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Original Research ARTICLE
Mutagenesis of PhaR, a Regulator Gene of Polyhydroxyalkanoate Biosynthesis of Xanthomonas oryzae pv. oryzae Caused Pleiotropic Phenotype Changes
- Department of Plant Pathology, College of Plant Protection, Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing Agricultural University, Nanjing, China
Polyhydroxyalkanoates (PHAs) are intracellular carbon and energy storage materials produced in various microorganisms under nutrient-limited conditions. PhaR is a regulatory protein involved in PHA synthesis. Xanthomonas oryzae pv. oryzae (Xoo) is one of the most important bacterial pathogens in rice and has PHA biosynthesis genes in its genome, but the biological function of phaR in Xoo is unknown. In this study, we investigated the effects of the mutagenesis of phaR gene in Xoo strain PXO99A. Compared to the wildtype, the PhaR gene knock-out mutant PXO99ΔphaR was hypermotile and showed decreased growth rates in both rich and limited nutrient media. PXO99ΔphaR also showed almost 75% decrease in extracellular polysaccharide (EPS) production. When inoculated in rice leaves by leaf-clipping method, PXO99ΔphaR displayed reduced virulence in terms of lesion length and bacterial multiplication compared with the wildtype strain. PXO99ΔphaR also showed enhanced hypersensitive response (HR) induction in the leaves of non-host Nicotiana benthamiana with elevated hpa1 gene expression. Introduction of a cosmid containing the phaR coding sequence restored the phenotypes of the mutant to those of the wildtype strain. These results suggest that PhaR gene is an important gene that affects multiple bacterial characteristics, including EPS production, growth rate, defense response induced harpin production and motility, related to its virulence in plant.
Bacterial blight on rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is a widely prevalent disease that causes serious rice production losses worldwide (Singh et al., 1978; Nino-Liu et al., 2006). Xoo invades rice leaf through wounds or natural openings like hydathodes, then colonizes the intercellular space, hijacking the nutrient from host and dispensing along vascular bundles, to cause leaf blight (Mew et al., 1984; Bezrutczyk et al., 2018). During interactions with host rice and non-host, like tobacco, Xoo largely depends on Type III secretion system (T3SS) to secrete different effector proteins to cause disease or induce hypersensitive response (HR) (Wei et al., 1992; Alfano and Collmer, 2004; Song and Yang, 2010). There are also other factors contributing to the virulence of Xoo, including extracellular polysaccharides (EPSs) and plant cell wall degrading enzymes. Typically, motility and bacterial virulence are positively related, but it has been reported that hypermotility could also cause reduced virulence (Ottemann and Miller, 1997; Meng et al., 2011).
Polyhydroxyalkanoates are biodegradable polyesters synthesized by most bacterial genera and some archaea under unbalanced source of carbon or nitrogen (Lee, 1996; Steinbuchel and Fuchtenbusch, 1998; Ciesielski et al., 2010). PHAs are water-insoluble granules stored in the cytoplasm as carbon-storage and energy storage materials, which bacteria degrade and use for energy when faced with starvation. Genes involved in PHA synthesis often form gene clusters on bacterial genomes (Maehara et al., 2001). These genes are cataloged into two groups, one encoding proteins for granule-associated compounds, the other encoding regulators involved in the regulation of structural proteins and PHA biosynthesis. Polyhydroxybutyrate (PHB) is perhaps the most common type of PHAs (Pötter and Steinbuchel, 2005; Rehm and Steinbüchel, 2005). The biosynthesis of PHB starts usually with the production of Acetoacetyl-CoA via the condensation of two molecules of acetyl-CoA catalyzed by a β-ketothiolase (PhaA); acetoacetyl-CoA is subsequently reduced by a stereospecific acetoacetyl-CoA-reductase (PhaB) to R-(-)-3-hydroxybutyryl-CoA, which finally is catalyzed by the PHA synthase and polymerizes the acyl moieties of 3-hydroxybutyryl-CoA to PHB with concomitant release of coenzyme A (Pötter and Steinbuchel, 2005). PhaR is a putative cytoplasmic regulator which can bind to the promoter of phaP, a PHB granule associated protein, and to the promoter of its own gene to repress transcription (Cai et al., 2015). PhaR is conserved in PHA-producing bacteria. In the model PHA-producing strain Ralstonia eutropha H16, PhaR functions as a repressor or autoregulator for the expression of PhaP and PhaR itself, both of which can tightly bind to PHB granules (York et al., 2002; Pötter et al., 2005). PHA synthesis related genes also exist in phytopathogenic bacteria, including Xanthomonas, but limited research exists on the biological functions of these genes during bacterial growth and host infection. In this study, we demonstrated that loss of phaR in Xoo strain PXO99A alters multiple physiological and biological functions which affect bacterial growth and virulence in plant, and provided new insights into the biological function of PhaR in X. oryzae pv. oryzae.
Materials and Methods
Bacterial Strains, Plasmids and Growth Conditions
The bacterial strains and plasmids used in this study are listed in Supplementary Table S1. Xoo strains were grown in either nutrient broth (NB) medium or hrp-inducing medium XOM2 (Xiao et al., 2007) at 28°C. The antibiotics used in this study were spectinomycin (50 μg mL-1), kanamycin (25 μg mL-1) and ampicillin (100 μg mL-1).
Construction of PhaR Knockout Mutant and Mutant Complementation
The PhaR gene knockout construct was made by overlap PCR to produce a chimeric DNA including 892 bp upstream flanked DNA of phaR gene, 1.4 kb kanamycin cassette from vector pKD13 and 729 bp downstream flanked arm of phaR gene. This chimeric DNA was then cloned into pMD19 through T-A cloning. The resulting plasmid was introduced into PXO99A cells by electroporation (Song and Yang, 2010). The single exchange mutant was screened on kanamycin and ampicillin-containing media. The second exchange occurred simultaneously during the culturing in a liquid medium without any antibiotics. The clones that could grow on NA medium supplemented with kanamycin and ampicillin-sensitive were chosen as phaR gene deficient mutant candidates for further PCR characterization.
To complement the phaR mutant, the cosmid pHMPhaR containing the coding sequence of phaR gene was transformed into PXO99ΔPhaR by the freeze-thaw method (Dityatkin et al., 1972) to make the complementary transformant C-ΔphaR. Primers used for construction are listed in Supplementary Table S2.
Bacterial Motility Assays
For motility assay, bacterial strains were grown in NB medium for 2 days. Bacterial cells were then harvested by centrifugation, re-suspended in sterilized distilled water, and adjusted to an optical density (OD600) of 1.0. 1 μL of bacterial suspension was then dropped onto the plates containing semisolid medium (containing per liter 5 g Bacto Peptone, 1 g yeast extract, 3 g beef extract, 10 g sucrose and 0.03 g agar). Plates were incubated at 28°C for 3 days. The diameters of the swimming zones indicated the ability of bacterial movement. The experiment was repeated four times with three replicates for each time.
Bacterial Extracellular Polysaccharides (EPS) Measurement
Bacterium was cultured in NB overnight at 28°C. An aliquot of 20 mL culture was taken out and added with KCl to a final concentration of 1%. Then two volumes of absolute ethanol were added to precipitate EPS at -20°C overnight. EPS was collected by centrifuging at 12000 rpm for 20 min, and the pellet was dried at 55°C overnight before weighed. The bacterial concentrations of the culture used for EPS extraction were measured by diluting serially and plating in triplicate on NA plates to count the colony numbers. The production of EPS was evaluated in terms of weight (g) per cfu. The experiment was repeated three times.
Virulence Assay in Rice and Hypersensitive Response in Tobacco
One-month-old rice cv. IR 24 seedlings were used for virulence assays. The top two fully expanded leaves were inoculated with Xoo strains at OD600 of 0.5 via the leaf-clipping method. Lesion lengths were measured 15 days after inoculation, and the inoculated leaves were then pooled and cut into small pieces and ground in sterile water (with 1 ml sterile water per leaf) using a sterilized mortar and pestle. The resulting suspension was diluted serially and plated in triplicate on NA agar with appropriate antibiotics. Counts were taken and converted into CFU per leaf.
One-month-old Nicotiana benthamiana was used for HR assays. A bacterial suspension at OD600 of 0.2 was infiltrated into leaf tissue using a needleless syringe. Symptoms were recorded 24 h after infiltration. The assays for micro-HR response, active oxygen burst, and ionic conductivity of tobacco leaf were performed 12 h after infiltration as previously described (Peng et al., 2003; Fu et al., 2007). For in situ detection of H2O2 at indicated time points, tobacco leaves were detached and vacuum infiltrated in a 3,3′-diaminobenzidene (DAB) solution (1 mg/ml, pH 3.8) for 10 min, and then incubated at 25°C for 7–8 h. The deposits formed were visualized under microscopy after discoloring leaves in boiled absolute ethanol. Micro cell death was monitored by staining with lactophenol–trypan blue and destaining in chloral hydrate (2.5 mg/ml) as described by Peng et al. (2003).
