Microbial rhodopsins are a superfamily of photoactive membrane proteins with the covalently bound retinal cofactor. Isomerization of the retinal chromophore upon absorption of a photon triggers conformational changes of the protein to function as ion pumps or sensors. After the discovery of proteorhodopsin in an uncultivated γ-proteobacterium, light-activated proton pumps have been widely detected among marine bacteria and, together with chlorophyll-based photosynthesis, are considered as an important axis responsible for primary production in the biosphere. Rhodopsins and related proteins show a high level of phylogenetic diversity; we focus on a specific class of bacterial rhodopsins containing the '3 omega motif.' This motif forms a stack of three non-consecutive aromatic amino acids that correlates with the B-C loop orientation and is shared among the phylogenetically close ion pumps such as the NDQ motif-containing sodium-pumping rhodopsin, the NTQ motif-containing chloride-pumping rhodopsin, and some proton-pumping rhodopsins including xanthorhodopsin. Here, we reviewed the recent research progress on these 'omega rhodopsins,' and speculated on their evolutionary origin of functional diversity.

In this study, we investigated the effects of linoleic acid (LA) treatment on Brucella abortus infection in professional phagocyte RAW264.7 cells, particularly during the pathogen's invasion and intracellular growth in these cells, as well as in murine model BALB/c mice focusing on bacterial splenic proliferation and immunoregulatory activities. LA inhibited the growth of Brucella in a dose- and time-dependent manner. The ability of the pathogen to enter the phagocytes was inhibited as was its survival within these cells. This was accompanied by increased nitrite accumulation in these cells at 24 h post-infection. The concentration of LA used in the present study did not affect the total body weight or liver function of the mice. During Brucella infection, the total splenic weight of these animals was not changed; rather, resistance to bacterial proliferation was enhanced in the spleen. Furthermore, mice treated with LA displayed elevated levels of IL-12 and IFN-γ but reduced levels of IL-10 during infection. The findings in this study showed the regulatory role of LA against B. abortus infection suggesting its potential use in designing intervention strategy for brucellosis.

This study examined the laxative effects of hot-water extracts of Hovenia dulcis Thunb. (HD), Phyllostachys pubescens Mazel (PM), and a 2:8 mixture of both (HP) in two chronic constipation models. For the loperamide-induced constipation model, animals were divided into an untreated group, negative control group (loperamide 4 mg/kg), positive control group (bisacodyl 4 mg/kg) group, and six treatment groups (HP 100 or 400, HD 50 or 100, and PM 100 or 400 mg/kg). For the low-fiber diet-induced constipation model, animals were divided into an untreated group (normal diet), negative control group (low-fiber diet), positive control group (Agio granule, 620 mg/kg), and the same treatment groups. Fecal number, weight, fecal water content, and intestinal transit ratio were higher in the groups treated with HP, HD, and PM than in the groups treated with loperamide or low-fiber diet. Thickness of colon mucosa and muscle layers were increased in the treated groups. Colon tension increased in the HP groups, and [Ca²⁺]i measurements using fura-2 as an indicator showed that HP inhibits ATP-mediated Ca²⁺ influx in IEC-18 cells. These results showed that the HP mixture has laxative activity by increased mucin secretion and inducing contractile activity and relaxation. It may be a useful therapeutic strategy for ameliorating in chronic constipation.

Epidermal growth factor receptor (EGFR) mutations are not only genetic markers for diagnosis but also biomarkers of clinical-response against tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC). Among the EGFR mutations, the in-frame deletion mutation in EGFR exon 19 kinase domain (EGFR exon 19-del) is the most frequent mutation, accounting for about 45% of EGFR mutations in NSCLCs. Development of sensitive method for detecting the EGFR mutation is highly required to make a better screening for drug-response in the treatment of NSCLC patients. Here, we developed a fluorometric tandem gene amplification assay for sensitive detection of low-abundance EGFR exon 19-del mutant genomic DNA. The method consists of pre-amplification with PCR, thermal cycling of ligation by Taq ligase, and subsequent rolling circle amplification (RCA). PCR-amplified DNA from genomic DNA samples was used as splint DNA to conjugate both ends of linear padlock DNA, generating circular padlock DNA template for RCA. Long stretches of ssDNA harboring multiple copies of G-quadruplex structure was generated in RCA and detected by thioflavin T (ThT) fluorescence, which is specifically intercalated into the G-quadruplex, emitting strong fluorescence. Sensitivity of tandem gene amplification assay for detection of the EGFR exon 19-del from gDNA was as low as 3.6 pg, and mutant gDNA present in the pooled normal plasma was readily detected as low as 1% fraction. Hence, fluorometric detection of low-abundance EGFR exon 19 deletion mutation using tandem gene amplification may be applicable to clinical diagnosis of NSCLC patients with appropriate TKI treatment.

