP.

The development of an efficient analytical method for the reliable detection and identification of the biosynthetic intermediates found in microbial cultures, which usually produce complex intermediates of the metabolites of interest, is essential for further biosynthetic investigations. This study developed a simple and highly selective method for detecting the biosynthetic intermediates involved in the FK506 pathway of Streptomyces clavuligerus KCTC 10561BP involving a cleanup procedure using a solid-phase extraction technique to provide reliable extraction of FK506-related compounds from a cell culture broth and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to separate and detect the FK506-related intermediates at concentrations as low as 0.2 microg/L in the broth. This method enabled the analytical profiling of the intermediates formed during the biosynthesis of FK506 in this S. clavuligerus strain, which produced FK506 as a main product. Eight FK506 intermediates--FK520, 37,38-dihydroFK506, prolylFK506, 9-decarbonyl-9-hydroxylFK506, 9-deoxoFK506, desmethylFK520, prolylFK520, and 9-deoxoFK520--were identified. This is the first report of the LC-ESI-MS/MS characterization of a wide range of FK506 analogs from a bacterial fermentation broth. The protocol employed in this study may be useful for estimating the structure of the metabolites without the need for a time-consuming isolation process and nuclear magnetic resonance (NMR) spectroscopy.

Elucidation of the Streptomyces coelicolor pathway to 2-undecylpyrrole, a key intermediate in undecylprodiginine and streptorubin B biosynthesis.

The red gene cluster of Streptomyces coelicolor directs production of undecylprodiginine. Here we report that this gene cluster also directs production of streptorubin B and show that 2-undecylpyrrole (UP) is an intermediate in the biosynthesis of undecylprodiginine and streptorubin B. The redPQRKL genes are involved in UP biosynthesis. RedL and RedK are proposed to generate UP from dodecanoic acid or a derivative. A redK(-) mutant produces a hydroxylated undecylprodiginine derivative, whereas redL(-) and redK(-) mutants require addition of chemically synthesized UP for production of undecylprodiginine and streptorubin B. Fatty acid biosynthetic enzymes can provide dodecanoic acid, but efficient and selective prodiginine biosynthesis requires RedPQR. Deletion of redP, redQ, or redR leads to an 80%-95% decrease in production of undecylprodiginine and an array of prodiginine analogs with varying alkyl chains. In a redR(-) mutant, the ratio of these can be altered in a logical manner by feeding various fatty acids.

Enzymatic properties of an extracellular phospholipase C purified from a marine streptomycete.

A novel extracellular phospholipase C (PLC) was purified from a marine streptomycete. It had a molecular mass of 28 kDa as estimated by SDS-polyacrylamide gel electrophoresis. Its enzyme activity was optimal at pH 8.0 at 45 degrees Celsius. The PLC hydrolyzed only phosphatidylcholine. Its activity was enhanced 300% by Na(+) (200 mM), suggesting that the purified PLC is a typical marine-type enzyme.

A novel extracellular phospholipase C purified from a marine bacterium, Pseudoalteromonas sp. J937.

Marine bacterial isolates were screened for phospholipase C (PLC) activity on PCY agar plates containing phosphatidylcholine (PC) as substrate. The strain that showed the highest activity on a PCY screening agar plate and a thin-layer chromatography was identified as a strain of Pseudoalteromonas and subsequently designated Pseudoalteromonas sp. J937. The extracellular PLC of the strain J937 was purified to a specific activity of 33 U mg(-1) protein by serial ion exchange and gel filtration column chromatography. It had a molecular mass of 32 kDa estimated by SDS-PAGE. The optimal pH and temperature of the enzyme were about pH 8 and 45 degrees C, respectively. The PLC hydrolyzed phosphatidylethanolamine as well as PC but not other glycerophospholipids. Its activity was enhanced by 150% with Ca2+ (200 mM) and by 180% with Na+ (500 mM), suggesting that the purified PLC is a marine-type enzyme.

Enhanced FK506 production in Streptomyces clavuligerus CKD1119 by engineering the supply of methylmalonyl-CoA precursor.