RNA Isolation and qRT-PCR
Bacteria strains were grown either in nutrient broth (NB) medium or in hrp-inducing medium XOM2 overnight at 28°C (Xiao et al., 2007). 1 mL culture was collected for RNA isolation using SV Total RNA Isolation System (Promega, Madison, WI, United States). Reverse transcription was carried out using PrimeScript RT Master Perfect Real Time Kit (TakaRa Bio. Inc., Dalian). Quantitative real-time PCR (qRT-PCR) was performed using SYBR Premix Ex TaqTM Kit (TaKaRa Bio. Inc., Dalian). 16s rDNA was used as internal reference gene. The primer sets used are listed in Supplementary Table S2.
Phylogenetic Analysis of PhaR Family
Phylogenetic analysis of the aligned full-length sequence of phaR gene was conducted with MEGA 3.1 using neighbor joining and the Poisson correction model (Saitou and Nei, 1987; Kumar et al., 2004). Pairwise deletion was used for handling of sequence gaps. Members of the PhaR effector family used for phylogenesis analysis are indicated as their bacterial origins. The responding locus tags and bacterial origins are listed in Supplementary Table S3. The sequences used for phylogenetic tree generation are listed in Supplementary Table S4.
For relative gene expression assays, Student’s t-test at the 99% confidence level was performed. One-way analysis of variance (ANOVA) statistical analyses were performed on the motility assay, EPS production assay and virulence assay. The Tukey honest significant difference test was used for post-ANOVA pairwise tests for significance, set at 5% (P < 0.5).
Identification of phaR Gene in PXO99 and Its Homologs in Several Phytopathogenic Xanthomonas Strains
Based on the genomic sequence annotations of Xoo strain PXO99, five PHA metabolism related genes were found (Figure 1A), which include a beta-ketoacyl-ACP reductase (KACPR, PXO_RS09075) gene, a polyhydroxyalkanoate synthesis repressor (phaR, PXO_RS09070) gene, a poly(R)-hydroxyalkanoic acid synthase subunit PhaC (phaC, PXO_ RS11155) gene, a PHA synthase subunit (phaE, PXO_RS11160,) gene, and PHB depolymerase (phaZ, PXO_RS14020) gene. Among these, phaC and phaE were adjacent. To investigate the relationship of phaR with other related genes, we qualified the transcriptional expression levels of KACPP, phaC, phaZ, and phaE genes in the phaR mutant and the wildtype. Considering phaR gene is a stress responsible gene, we selected two time points, the first at 12 h representing the early stage of growth with the equal growth rates of mutant and wildtype strains, the other at 22 h representing the later stage with differential growth rates of mutant and wildtype strains. The results showed that mutagenesis of phaR gene affected the expression of other PHA metabolism related genes (Figures 1B,C). At both stages, the most significantly affected was the expression of phaZ, responsible for the degradation of PHB granule. The relative expression levels of phaZ for phaR mutant were 13.9 and 65.8 folds of those in the wildtype at 12 and 22 h after inoculation, respectively. The expression of phaC and phaE were also induced in the mutant, with 5.9 and 5.7 folds of induction for phaC at 12 and 22 h, respectively, and 4.7 and 2.1 folds of induction for phaE at 12 and 22 h, respectively. These results indicate that the bacteria may produce poly(3-hydroxybutyrate) (PHB), a type of PHA, and that phaR gene of PXO99 negatively regulated the degradation of PHB, which was more apparent in the late stage of growth with insufficient oxygen and nutrients.
Figure 1. The effect of phaR mutagenesis on the expression levels of other genes. (A) Genetic organization of the pha gene cluster. The arrows indicate the direction of gene transcription. (B,C) The relative expression levels of phaC, phaE, KACPR, phaZ, and phaR in phaR mutant and wildtype cultured in NB medium at 12 h (B) and 22 h (C) respectively, after inoculation. Error bars represent ±SD. The asterisk (∗) indicates a significant difference in Student’s t-test at p < 0.01.
The Xoo phaR gene is commonly found in plant pathogenic Xanthomonas species, but the gene sequences from different strains are polymorphic. The phylogenetic relationship was established based on phaR gene nucleotide sequences of 43 strains of Xanthomonas (Figure 2). As shown in Figure 2, the phylogenomic relationship displayed host, tissue, and geographic specificity. There are two main phylogenetic groups. One consists of strains of Xoo and X. oryzae pv. oryzicola (Xoc), which infect monodicot. The other group, with the exception of X. axonopodis pv. dieffenbachiae LMG 695 (Xad LMG695), a pathogen of monocot Ariod plants (Robène et al., 2016), consists of dicot plant parasites, including X. axonopodis pv. phaseoli (XapCFBP6982, pathogen of bean (Gent et al., 2005), X. perforans (Xp91-118, pathogen of Solanacearum plant (Itako et al., 2015), X. euvesicatoria LMG930 (Xe LMG930, pathogen of pepper Vancheva et al., 2018), X. campestris pv. vesicatoria (Xcv85-10, pathogen of pepper and tomato; Jones et al., 2004; Quelas et al., 2016), X. axonopodis pv. citrumelo F1 (XacF1, pathogen of citrus Jalan et al., 2011), X. citri pv. vignicola strain CFBP7111 (XcivCFBP7111, pathogen of cowpea Ruh et al., 2017a), X. fuscans subsp. aurantifolii strain 1566 (Xfa1566, pathogen of citrus Rigano et al., 2007), X. phaseoli pv. phaseoli (XppCFBP6988, pathogen of common bean Ruh et al., 2017b), X. fuscans subsp. fuscans str. 4834-R (Xff4834-R, pathogen of common bean Aritua et al., 2015). Within the first group, 11 Xoc strains infecting the mesophyll tissue formed a clade, and had identical sequences. Interestingly, 22 Xoo strains were divided into two clades, one for 9 African strains with identical sequences, the other for 13 Asian strains originated from Japan, Korea and the Philippines having almost identical sequences except one sequence from PXO99 strain with a 1 bp difference.
Figure 2. Unrooted phylogenic tree of phaR homologs from different Xanthomonas species. Bootstrap values shown at nodes were obtained from 1000 trials, and branch lengths correspond to the divergence of sequences, as indicated by the relative scale (20% weighted sequence divergence).
Loss of phaR Reduced the Growth Rate and EPS Production in Medium
To examine the effects of phaR mutagenesis on the morphology of PXO99A, we spotted the wildtype PXO99A, the mutant PXO99ΔphaR and the complemented strain C-ΔphaR on NA plates. 3 days later, PXO99ΔphaR developed dry colonies with reduced slime on the surface, while PXO99A and C-ΔphaR had normal mucoid colonies (Figure 3A). This indicated that phaR gene might regulate the growth rate of the bacteria or affect the production of EPS.
Figure 3. Colony morphology and EPS production of phaR mutant. (A) Colony morphology on NA plates. 1 μL bacterial suspension with OD600 of 0.5 was spotted on NA plate, and the photo was taken after the plate was incubated at 28°C for 3 days. (B,C) Bacterial growth in XOM2 (B) and NB liquid media (C). The bacterial strains were inoculated with the final concentration of OD600 of 0.05, and the OD600 of the culture was measured at different time points. The experiment was repeated three times. (D) EPS production. EPS from 20 ml bacterial culture was precipitated with ethanol and KCl. The EPS mass was measured after drying the pellet under 65°C for 72 h. The EPS productivity was counted in terms of g/cell. Error bars represent ±SD. Columns with different letters represent significant difference by ANOVA (p < 0.05).
To test if phaR regulates bacterial growth, we examined the growth curves of phaR mutant both in rich (NB) and minimal (XOM2) nutrient medium. The results showed that, in both media, the growth rate of PXO99ΔphaR was reduced greatly compared with that of the wildtype (Figures 3B,C). When inoculated with the same starting concentrations in liquid NB and XOM2 medium, the wildtype PXO99A, mutant PXO99ΔphaR and the complementary strain PXO99ΔphaR/phaR all grew at the similar rate for the first few hours, and then started to differ about 12 h after inoculation. PXO99ΔphaR kept propagating steadily but at much lower level, with the population size about 15.9 and 14.1% of the wildtype at 32 h (in NB) and 60 h (in XOM2), respectively (Figures 3B,C). The complement strain grew similarly as the wildtype in both media.
Since PXO99ΔphaR displayed the dry colony phenotype, we next tested if the EPS production was affected in the mutant. To do this, we extracted and compared the EPS mass from the 46 h NB culture supernatant of PXO99A and PXO99ΔphaR. With the same volume of culture supernatant, significantly less EPS pellet was precipitated by ethanol from the phaR mutant than the wildtype. Since the growth of the mutant was reduced compared to the wildtype and the complement strain, we measured the bacterial numbers and determined the EPS production per cell, in order to deduce if the reduced EPS production was caused by a reduced amount of bacteria or by the reduced EPS production efficiency. The results showed that EPS productivity of the mutant was greatly impaired and was about 22% of the wildtype (Figure 3D). This suggests that phaR positively regulates EPS production in PXO99A.