Bacillus velezensis is an important plant growth-promoting rhizobacterium with immense potential in agriculture development. In the present study, Bacillus velezensis Lle-9 was isolated from the bulbs of Lilium leucanthum. The isolated strain showed antifungal activities against plant pathogens like Botryosphaeria dothidea, Fusarium oxysporum, Botrytis cinerea and Fusarium fujikuroi. The highest percentage of growth inhibition i.e., 68.56±2.35% was observed against Fusarium oxysporum followed by 63.12 ± 2.83%, 61.67 ± 3.39% and 55.82 ± 2.76% against Botrytis cinerea, Botryosphaeria dothidea, and Fusarium fujikuroi, respectively. The ethyl acetate fraction revealed a number of bioactive compounds and several were identified as antimicrobial agents such as diketopiperazines, cyclo-peptides, linear peptides, latrunculin A, 5α-hydroxy-6-ketocholesterol, (R)-S-lactoylglutathione, triamterene, rubiadin, moxifloxacin, 9-hydroxy-5Z,7E,11Z,14Z-eicosatetraenoic acid, D-erythro-C18-Sphingosine, citrinin, and 2-arachidonoyllysophosphatidylcholine. The presence of these antimicrobial compounds in the bacterial culture might have contributed to the antifungal activities of the isolated B. velezensis Lle-9. The strain showed plant growth-promoting traits such as production of organic acids, ACC deaminase, indole-3-acetic acid (IAA), siderophores, and nitrogen fixation and phosphate solubilization. IAA production was accelerated with application of exogenous tryptophan concentrations in the medium. Further, the lily plants upon inoculation with Lle-9 exhibited improved vegetative growth, more flowering shoots and longer roots than control plants under greenhouse condition. The isolated B. velezensis strain Lle-9 possessed broad-spectrum antifungal activities and multiple plant growth-promoting traits and thus may play an important role in promoting sustainable agriculture. This strain could be developed and applied in field experiments in order to promote plant growth and control disease pathogens.

Bacterial cell-based biosensors, or whole-cell bioreporters (WCBs), are an alternative tool for the quantification of hazardous materials. Most WCBs share similar working mechanisms. In brief, the recognition of a target by sensing domains induces a biological event, such as changes in protein conformation or gene expression, providing a basis for quantification. WCBs targeting heavy metal(loid)s employ metalloregulators as sensing domains and control the expression of genes in the presence of target metal(loid) ions, but the diversity of targets, specificity, and sensitivity of these WCBs are limited. In this study, we genetically engineered the metal-binding loop (MBL) of ZntR, which controls the znt-operon in Escherichia coli. In the MBL of ZntR, three Cys sites interact with metal ions. Based on the crystal structure of ZntR, MBL sequences were modified by site-directed mutagenesis. As a result, the metal-sensing properties of WCBs differed depending on amino acid sequences and the new selectivity to Cr or Pb was observed. Although there is room for improvement, our results support the use of currently available WCBs as a platform to generate new WCBs to target other environmental pollutants including metal(loid)s.

Brevibacillus brevis GZDF3 is a gram-positive, plant growth-promoting rhizosphere bacterium (PGPR) isolated from the rhizosphere soil of Pinellia ternata (an important herb in traditional Chinese medicine). The GZDF3 strain produces certain active compounds, such as siderophores, which are the final metabolite products of non-ribosomal peptide synthetase (NRPS) and independent non-ribosomal peptide synthetase (NIS) activity. With the present study, we attempted to investigate the siderophore production characteristics and conditions of Bacillus sp. GZDF3. The antibacterial activity of the siderophores on pathogenic fungi was also investigated. Optimal conditions for the synthesis of siderophores were determined by single factor method, using sucrose 15 g/l, asparagine 2 g/l, 32℃, and 48 h. The optimized sucrose asparagine medium significantly increased the production of siderophores, from 27.09% to 54.99%. Moreover, the effects of different kinds of metal ions on siderophore production were explored here. We found that Fe³⁺ and Cu²⁺ significantly inhibited the synthesis of siderophores. The preliminary separation and purification of siderophores by immobilized-metal affinity chromatography (IMAC) provides strong antibacterial activity against Candida albicans. The synergistic effect of siderophores and amphotericin B was also demonstrated. Our results have shown that the GZDF3 strain could produce a large amount of siderophores with strong antagonistic activity, which is helpful in the development of new biological control agents.