FK506 is a 23-membered polyketide macrolide with immunosuppressant activity produced by Streptomyces species. The production of FK506 in S. clavuligerus CKD1119 (KCTC 10561BP) was improved by enhancing the supply of biosynthetic precursors. This improvement was approximately 2.5-fold (15 mg/l) with the supplementation of 10 mM methyl oleate, which is the probable source of acyl-CoAs, to R2YE medium. When the level of FK506 production reached its maximum, the intracellular concentration of methylmalonyl-CoA in S. clavuligerus CKD1119 supplemented with methyl oleate was 12.5-fold higher than that of the unsupplemented strain, suggesting that an increased methylmalonyl-CoA level caused the high-level production of FK506. The following three pathways for the production of (2S)-methylmalonyl-CoA were evaluated to identify the effective precursor supply pathway that can support the high production of FK506 in S. clavuligerus CKD1119: propionyl-CoA carboxylase, methylmalonyl-CoA mutase (MCM), and malonyl/methylmalonyl-CoA ligase. Of the three pathways examined, the MCM pathway supported the highest levels of FK506 production. The expression of MCM in S. clavuligerus CKD1119 led to a threefold and 1.5-fold increase in the methylmalonyl-CoA pool and FK506 production, respectively. Supplementing the culture broth of S. clavuligerus CKD1119 expressing MCM with methyl oleate resulted in an additional twofold increase in the FK506 titer (17.8 mg/l). Overall, these results show that the methylmalonyl-CoA supply is a limiting factor for FK506 biosynthesis and that among the three pathways analyzed, the MCM pathway is the most effective precursor supply pathway supporting the highest titer of FK506 in S. clavuligerus CKD1119.

Light-emitting diode irradiation using 660 nm promotes human fibroblast HSP90 expression and changes cellular activity and morphology.

We evaluated changes in cell viability and morphology in response to low-level light irradiation and underlying variations in the levels of heat shock proteins (HSPs). Human fibroblasts were irradiated with a light-emitting diode (LED) array at 660 nm (50 mW for 15, 30, and 60 minutes). Cell viability and morphological changes were evaluated via epifluorescence analysis; we also assessed cell viability and length changes. The expression levels of adenosine triphosphate (ATP) and various HSPs (HSP27, 60, 70, and 90) were analyzed by immunohistochemical staining, Western blotting and microarray analysis. After LED irradiation, cellular viability and morphology changed. Of the several HSPs analyzed, the HSP90 level increased significantly, suggesting that this protein played roles in the morphological and cellular changes. Thus, low-level irradiation triggered cellular changes mediated by increased HSP90 expression; this may explain why skin irradiation enhances wound-healing.

Production of branched-chain alkylprodiginines in S. coelicolor by replacement of the 3-ketoacyl ACP synthase III initiation enzyme, RedP.

The enzyme RedP is thought to initiate the biosynthesis of the undecylpyrolle component of the antibiotic undecylprodiginine produced by Streptomyces coelicolor. RedP has homology to FabH, which initiates fatty acid biosynthesis by condensing the appropriate acyl-CoA starter unit with malonyl ACP. We have generated a redP-deletion mutant of S. coelicolor M511 (SJM1) and shown that it produces reduced levels of prodiginines and two new analogs, methylundecylprodiginine and methyldodecylprodiginine. Incorporation studies with perdeuterated valine were consistent with these being generated using methylbutyryl-CoA and isobutyryl-CoA as starter units, respectively. Plasmid-based expression of a streptomycete fabH in the SJM1 mutant led to restoration of overall prodiginine titers but the same overall ratio of undecylprodiginines and novel prodiginines. Thus, the redP FabH can be replaced by FabH enzymes with different substrate specificities and provides a method for generating novel prodiginines.

Interspecies Complementation of the LuxR Family Pathway-Specific Regulator Involved in Macrolide Biosynthesis.

PikD is a widely known pathway-specific regulator for controlling pikromycin production in Streptomyces venezuelae ATCC 15439, which is a representative of the large ATP-binding regulator of the LuxR family (LAL) in Streptomyces sp. RapH and FkbN also belong to the LAL family of transcriptional regulators, which show greatest homology with the ATP-binding motif and helix-turn-helix DNA-binding motif of PikD. Overexpression of pikD and heterologous expression of rapH and fkbN led to enhanced production of pikromycin by approximately 1.8-, 1.6-, and 1.6-fold in S. venezuelae, respectively. Cross-complementation of rapH and fkbN in the pikD deletion mutant (ΔpikD) restored pikromycin and derived macrolactone production. Overall, these results show that heterologous expression of rapH and fkbN leads to the overproduction of pikromycin and its congeners from the pikromycin biosynthetic pathway in S. venezuelae, and they have the same functionality as the pathwayspecific transcriptional activator for the pikromycin biosynthetic pathway in the ΔpikD strain. These results also show extensive "cross-communication" between pathway-specific regulators of streptomycetes and suggest revision of the current paradigm for pathwayspecific versus global regulation of secondary metabolism in Streptomyces species.