Loss of PhaR Reduced the Multiplication of PXO99A in Rice
Since phaR mutagenesis affected the bacterial growth in media, esp. in XOM2, a medium mimicking the host nutrient state in vitro, we hypothesized that it might also affect the bacterial propagation of Xoo in rice. To test this, we inoculated rice cv. IR24 with PXO99A, PXO99ΔphaR, and PXO99ΔphaR/ΔphaR by the leaf-clipping method. 14 days after inoculation, the average lesion length caused by PXO99ΔphaR inoculation was about 4 cm, which was about one third of that caused by wildtype strain PXO99A (Figures 4A,B). The bacterial multiplication in the inoculated leaves was measured by counting the colonies grown on media. Compared with PXO99A, the population size of PXO99ΔphaR in rice was reduced about 68% (Figure 4C). Complementation of the mutant with phaR gene restored the phenotype similar to the wildtype level. This data showed that the growth of PXO99ΔphaR is drastically restrained due to the loss of the phaR gene, which then reduces the overall virulence of Xoo in rice.
Figure 4. PhaR mutant had reduced virulence in rice. (A) Lesions formed in rice IR24 leaves 14 days after leaf-clipping inoculation with different strains. (B) Lesion lengths caused by different strains 14 days post-inoculation. (C) Bacterial population sizes of different strains in rice. Error bars represent ±SD. Columns with different letters above were significantly different by ANOVA (p < 0.05).
PXO99ΔphaR Mutant Was Hypermotile
The fact that the phaR mutant failed to travel to long distance to cause leaf lesion suggested that phaR might be involved in bacterial movement. To test this hypothesis, we evaluated the swimming motility of Xoo strains in semisolid motility agar. Unexpectedly, the results showed that PXO99ΔphaR was hypermotile, producing swimming haloes 1.36-fold in diameter of those of the wildtype PXO99A. The complementary strain restored motility to wildtype level (Figure 5). Thus, phaR might negatively affects motility in PXO99A.
Figure 5. Motility phenotypes of different strains. (A) Typical bacterial halos, formed on semisolid 0.3% agar plates after incubation at 28°C for 3 days. (B) Diameters of motility halos, formed on semisolid 0.3% agar plates. Error bars represent ±SD. Columns with different letters above were significantly different by ANOVA (p < 0.05).
Loss of PhaR Enhanced the Elicitation of HR in Tobacco
In Xoo, pathogenesis on host plants and HR on non-host plants are correlated properties regulated by the hrp genes. Because PXO99ΔphaR displayed attenuated virulence in the host plant rice, we next tested if the HR on non-host tobacco would be changed. To do this, a bacterial suspension was infiltrated into tobacco leaves to compare the ability of different strains to induce HR. 24 h after infiltration, PXO99ΔphaR induced an intensive HR and produced a larger necrosis area than that caused by PXO99A (Figure 6A).
Figure 6. Loss of PhaR increased HR, H2O2 accumulation and ion leakage in leaves of Nicotiana benthamiana. (A) Macroscopic views of HR. Leaves of 30-day-old tobacco plants were infiltrated with indicated bacterial suspensions. Photos were taken at 24 h post-treatment (hpt). (B) ROS detection using the DAB infiltration method. Tobacco leaves infiltrated with bacterial suspension were detached and infiltrated with DAB (1 mg/mL, pH 3.8) by vacuum pump and decoloration. Deposits formed were visualized after discoloring leaves in boiled absolute ethanol. (C) Binocular microscope views of micro-HR in tobacco leaves. Leaves of 30-day old tobacco plants were infiltrated with water or indicated bacterial suspension. Leaves were excised at 12 hpt and stained with trypan blue, which stains dead cells blue. (D) Histograms of ionic conductivity, which is an indicator of ionic leakage. Ionic conductivity was determined 12 h after tobacco was infiltrated with bacterial suspension. Columns with different letters above were significantly different by ANOVA (p < 0.05). Error bars represent ±SD.
To further measure the HR reaction strength, two landmark events of HR, the oxidative burst and micro-HR cell death were examined at 12 hpi through directly staining the biochemical component H2O2 with DAB or the dead cells with the trypan blue. DAB is oxidized by hydrogen peroxide to give a dark-brown color, and trypan blue selectively stains dead tissues or cells. The DAB staining results showed that PXO99ΔphaR induced extensive and intensive oxidative burst (dark brown spots) around the infection site (Figure 6B). Trypan blue staining of the leaf disks showed PXO99ΔphaR inoculation induced obvious cell death (blue spots), indicating micro-HR occurred around the infiltration site; while for PXO99A treatment, no obvious cell death was found (Figure 6D). Loss of phaR gene also increased the ability of PXO99A to induce cell membrane permeability of tobacco (Figure 6C). The reintroduction of the phaR gene restored the mutant’s phenotypes similar to those of the wildtype strain (Figure 6). These results indicated that the phaR gene negatively regulated HR induction in tobacco.
Loss of PhaR Gene Increased the Expression of Harpin-Encoding Gene Hpa1
In Xoo, HR induction in non-host plants is associated with hrp genes, esp. the hpa1 gene which encodes HR inducing harpin protein. To evaluate if the increased induction of HR by PXO99ΔphaR in tobacco is related to the changes of harpin expression, we analyzed the expression levels of hpa1 gene in different strains under hrp inducing medium via quantitative real time PCR (qPCR). The results showed that when cultured in XOM2 medium, which mimics the nutrient state of the plant, the expression levels of hpa1 in both mutant and wildtype strains increased significantly compared with those in nutrient rich medium NA broth. Moreover, compared with wildtype, the expression of hpa1 increased significantly in PXO99ΔphaR both in NA and XOM2 broth media (Figure 7). These results confirmed that hpa1 gene is induced by plant and suggested that phaR gene negatively regulated the expression of hpa1 in Xoo.
Figure 7. The transcriptional levels of hpa1 in XOM2 and NB liquid media. The hpa1 gene expression was measured by quantitative real time PCR and compared between wildtype strain PXO99A and the mutant ΔphaR. The asterisk (∗) indicates a significant difference in Student’s t-test at p < 0.01. Error bars represent ±SD from three replicates.
Polyhydroxyalkanoates serve as energy and carbon storage compounds under starvation conditions (Anderson and Dawes, 1990). The existence of PHAs metabolism enables the bacterial survival adaptation under poor nutritional condition. In the genome database of PXO99, we analyzed the gene cluster for PHA biosynthesis and metabolism. Searching for phaA and phaB homolog sequences by BLASTn showed no significant similarity, which indicated that there were no homologs of phaA and phaB in PXO99. However, adjacent to phaR, there is a gene encoding beta-ketoacyl-ACP reductase catalyzing the conversion of 3-hydroxyacyl-CoA to 3-oxyacyl-CoA. Similar as in other PHB producing bacteria, there are phaC and phaE genes responsible for the biosynthesis of PHB, phaR gene for regulation and phaZ for PHB degradation in PXO99. The above different compositions of PHB metabolism suggest that there might be different pathways to start the PHB synthesis because of the different nutritional provisions between pathogenic and non-pathogenic bacteria. More interestingly, the phylogenetic analysis based on phaR gene sequences suggests that, even though phaR gene is widely conserved in phytopathogenic genera of Xanthomonas, the polymorphism of nucleotide acid reflects the host specificity and even tissue and geographic region specificity. Bogdanove et al. (2011) previously identified hundreds of tissue and host specificity genes from different completely sequenced Xanthomonas strains, but all of these candidate genes were unique genes expressed in strains showing host or tissue infection specificity. However, PhaR is a conserved microbe nutrition related metabolism regulator encoding gene in Xanthomonas strains. The phylogenetic relationship of phaR gene in different phytopathogenic Xanthomonas strains displayed host and tissue specificity (Figure 2). This suggests that it may be correlated with the bacterial nutritional acquisition nature for bacteria infecting monocot or dicot plants, and for monocots, the phaR gene may be related with different host survival environments. For example, water supply and temperature for rice cultivated in Africa and Asia are very different. In the nitrogen-fixing bacterium Bradyrhizobium diazoefficiens, phaR was found to be localized at a higher hierarchy of a global regulator which activates the expression of more than 200 genes needed for bacterial life under low-oxygen tension (Quelas et al., 2016). Although the phaR gene is commonly found in different phytopathogenic Xanthomonas spp. (Figure 2), the way phaR affects bacterial traits related to its pathogenesis in Xanthomonas spp. is yet to be explored.