The root-knot nematode (Meloidogyne incognita) is an important pathogen in crop cultivation, however, few methods are available to control this parasitic roundworm. In this study, the nematicidal effects of approximately 30 Streptomyces strains isolated from soil samples of Mt. Naejang (Korea) were tested against Meloidogyne incognita, and the culture broth of the strains KRA-24 and KRA-28 exhibited approximately 75% and 85% insecticidal activity, respectively, in in vitro assays. In in vivo pot experiments, these strains reduced the number of nematodes in the soil and the number of egg masses in the roots of red peppers. The two strains also survived in the presence of insecticidal agents (0.1 to 3.0%) such as fosthiazate, ethoprophos and terbufos when they were used in parallel. The mixture of KRA-24 or KRA-28 culture broth and fosthiazate exhibited nematicidal effects that were similar to those observed when KRA-24 or KRA-28 were used alone. Our results clearly suggest that the Streptomyces strains KRA-24 and KRA-28 should be promoted as a biocontrol agent against Meloidogyne incognita.

The purpose of this study was to identify strawberry wilt pathogens and evaluate the efficacy of Chlorella fusca CHK0059 for improving plant growth and suppressing Fusarium wilt. We identified 10 isolates of wilt pathogens of non-pesticide Seolhyang strawberry plant, including Fusarium oxysporum f. sp. fragariae, using morphological and molecular analysis. On the 15^th day after 0.4% CHK0059 treatment, the plant height of the untreated control strawberry plants was significantly greater than that of the CHK0059-treated strawberry plants. After 85 days, both treatments showed a similar tendency regarding the height of the strawberry plants. However, the thickness of strawberry leaves treated with the CHK0059 was found to be 1 mm thicker than that of the untreated control. The flowering percentage of the CHK0059 plants was also 40.2% higher on average than that of the untreated control. The chlorophyll content of strawberry leaves treated with the CHK0059 was also, on average, 6.63% higher than that of the untreated control. After 90 days of the CHK0059 treatment, the incidence of Fusarium wilt in the CHK0059-treated plants had reduced by 9.8% on average compared to the untreated control. The population density of F. oxysporum f. sp. fragariae was also reduced by approximately 86.8% in the CHK0059-treated plants by comparison to the untreated control at 70 days after treatment. The results indicate that the microalga C. fusca CHK0059 is an efficient biological agent for improving strawberry plant growth and suppressing Fusarium wilt disease in organic strawberries.

The use of plant growth-promoting rhizobacteria is economically viable and environmentally safe for mitigating various plant stresses. Abiotic stresses such as flood and drought are a serious threat to modern agriculture. In the present study, the indole-3-acetic acid-producing rhizobacterium R. sphaeroides KE149 was selected, and its effects on the growth of adzuki bean plants under flood stress (FS) and drought stress (DS) were investigated. IAA quantification of bacterial pure culture revealed that KE149 produced a significant amount of IAA. Moreover, KE149 inoculation notably decreased stress-responsive endogenous abscisic acid and jasmonic acid and increased salicylic acid in plants under DS and FS. KE149 inoculation also increased proline under DS and methionine under FS. In addition, KE149 inoculation significantly increased the levels of calcium (Ca), magnesium (Mg), and potassium (K) while lowering the sodium (Na) content in the plant shoot under stress. KE149-treated plants had markedly greater root length, shoot length, stem diameter, biomass, and higher chlorophyll content under both normal and stressed conditions. These results suggest that KE149 could be an efficient biofertilizer for mitigating water stress.

Indonesian kefir grains are potential sources of lactic acid bacteria (LAB) that may act as probiotics with specific functional properties. In this study we explored the potential of the probiotic and cholesterol-lowering effect of LAB isolated from Indonesian kefir grains obtained from Bogor, Bandung, Jakarta, and Yogyakarta. The results revealed that 10 isolates showed considerable survivability at low pH and bile salt with total cell reduction of ~3 log colony-forming units per milliliter after exposure to pH 2.5 and 0.5% (w/v) bile salt for 1 and 3 h, respectively. All strains exhibited strong antimicrobial activities against pathogenic bacteria and were sensitive to a wide spectrum of antibiotics but exhibited weak bile salt hydrolase activity. Identification based on 16S RNA suggested that nine isolates were Lactobacillus kefiri and one was Lactobacillus rhamnosus. The ability of the isolates to reduce cholesterol from the media varied, ranging from 22.08% to 68.75% with the highest reduction shown by L. kefiri JK17. The ability to remove cholesterol from the media decreased greatly in resting and dead cells, ranging from 14.58% to 22.08% in resting cells and from 7.89% to 18.17% in dead cells. It can be concluded that Indonesian kefir grains contain LAB potentially acting as probiotics capable of reducing cholesterol. The cholesterol-lowering effect especially occurs when the cells are metabolically active.