In this study, we made a phaR gene knock-out mutant in Xoo strain PXO99 using marker exchange method. By comparing the phaR mutant with the wildtype, we discovered that the phaR gene is involved in multiple bacterial traits related with bacterial physiology and its pathogenicity on host plants.
Bacterial motility is advantageous as it supports movement toward the host to obtain more nutrients and to avoid toxic environments. Thus, swimming ability is an important virulence factor for phytopathogenic bacteria, such as Erwinia spp., Ralstonia spp., Pseudomonas spp., and Xanthomonas spp (Meng et al., 2011; Pique et al., 2015; Kan et al., 2018; Markel et al., 2018). But in this study we found that loss of phaR increased the motility of Xoo, and the hypermotile phaR mutant displayed decreased virulence in rice. The hypermotility of bacteria may be caused by more flagella or speedy bacterial distribution under irregular environment. While having more flagella may promote the movement of bacteria in the environment, it may also produce abundant flagellin triggering the basal defense response of host. A previous study on Erwinia amylovora showed that E. amylovora lost motility under starvation condition with no change of flagella (Santander et al., 2014). Also the limited nutrient state in planta, XOM2 medium, and xylem sap induced genes related to motility (Kumar Verma et al., 2018). The global regulator CsrA gene of the carbon storage constituent of Escherichia coli was identified to positively regulate bacterial motility (Wei et al., 2001). The two-component regulatory system response regulator gene mutation studies of X. oryzae pv. oryzicola showed that, various mutants had similar tendency in EPS production and swarming ability, either increasing together or reducing together, but there was one mutant of GX_XOC2013 showed decreased EPS production and enhanced swarming ability and significantly reduced virulence on rice (Zhang et al., 2018). We thus speculated that the intracellular nutrient condition altered by the loss of phaR affected the flagella’s function. However, the exact regulatory mechanism remains unknown.
Bacterial leaf blight caused by Xoo is a typical vascular disease of rice. For vascular pathogen, EPS plays an important role in virulence. EPS promotes bacterial survival and colonization in planta by protecting the pathogen from environmental stresses and host defenses (Buttner and Bonas, 2010; Wu et al., 2012). The phaR mutant of PXO99 displayed reduced EPS production and altered colony phenotype as well as an attenuated growth rate. PhaR is a regulator of PHAs metabolism, its dysfunction greatly promoted the expression of phaZ gene, increasing the degradation of PHB in PXO99 (Figure 1B), which may affect the biosynthesis of EPS. It was reported that in Sinorhizobium meliloti Rm1021, the PHB synthase knockout mutants showed reduced production of the EPS succinoglycan (Aneja et al., 2004). Contrary to our findings, in the nitrogen fixing bacteria B. diazoefficiens and Ensifer meliloti, the phaR mutants produced more EPS (Encarnacion et al., 2002; Quelas et al., 2016). However, the PHB biosynthesis systems of Ensifer and Bradyrhizobium are quite different in terms of composition from that of PXO99, which suggested that there were different regulation models of phaR. For other bacteria, reduced EPS production affected the formation of biofilms, exhibited reduced growth and survival rates on leaf surfaces and reduced disease symptoms (Rigano et al., 2007), or impaired survival under oxidative stress (Dunger et al., 2007). For Xoo, according to the above results, the phaR mutant displayed reduced EPS production and relatively low multiplication ability both in nutrient-rich NB or nutrient-limited XOM2 media, or in vivo of plant host (Figures 3B,C, 4C), which may result in reduced virulence in rice.
Our experiments also revealed that the phaR mutant elicited increased plant defense resistance. Specifically, it induced a strong HR reaction when infiltrated into non-host tobacco leaf. For Xoo, the main effector inducing HR in non-hosts is a proteinaceous elicitor, harpin protein. Harpins are multifunctional and function not only as a part of translocator complexes for effector translocation into plant cells to promote bacterial virulence, but also as inducers of defense responses and as elicitors of HR cell death in the apoplast of plant tissues (Choi et al., 2013). The expression of harpin was greatly increased in the phaR mutant compared with the wildtype. The enhanced harpin expression caused by the loss of phaR may result from a physiological inability to respond to nutrients. The expression of the harpin protein in Xoo strains is highly related to the nutrition condition that the strain is exposed to Xiao et al. (2007). When the strain is cultured in nutrient-rich medium such as beef broth medium, the expression of harpin protein is suppressed, whereas the expression of harpin protein is induced when there is an interaction between the pathogen and host plant or when pathogen grows in a nutrient-deprived medium, such as XOM2. When PhaR is unfunctional in Xoo, it will reduce the carbon and energy storage by restraining PHA synthesis, or degrading PHB granules, which will in turn cause a state of nutrient deficiency and induce the expression of type III effectors including harpin protein. Our results showed that in both XOM2 and NB media, expression of harpin was induced for phaR knock-out mutant (Figure 4). This data indicates that PhaR is a negative regulator for harpin expression. Additionally, the enhanced harpin protein expression maybe a major contributing factor of the phaR mutant with enhanced HR inducing ability.
Carbohydrates are one kind of major nutrient sources for Xoo, impairing their corresponding metabolic pathway will result in abnormal bacterial phenotype. In this research we characterized a phaR mutant showing impaired EPS production which may reduce the environment viscosity to result in increased motility.
The poor survival adaption indicated by reduced growth rates in both medium and plant as well as increased defense response induced by high expression of harpin and reduced virulence factor EPS production likely resulted in attenuated virulence in rice. Our data suggests that phaR is a key gene coupling multiple bacterial characteristics related with its virulence in plant through regulation of carbohydrate metabolism.
C-FS and J-YL conceived the study. K-LS, XH, BZ, and X-FC performed the experiments. J-YL and K-LS analyzed the data. C-FS and J-YL wrote the manuscript.
This research was supported by the Natural Science Foundation of China (31471744) and National Special Fund for Agro-Scientific Research in the Public Interest (201303015).
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We are grateful to Dr. F.H. Meng for comments on the manuscript.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2018.03046/full#supplementary-material
Alfano, J. R., and Collmer, A. (2004). Type III secretion system effector proteins: double agents in bacterial disease and plant defense. Annu. Rev. Phytopathol. 42, 385–414. doi: 10.1146/annurev.phyto.42.040103.110731
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Quelas, J. I., Mesa, S., Mongiardini, E. J., Jendrossek, D., and Lodeiro, A. R. (2016). Regulation of polyhydroxybutyrate synthesis in the soil bacterium Bradyrhizobium diazoefficiens. Appl. Environ. Microbiol. 82, 4299–4308. doi: 10.1128/AEM.00757-16
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Zhang, Z. C., Zhao, M., Xu, L. D., Niu, X. N., Qin, H. P., Li, Y. M., et al. (2018). Genome-wide screening for novel candidate virulence related response regulator genes in Xanthomonas oryzae pv. oryzicola. Front. Microbiol. 9:1789. doi: 10.3389/fmicb.2018.01789
Keywords: Xanthomonas oryzae pv. oryzae, polyhydroxyalkanoate regulator, virulence, motility, hypersensitive response, hpa1 gene, extracellular polysaccharide
Citation: Long J-Y, Song K-L, He X, Zhang B, Cui X-F and Song C-F (2018) Mutagenesis of PhaR, a Regulator Gene of Polyhydroxyalkanoate Biosynthesis of Xanthomonas oryzae pv. oryzae Caused Pleiotropic Phenotype Changes. Front. Microbiol. 9:3046. doi: 10.3389/fmicb.2018.03046
Received: 09 August 2018; Accepted: 27 November 2018;
Published: 17 December 2018.
Edited by:Camille Eichelberger Granada, University of Taquari Valley, Brazil
Reviewed by:Luiz Gustavo Dos Anjos Borges, Icahn School of Medicine at Mount Sinai, United States
Kaijun Zhao, Institute of Crop Sciences (CAAS), China
Akanksha Singh, Central Institute of Medicinal and Aromatic Plants (CIMAP), India
Copyright © 2018 Long, Song, He, Zhang, Cui and Song. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Cong-Feng Song, email@example.com
†These authors have contributed equally to this work

domain: biology


text:
Events have been banned from taking place on land in Sarrat following a High Court injunction. The High Court granted the application of an injunction which will permanently prevent events from being held on land at Bucks Hill, Sarratt.
Three Rivers District Council applied for the permanent injunction following an earlier interim order granted in May. The injunction will remain in place, unless varied or discharged, and prevents the unauthorised use of a parcel of land within Bucks Hill.
The council sought the injunction after it was inundated with complaints from local residents last summer when a religious traveller festival, known as the Light and Life event, descended on land within the small hamlet. The council stated that the festival lasted for a week and caused "significant distress and unacceptable levels of disturbance to local residents."
Three Rivers District Council said that they have always been of the view that the use of the land during the festival was as a caravan site, a use excluded from the 28 day temporary use rule. On receiving information that a similar festival was to take place this summer, the council moved to secure the order.
Cllr Stephen Giles-Medhurst, the council’s Lead Member for Infrastructure and Planning Policy, said: “We will not tolerate the sort of intrusion and disturbance experienced by residents of Bucks Hill and the surrounding area last summer. Rules and procedures for holding such large scale events are there for a reason, and they must be followed. In this example, those rules were completely ignored and we have now ensured no such event can happen again on that land.
“I am delighted we have been successful in obtaining the permanent order from the High Court. Let this serve as an example to others who may ever consider such blatant disregard for residents of Three Rivers again.” The order prohibits bringing any caravans on to the land, mobile homes or any other structures intended for or capable of habitation, the use of the land as a caravan site as well as other restrictions.
Contact us with your stories.

domain: news


text:
Who Is the Virgin Atlantic White Card Good For?
Virgin Atlantic offers two credit cards to loyal flyers in the United States: the White Card and the Black Card. The White Card is good for those who are collecting Virgin Atlantic miles but don’t want to pay a big annual fee. The Black Card comes with a few more benefits, but also a higher annual fee and more conditions.
You’ll earn the same three miles per dollar on Virgin Atlantic purchases with both the White and Black Cards. With the White Card, you’ll also get an anniversary bonus and a 50 percent off companion award ticket if you charge $25,000 in a year.
The White Card is a premium World Elite MasterCard, which means that you’ll also get an excellent suite of
Virgin Atlantic is a transfer partner with Hilton Hotels and Resorts. You can transfer your miles to Hilton HHonors at a 1:1.5 ratio.
|AIRLINE TRANSFER PARTNER
|RECOMMENDED CREDIT SCORE
|TYPE OF CARD
|Bank of America
You get a 12,500-mile sign-up bonus after your first purchase. You can also get 2,500 miles when you add an authorized user, and 7,500 miles annually after charging $15,000 in a year.
- Three miles per dollar spent on Virgin Atlantic purchases
- One mile on everything else
The Virgin Atlantic White Card is one of the few airline credit cards offering three miles per dollar on flights. Most other co-branded credit cards offer 1.5 or 2 miles.
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Besides innovative service, mood lighting and fashionable flight attendant uniforms, Virgin Atlantic has a very versatile frequent flyer program called Flying Club.
The airline has the following partners:
- Air China
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While the sign-up bonus won’t get you too far, it can be used for short domestic flights in many countries. There are many options; for example, 15,000 miles or less can get you:
- Two round-trip flights within the U.K.
- One round-trip ticket between Los Angeles and Seattle or Portland
- Anywhere within New Zealand and back on Air New Zealand
- A roundtrip between Tokyo and most other Japanese cities on ANA
- A 620-mile or less flight in India on Jet Airways
- No minimum spend for the sign-up bonus
- Good earning potential on Virgin Atlantic purchases
- Several airline booking partners
- Miles transfer to Hilton HHonors at 1:1.5
- Half off a companion award ticket after spending $25,000 a year
- Miles don’t expire for three years
- Low sign-up bonus
- Fuel surcharges on Virgin Atlantic and most partners
- Some booking restrictions on partners
- High spending requirement for the 7,500-mile annual bonus
How Far Do Your Miles Go?
If you fly on Virgin Atlantic several times a year, the Virgin Atlantic White Card would be a great option thanks to the generous triple miles earned on Virgin Atlantic purchases. The 15,000-mile sign-up bonus can only take you so far, but if you’re interested in domestic travel in the U.S. and some other countries, the $49 annual fee might be a worthwhile investment.
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domain: travel


text: Alan Ruiz (b. 1984, Mexico City, Mexico) is an artist living and working in New York City. His work addresses the intersection of site-reflexivity, architectural discourse, and urban policy. He received an MFA from Yale University, a BFA from Pratt Institute, and is a 2015–2016 participant in the Whitney Museum Independent Study Program. Engaging architecture as a perceptual and political medium, his work explores ways in which our built environment may reproduce social hierarchies. His work has been shown both nationally and internationally, including exhibitions at The Bronx Museum of the Arts, Abrons Art Center, Y Gallery, Horatio Jr., and Johannes Vogt Gallery. His writing has been featured in TDR (The Drama Review, MIT Press), InVisible Culture: an Electronic Journal for Visual Culture, and BOMB Magazine. In 2015 Ruiz was an Artist-in-Residence with the Youth Insights Program at the Whitney Museum of American Art.