Glucose deprivation and hypoxia frequently occur in solid tumor cells, including pancreatic cancer cells. Glucose deprivation activates the unfolded protein response (UPR) and causes the up-regulation of glucose-regulated protein 78 (GRP78). Induction of GRP78 has been shown to protect cancer cells. Therefore, shutting down of GRP78 expression may be a novel strategy in anticancer drug development. Based on this understanding, a screening system established for anticancer agents that exhibit selective cytotoxicity on pancreatic cancer cells under glucose-deprived conditions. To test this hypothesis, the new compounds isolated, pancastatin A (PST-A) and B (PST-B), from Ponciri Fructus. PST-A and B were identified as glabretal triterpenoid moieties by electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopic methods. PST-A and B suppressed the accumulation of the UPR hallmark gene, GRP78, during glucose deprivation. Furthermore, PST-A and B showed selective cytotoxicity on PANC-1 pancreatic cancer cells under glucose deprivation. Interestingly, PST-A and B had no effect on these cells under normal growth conditions. Our results suggest that PST-A and B act as novel therapeutic agents to induce selective cell death in glucose-deprived pancreatic cancer cells.

In this study, a method of heat adaptation was implemented in an attempt to increase the upper thermal threshold of two Streptococcus thermophilus found in South Korea and identified the alterations in membrane fatty acid composition to adaptive response to heat. In order to develop heat tolerant lactic acid bacteria, heat treatment was continuously applied to bacteria by increasing temperature from 60℃ until the point that no surviving cell was detected. Our results indicated significant increase in heat tolerance of heat-adapted strains compared to the wild type (WT) strains. In particular, the survival ratio of basically low heat-tolerant strain increased even more. In addition, the strains with improved heat tolerance acquired cross protection, which improved their survival ratio in acid, bile salts and osmotic conditions. A relation between heat tolerance and membrane fatty acid composition was identified. As a result of heat adaptation, the ratio of unsaturated to saturated fatty acids (UFA/SFA) and C18:1 relative concentration were decreased. C6:0 in only heat-adapted strains and C22:0 in only the naturally high heat tolerant strain were detected. These results support the hypothesis, that the consequent increase of SFA ratio is a cellular response to environmental stresses such as high temperatures, and it is able to protect the cells from acid, bile salts and osmotic conditions via cross protection. This study demonstrated that the increase in heat tolerance can be utilized as a mean to improve bacterial tolerance against various environmental stresses.

In the search for novel, natural melanogenesis inhibitors, a new sesquiterpene, inularin, was isolated from the flowers of Inula britannica, and the structure was determined using spectroscopic and chemical methods. The antimelanogenic effects of inularin on B16F10 melanoma cells and zebrafish embryos were evaluated. Inularin dose-dependently reduced melanocyte-stimulating hormone-induced melanin production and L-DOPA oxidation in B16F10 cells. Zebrafish embryos were used to confirm the antimelanogenic activity. Inularin significantly decreased the pigmentation of embryos compared with untreated controls.

To determine the role of pyruvate dehydrogenase complex (PDHC) in Klebsiella pneumoniae, the growth and metabolism of PDHC-deficient mutant in glycerol-based medium were analyzed and compared with those of other strains. Under aerobic conditions, the PDHC activity was fourfold higher than that of pyruvate formate lyase (PFL), and blocking of PDHC caused severe growth defect and pyruvate accumulation, indicating that the carbon flux through pyruvate to acetyl coenzyme A mainly depended on PDHC. Under anaerobic conditions, although the PDHC activity was only 50% of that of PFL, blocking of PDHC resulted in more growth defect than blocking of PFL. Subsequently, combined with the requirement of CO₂ and intracellular redox status, it was presumed that the critical role of PDHC was to provide NADH for the anaerobic growth of K. pneumoniae. This presumption was confirmed in the PDHC-deficient mutant by further blocking one of the formate dehydrogenases, FdnGHI. Besides, based on our data, it can also be suggested that an improvement in the carbon flux in the PFL-deficient mutant could be an effective strategy to construct high-yielding 1,3-propanediol-producing K. pneumoniae strain.