domain: artistic


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text:
The Westbrass Velosah Single-Handle Hot Water Dispenser in Polished Chrome with Hot Water Tank features an automatic turn-off feature for your safety, along with a high-arc spout for great sink clearance. To clean the water before it gets heated, install a filter (not included) between the water supply and the tank. A hot water tank is included with the faucet. This faucet is designed for single-hole installation (mounting materials included).
- Polished chrome finish for an attractive look
- Single-handle design for quick and easy water control
- High-arc spout provides great sink clearance
- Auto-off hot handle for your safety
- Install a filter (not included) between the water supply and tank to clean water before heating
- Includes a hot water tank
- Designed for single-hole installation (mounting materials included)

domain: design


text:
TO GET THE COMPLETE JOURNAL/THESIS FOR TOPIC BELOW,
CALL: 08168759420, 08068231953
CONTEMPORARY SECURITY THREATS AND TERRORISM ON SOCIOECONOMIC AND POLITICAL DEVELOPMENT OF NIGERIA
Security is not just a scared value for humans, groups and nations, it is the ultimate value. Security in the world today has become a seriously endangered value. Nigeria’s contemporary constitution actually asserts that the security and welfare of Nigerians shall be primary duty of government. Today, Nigeria has become the country where citizens are most endangered. Apart from the dangers posed by corruption, citizens are daily exposed to the fires from armed robbers, rapists, assassins, thugs, ritualists, human traffickers, arms traffickers cross-border bandits, drug traffickers, kidnappers, militants and above all terrorists. This paper examines the implication of contemporary security treats and terrorism of Boko Haram and Ansaru on the socio-economic and political development of Nigeria, then, discovers that the constitutional provision of the federal system of government which gave enormous power to the federal government at the expense of the federating units led to series of instability, insecurity and terrorism in Nigeria, and concludes that to curtail instability, insecurity and terrorism, there is a need to embrace a political system of government that gives more power to the federating units rather than concentrating so much power at the centre.
Key words: Security treats, terrorism, economy, politics, development.

domain: sociology


curated by Cristiana Perrella
Gian Ferrari Hall
On the occasion of the Architecture in Uniform
On the occasion of the exhibition Architecture in Uniform Adrian Paci and Roland Sejko reflect on this episode.
A video-installation shows international actors interpreting fragments of some of those letters.
A space, which accommodates prints of the letters, becomes a map, a physical presence of these traces of communications that were never received.
The authors would like to thank The State Archives of Albania


text:
JUSTICE BEHAVIORAL HEALTH COORDINATOR
Region 3 Behavioral Health Services is seeking qualified applicants for a Justice Behavioral Health Coordinator position. Region 3 is one of the six Regional Behavioral Health Authorities in Nebraska responsible for the planning, developing, coordinating, funding, and monitoring the provision of mental health and substance use disorder treatment, rehabilitation, recovery, and prevention services across central and south central Nebraska.
Qualified applicants will have:
- Knowledge in the behavioral health field and current theories and practices regarding service development and delivery of emergency behavioral health services
- Skill in working effectively in team/group situations and ability to present ideas clearly and to build consensus
- Exceptional verbal, written, interpersonal and organizational communication skills
- Ability to use sound judgment and timing in handling situations of a sensitive and difficult nature
- Excellent time management skills and ability to prioritize multiple, competing tasks.
Education and Qualifications:
- Bachelor’s Degree in criminal justice, social work, psychology, or a related field
- 3+ years experience in health and human services
- Promote services and activities designed to divert individuals experiencing a behavioral health disorder from the justice system.
- Collaborate with forensics, judicial, justice, and behavioral health systems and services.
- Explore strategies to better serve individuals with complex behavioral health needs who cycle in and out of the justice system.
- Support individuals, families, and stakeholders in understanding and connecting to various continuums of treatment and recovery options within our system.
- Build community awareness to decrease the use of corrections instead of behavioral health treatment increasing connections to community resources as a response to unmet behavioral health treatment needs in communities.
- Utilize a collaborative, cross systems measurable process to identify opportunities for intervention and community level behavioral health interventions and engagement.
Region 3 Behavioral Health Services is a family friendly organization, offering competitive salary and generous benefits package including paid employee health and dental insurance with partial paid family insurance coverage, retirement plan with six percent company contribution, four personal days per year, eight paid vacation days per year, eight paid holidays per year, twelve paid sick days per year, flexible scheduling, casual dress code, and partial remote work option. Persons of all colors, creeds, and ethnic backgrounds are encouraged to apply. Interested individuals should submit a letter of application, resume, and names of three professional references to: Tammy Burgeson, Region 3 Behavioral Health Services, PO Box 2555, Kearney NE, 68848. Application deadline is July 7, 2022. www.region3.net. EOE/AA

domain: medical


text:
Mark Stephens lives in the Los
Angeles area and has been a
vital contributor to the music industry for years. He is in possesion
of a rare sensibility which allows him to interpret diverse styles
with equal authenticity and yet his Jazz improvisational style
remains unique and recognizable.
Among the luminaries he has worked with are
Alanis Morissette, Prince, Diana Ross, Faith Hill, Jeffrey Osbourne,
Mariah Carey, Vanessa Williams, Take 6, Marcus Miller, Michael
Brecker, Stanley Clarke, Lee Ritenour, George Duke, Larry Carlton,
Kirk Whalum, Boney James, Jonathan Butler, Rachelle Ferrell,
Herbie Mann and James Moody.
Mark Stephens is the quintessential musician
whose talents as
a pianist / keyboardist and arranger / composer continue to carve
an exclusive place in the world of music. His eagerly anticipated
debut project features some of todays' most exciting Jazz Artists.

domain: entertainment


text:
HCMC – Dragon Capital has raised its stake in Hoa Sen Group, which trades its HSG shares on the Hochiminh Stock Exchange, becoming one of the steelmaker’s major shareholders.
On May 25, Dragon Capital’s members sold 1.75 million HPG shares and cut its ownership in the steelmaker from 5.12% to 4.86%, making it no longer a major shareholder of Hoa Sen Group.
However, the fund manager acquired a combined seven million HSG shares on June 12 and 14, increasing its ownership in Hoa Sen Group to 6.38%.
Norges Bank, a member of Dragon Capital, bought the highest amount, at two million shares, while Hanoi International Limited acquired 1.5 million shares.
Other members also bought more HSG shares. Grinling International Limited, Hanoi Investments Holdings Limited and Vietnam Enterprise Investments Limited purchased one million shares each, while Amersham Industries Limited got 500,000 shares.
HSG shares of Hoa Sen Group have been included in the list of stocks ineligible for margin trading due to its net losses in the first half of the fiscal year 2022-2023.
Hoa Sen’s revenue plunged by a staggering 49.7% to VND14.9 trillion, according to its reviewed consolidated semi-annual financial statements for FY2022-2023.
It posted VND424.2 billion in net losses, compared to a profit of VND873 billion in the year-ago period.
Meanwhile, the steelmaker’s gross profit plummeted 69.2% year-on-year.
Hoa Sen has worked out two scenarios for this fiscal year. In its first scenario, the company looks to produce 1.4 million tons of products and obtain VND34 trillion in revenue and VND100 billion in profit, down 32% and 60% year-on-year.
In the second scenario, in which the firm’s output reaches 1.5 million tons, its revenue would drop by 28% year-on-year to VND36 trillion, while profit increased 20% over the same period in 2022 to VND300 billion.
HSG shares extended the losing streak to the fourth straight session at the close today, June 19, at VND16,250 per share, with a matching volume of eight million shares.
On the southern bourse, the number of decliners tripled that of advancers by a ratio of 315 to 113, sending the VN-Index of the HOSE down 9.82 points, or 0.88% from last Friday, to 1,105.4 points.
Trade on the southern market plunged by 30.1% in volume and 36.1% in value, with over 811.3 million shares worth VND14.3 trillion changing hands. Nearly 87.7 million shares were transacted in block deals, contributing more than VND1.8 trillion to the overall value.
There were 10 bluechips rising and 18 others dipping in the VN30 basket. The VN30-Index slid 10.12 points, or 0.91% lower than the session earlier, to 1,098.94 points.
Lender VCB was the main drag as it lost 3.1%, contributing nearly 3.8 negative points to the benchmark VN-Index.
Many real estate stocks finished the day in the red. NVL crashed to the floor, losing 6% to VND14,000 per share and took the lead by liquidity on the southern bourse with a matching volume of 67.2 million shares.
PDR, HQC, LDG, DRH, SCR, and HPX lost between 3.1% and 4.2%, with trading volume ranging from 5.5 million shares to 25.4 million shares.
The HNX-Index of the Hanoi Stock Exchange followed the market downward trend, ending the day down 1.93 points, or 0.84% from the previous session, 226.52 points.
There were 59 winners and 125 losers in the northern market. Trade on the Hanoi bourse amounted to VND1.9 trillion with 127.5 million shares changing hands.

domain: finance


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Skype Security | City Vision University
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Freida's 'Trishna' at IIFI
Freida Pinto's movie has received rave reviews world-over
Hollywood-based Indian actress Freida Pinto-starrer 'Trishna' will be screened Friday at the 42nd International Film Festival of India here.
'Trishna' is about a woman whose life is destroyed by a combination of love and circumstances. Freida, along with director Michael Winterbottom, co-producers Anurag Kashyap and Sunil Bohra as well as composer Amit Trivedi, will attend the screening.
The story is based on Thomas Hardy's classic novel 'Tess of the D'Urbervilles'. Set in contemporary Rajasthan, the film has Trishna (Freida) meeting a wealthy young British businessman Jay Singh (Riz Ahmed) who has come to India to work in his father's hotel business. After an accident destroys her father's jeep, Trishna goes to work for Jay, and they fall in love.
But despite their feelings for each other, they cannot escape the conflicting pressures of a rural society which is changing rapidly through industrialisation, urbanisation and, above all, education. Trishna's tragedy is that she is torn between the traditions of her family life and the dreams and ambitions that her education has given her.
The film had its international premiere at the Toronto International Film Festival (TIFF) and received rave reviews from critics.
The film will have worldwide release in 2012. For the India release, it will be dubbed in eight languages.


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Places In Cape CityFor The Greatest Cape City Holiday That Are Worth A Go To
The urban area of Cape City enters your mind as the front runner whenever you think about a traveler destination with wealthy historical sights as well as social ancestry. Being actually South Africa's earliest town, the urban area supplies an excellent option to take pleasure in with family and friends. The white colored seasides, long extent of coastline, as well as alright dine seaside restaurants provide enough entertainment to sunshine seekers as well as beach front enthusiasts, Read This.
The many wine paths is another crucial explanation for the improving popularity of Cape Town as a well-known tourist location. A travel to Cape City supplies you possibilities to explore the different red wine options, and also savour some of the finest red wines. Wintertime is actually the suitable season if you want to discover Cape Wine Lands.
Being among the absolute most stunning urban areas in South Africa, Cape Town has the Table Mountain standing majestically on its own background, as well as a collection of Edwardian and Victorian structures that reflect the building style of the metropolitan area's previous totalitarians. For folks that have a craze for crafts and also arts, there are actually a lot of options including antique outlets, exquisite malls, and also craft markets that are capable of fully pleasing their necessities.
Long just before the Table Mountain range was actually deemed a traveler destination, Cape Cape was actually recognized all over the world, for its vast variety of attractive flora. Along with thousands of rare types, the green lowlands as well as ocean cliffs produces this spot among the wonderful natural marvels of the world. A see to the National Botanical Gardens at Kirstenbosch, makes certain to assist you find out the indigenous types of flora and also is the most ideal selection for family travels.
The Table Mountain Range National forest that stretches from the V&A Waterfront to Cape Point offers the one-of-a-kind Hoerikwaggo Route and also is utilized by countless guests daily. Due to the steep high cliffs, spectacular sea perspectives and forests, Cape City is taken into consideration a gorgeous destination that supplies distinct trip encounters for site visitors.
The urban area offers an advanced scenery, along with the lengthy selection of hotels that position absolute best on the planet. Museums, galleries, and also home structures utilize a majority of the yard making it possible for website visitors to outline the effect of the Dutch and also the English.
Accommodation varies and also abundant, coming from 5 star hotels and resorts to Back-packers, flats or even houses. The fantastic V & A waterside is actually a knowledge, to enjoy most of the unique boutique, dining establishments and coffee shops. In the calm of morning or even dusk, resting on the harbour wall surface along with fish and also potato chips, will definitely give you a brand new impression this contrasting city and wonderful memories. As a choice, there is actually a sizable selection of movie houses and also events, to maintain you amused and fascinated.
Another fascinating feature of Cape Community is its pleasant weather condition with wintertime rains in July, and mild temperature level throughout the months off November to April and a gently very hot summer months, Read More Here.
Whether on a business see, family excursion, or a weekend break travel along with good friends, hotels in Cape City provide specials that are made specifically to suit your requirements and budget plan criteria. From deluxe facilities to cheap-budget accommodations, Cape Town has a variety of options to deliver you the excellent keep in this attractive urban area known for its ancient lifestyles and also organic scenery.


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Whaling and Sealing in Maori Times By Jasleen Oberoi, Danielle Gamlena, Hubert Januszczak, Andrew Power and Rama Yogakumar!!!!!!!!!
What is Whaling and Sealing • Whaling is the hunting of whales primarily for meat and oil. Its earliest forms date to at least 3000 BC. Various coastal communities have long histories of sustenance whaling and harvesting beached whales. Industrial whaling emerged with organized fleets in the 17th century; competitive national whaling industries in the 18th and 19th centuries; and the introduction of factory ships along with the concept of whale harvesting in the first half of the 20th century. • Seal hunting, or sealing, is the personal or commercial hunting of seals.
Before Whaling and Sealing & How they Whaled and Sealed • Before whaling and sealing New Zealand had larger population of whales and seals that lived throughout the coasts. Maoris started sealing as soon as they made contact with Pakeha. • Some ways that they whaled and sealed: • They had a group of boats, which moved in on to the whales and seals and herded them on to the beach so they couldn’t swim. • Another way they caught whales was to throw harpoons with a semi-floating object like a blown-up seal bladder and followed the whale around until it got too tired and killed it.
Whaling and Sealing and some effects • Whaling:Whaling in New Zealand covered a period of about 172 years from 1792 to1964. During the first 50 years the industry was of genuine significance toNew Zealand, as it was the first externally financed and controlled staple industry in the country.It was significant for the symbiosis which developed between European/Pakeha New Zealanders and Maori.Through whaling, Maori were able to travel the oceans of the world and absorb a great range of skills which they brought back home. Above all, they learned English, the language of Pacific Whaling. Maori also traded using whaling by providing the wants of the whale fleets and therefore acquired iron tools, musket and gunpowder.
Whaling and Sealing and some effects • Sealing:Sealing and whaling were the earliest Pakeha commercial activities to flourish in New Zealand and were significant socially and politically as the ‘contact’ period between Maori and other cultures. From an economic perspective New Zealand sealing and whaling were a tiny part of a global commodity market. For example, many seal skins were sold on Asian markets. Sealing and whaling were operated by offshore companies and were largely ship based. The saleable products were skins, bones and oil. Seals in New Zealand had been hunted to the verge of extinction by 1830 and sealing was outlawed in 1926. Whaling continued and some large land based stations were built. The last station, Perano, closed after the killing of its last whale in December 1964 as Japanese and Russian whale fleets were over-fishing.
Uses of Whaling and Sealing • After Cook visited New Zealand (NZ), many ships arrived into the South Seas, in search of whales and seals. • Whales were slaughtered in thousands. Their carcasses were dragged ashore and the blubber was boiled to melt it down into oil. The oil then got used as lamp oil. This was a important job, because it provided people with light at the time where there was no electricity. • On the other hand, seals had a completely unnecessary reason. They were used for- Fashion. Seals were killed to death on rocks along the coasts of NZ. After this, their skins were salted and cured to be sent to Britain; where they were made into hats and cloaks. • The flubber inside the whale is usually the main cause for whaling. To insulate them from the cold, whales have a thick, dense layer of connective tissue and fat under their skin called blubber. Blubber can be used for all types of products, but is now not as commonly used. It is also a source of Vitamin D. • Whaling and Sealing were amongst the busiest industries in the late 18th Century and early 19th Century.
Other information • Europeans of all description came to New Zealand during these periods: Dutch, French, Russian, German, Spanish, Portuguese, British and even North Americans. • Whalers and sealers mainly caught fur seals, humpback whales, sperm whales, minke whales bryde’s whales and southern right whales. • Locations of sites include: Northland, Auckland, East Coast/Hawkes Bay, Wellington, Nelson/Marlborough, Otago and Southland. As whales, would pass here through different seasons of the year, Pakehas would set up stations in these places with the consent of the Maori chief. • Dragging whales back to shore could take up to 14 hours of hard rowing depending on the size and the weight.
Species of Whales and Seals Humpback Whale Southern Right Whale Sperm Whale Fur Seal Colony
New Zealand Map Shows where the species where located in Light Blue: Humpback Whales Grey: Sperm Whales Green: Bryde’s Whales Dark Blue: Southern Right Whales

domain: history


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ONE: Lubavitch UK Drinks to Revival
After 6 years of not holding a gala celebration, Lubavitch UK held its largest dinner yet. They had a good reason to do so.
By Simon Rocker, the Jewish Chronicle
The Sukkah on New Street Square
With tall buildings, glass windows, and geometric angles, New Street Square has some of the most contemporary architecture of London. So when a single-story structure made of wooden panels and a leafy-green roof appeared one day, well… it attracted more than a few stares. Erected by ...
Chabad Summer Camp Fun!
Camp Gan Israel UK camps for boys and girls recently ended a successful summer of fun and inspiration.
"Camp Gan Israel UK overnight camps were this year an outstanding success, and it was all due to the amazing group of Head staff and councellors who created a super chassidish ...
Amazing Success At The Shluchim Conference
From a single house to a household name, Lubavitch UK has grown over the past 6 decades to a network of 115 Shluchim who span the length and breadth of the United Kingdom.
This week 92 of those Shluchim gathered for the annual Chabad Lubavitch UK Kinus Hashluchim that was held in the magnificent ...