Vitamin K2 (menaquinone) is an essential vitamin existing in the daily diet, and menaquinone-7 (MK-7) is an important form of it. In a recent work, we engineered the synthesis modules of MK-7 in Bacillus subtilis, and the strain BS20 could produce 360 mg/l MK-7 in shake flasks, while the methylerythritol phosphate (MEP) pathway, which provides the precursor isopentenyl diphosphate for MK-7 synthesis, was not engineered. In this study, we overexpressed five genes of the MEP pathway in BS20 and finally obtained a strain (BS20DFHG) with MK-7 titer of 415 mg/l in shake flasks. Next, we optimized the fermentation process parameters (initial pH, temperature and aeration) in an 8-unit parallel bioreactor system consisting of 300-ml glass vessels. Based on this, we scaled up the MK-7 production by the strain BS20DFHG in a 50-l bioreactor, and the highest MK-7 titer reached 242 mg/l. Here, we show that the engineered strain BS20DFHG may be used for the industrial production of MK-7 in the future.

Genistein is a type of isoflavonoid found predominantly in leguminous plants. Genistein has diverse biological activities, such as anthelmintic and antioxidant effects, as well as inhibitory effects on the growth of several cancers. In addition, genistein is well known as a phytoestrogen. In this study, we attempted to biologically synthesize genistein from either p-coumaric acid or naringenin using Escherichia coli as a biotransformation host. Four genes, Os4CL, PeCHS, RcIFS, and OsCPR, were used for genistein production. To functionally express RcIFS and OsCPR, two members of the cytochrome P450 family, in E. coli, the membrane-binding anchor domain of each gene was removed, and RcIFS and OsCPR were translationally fused to generate an RcIFS-OsCPR hybrid. Os4CL and PeCHS, or the RcIFS-OsCPR hybrid, were then transformed into E. coli BL21(DE3). Using these strains, we optimized our culture system at a laboratory scale in terms of the cell density, concentrations of substrate and isopropyl-β-D-thiogalactoside, temperature, and culture medium. Under the optimized culture conditions, genistein was produced at up to 35 mg/l and 18.6 mg/l using naringenin and p-coumaric acid, respectively.

Self-sufficient P450s, due to their fused nature, are the most effective tools for electron transfer to activate C-H bonds. They catalyze the oxygenation of fatty acids at different omega positions. Here, two new, self-sufficient cytochrome P450s, named 'CYP102A15 and CYP102A170,' from polar Bacillus sp. PAMC 25034 and Paenibacillus sp. PAMC 22724,respectively, were cloned and expressed in E. coli. The genes are homologues of CYP102A1 from Bacillus megaterium. They catalyzed the hydroxylation of both saturated and unsaturated fatty acids ranging in length from C₁₂-C₂₀, with a moderately diverse profile compared to other members of the CYP102A subfamily. CYP102A15 exhibited the highest activity toward linoleic acid with K_m 15.3 μM, and CYP102A170 showed higher activity toward myristic acid with Km 17.4 μM. CYP10A170 also hydroxylated the Eicosapentaenoic acid at ω-1 position only. Various kinetic parameters of both monooxygenases were also determined.

ʟ-Theanine, found in green tea leaves has been shown to positively affect immunity and relaxation in humans. There have been many attempts to produce ʟ-theanine through enzymatic synthesis to overcome the limitations of traditional methods. Among the many genes coding for enzymes in the ʟ-theanine biosynthesis, glutamylmethylamide synthetase (GMAS) exhibits the greatest possibility of producing large amounts of production. Thus, GMAS from Methylovorus mays No. 9 was overexpressed in several strains including vectors with different copy numbers. BW25113(DE3) cells containing the pET24ma::gmas was selected for strains. The optimal temperature, pH, and metal ion concentration were 50℃, 7, and 5 mM MnCl₂, respectively. Additionally, ATP was found to be an important factor for producing high concentration of ʟ-theanine so several strains were tested during the reaction for ATP regeneration. Baker's yeast was found to decrease the demand for ATP most effectively. Addition of potassium phosphate source was demonstrated by producing 4-fold higher ʟ-theanine. To enhance the conversion yield, GMAS was additionally overexpressed in the system. A maximum of 198 mM ʟ-theanine was produced with 16.5 mmol/l/h productivity. The whole-cell reaction involving GMAS has greatest potential for scale-up production of ʟ-theanine.