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"Hiya, Georgie!" See if they float, too, for yourself with new Vynl. figures from Funko. Both Georgie and Pennywise from the new film adaptation of Stephen King's IT each come with specific accessories (including that eerie red balloon and the doomed S.S. Georgie, and will be available this winter!
Keep an eye out for Funko's IT movie Vynl. figures to emerge from the sewers of Derry sometime in the next several months, and check out official images and details on the creepy collectibles below. Will you be adding them to your shelf?
From Funko: “Vynl.: It – Pennywise and Georgie
There’s no I in Vynl. – because there’s always two of them. The newest Vynl. features characters from the terrifying film It! Will Pennywise convince Georgie to float?
This Vynl. pair features Pennywise holding his enticing red balloon, and Georgie - complete with his raincoat and SS Georgie boat!
Add them to your collection this Winter!
Coming this Winter!”
Image via Funko:


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domain: food


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Sweet and savory is the best way I can describe this guacamole. The buttery and chunky avocado, the acidic touch from the lime, the crunch from the fresh vegetables, and the sweet, ripe avocado make a delicious dip or topping for your tacos.
When I made this recipe I had simply eaten too much regular guacamole and eventually grew tired of it. Even the good things get old after a while if that’s all you’re having… everything in moderation! So I tried making it with different ingredients, like a mango.
Thank you for reading my recipe, if you decide to try it out tag me on instagram @breakthrukitchen. Be sure to subscribe down below to get notified when I post more recipes like this!
Sweet Mango Guacamole
A sweet and savory twist to traditional guacamole.
- 2 ripe avocados
- 1/2 ripe mango
- 1/2 onion
- 1 clove garlic
- 1/2 jalapeño pepper
- 1 tsp salt
- 1/2 tsp cayenne pepper
- 1 lime
- 1/8 cup cilantro
Directions1.Finely chop onion, garlic, and jalapeño pepper and squeeze your one half of lime juice into the mixture.
2.Add avocado and use a fork to mush your avocado to your preferred consistency.
3.Add diced mango and mix. Season with salt and add your other half of lime.
4.Finish it off with fresh cilantro and give it one last good mix. Enjoy!


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Scientists at the Montreal Neurological Institute and the Montreal Proteomics Network at McGill University have published the most complete picture to date of the components of the molecular machinery that controls the entry of nutrients and other molecules into cells. In a study published in the Proceedings of the National Academy of Sciences of the USA (PNAS), Dr. Peter McPherson and colleagues used proteomics, the large-scale study of proteins, to identify the protein complement of clathrin-coated vesicles. These vesicles are the vehicles by which cells are able to take up nutrients, such as cholesterol, from their environment. Defects in this uptake process have profound repercussions on cellular function and human health. For example, genetic diseases that lead to deficiencies in cholesterol uptake cause elevations in plasma cholesterol levels and early-onset coronary atherosclerosis. In the brain, problems in the uptake process involving clathrin-coated vesicles can disrupt the transmission of signals between nerve cells. This can lead to a number of disorders including defects in the ability to form new memories.
“Proteins are the workhorses in our cells,” explained Dr. McPherson, Associate Professor of Neurology and Neurosurgery, and Anatomy and Cell Biology at the Montreal Neurological Institute (MNI) at McGill University. “Increasingly, we are learning that proteins don’t work in isolation, but function in large arrays that form protein machines. Proteomics is exciting because it allows us to breakdown this complex machine into its component parts. We can then figure out how it is assembled, how the proteins interact with one another, and what goes wrong in disease.
“The study from Dr. McPherson and his colleagues is fundamental to our understanding of the cellular uptake process because it provides a comprehensive molecular inventory of the clathrin-coated vesicle. Its results have broad implications for a variety of fields in biology and medicine,” said Dr. Pietro De Camilli, Professor of Cell Biology, Yale University School of Medicine and Investigator, Howard Hughes Medical Institute.
Sandra McPherson | McGill University
Molecular Force Sensors
20.09.2017 | Max-Planck-Institut für Biochemie
Foster tadpoles trigger parental instinct in poison frogs
20.09.2017 | Veterinärmedizinische Universität Wien
Whispering gallery mode (WGM) resonators are used to make tiny micro-lasers, sensors, switches, routers and other devices. These tiny structures rely on a...
Using ultrafast flashes of laser and x-ray radiation, scientists at the Max Planck Institute of Quantum Optics (Garching, Germany) took snapshots of the briefest electron motion inside a solid material to date. The electron motion lasted only 750 billionths of the billionth of a second before it fainted, setting a new record of human capability to capture ultrafast processes inside solids!
When x-rays shine onto solid materials or large molecules, an electron is pushed away from its original place near the nucleus of the atom, leaving a hole...
For the first time, physicists have successfully imaged spiral magnetic ordering in a multiferroic material. These materials are considered highly promising candidates for future data storage media. The researchers were able to prove their findings using unique quantum sensors that were developed at Basel University and that can analyze electromagnetic fields on the nanometer scale. The results – obtained by scientists from the University of Basel’s Department of Physics, the Swiss Nanoscience Institute, the University of Montpellier and several laboratories from University Paris-Saclay – were recently published in the journal Nature.
Multiferroics are materials that simultaneously react to electric and magnetic fields. These two properties are rarely found together, and their combined...
MBM ScienceBridge GmbH successfully negotiated a license agreement between University Medical Center Göttingen (UMG) and the biotech company Tissue Systems Holding GmbH about commercial use of a multi-well tissue plate for automated and reliable tissue engineering & drug testing.
MBM ScienceBridge GmbH successfully negotiated a license agreement between University Medical Center Göttingen (UMG) and the biotech company Tissue Systems...
Pathogenic bacteria are becoming resistant to common antibiotics to an ever increasing degree. One of the most difficult germs is Pseudomonas aeruginosa, a...
19.09.2017 | Event News
12.09.2017 | Event News
06.09.2017 | Event News
20.09.2017 | Life Sciences
20.09.2017 | Power and Electrical Engineering
20.09.2017 | Physics and Astronomy


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It is hard to believe it, but fourth-year is almost over. I’m on my last rotation now, which means less than three weeks left until I am finished with vet school. Fourth-year has gone by in the blink of an eye, and I’m sure these next three weeks will go even faster.
My last rotation here at the AVC is small animal surgery. While I’m not interested in specializing in surgery, and I’ll certainly never be performing total hip replacements in general practice, it is useful to learn about these complicated procedures so that if a patient of mine ever needs to be referred, I’ll be able to speak with an owner about the surgery.
My first day was quite the introduction to the rotation. I took over a case that went to surgery the first day. I think I quickly learned why I would never be a surgeon, and that’s my inability to stand in surgery for five hours without becoming a miserable person. It takes a large amount of patience and fine attention to detail to be a surgeon, and I respect anybody who can do that. I will happily refer complicated surgery cases in the future!
Over the next three weeks, I’m hoping to absorb all of the information I can about orthopedic and neurologic examinations, as those make up a large majority of surgical cases at the AVC. I’m excited to see some cool surgeries while I’m here, and I’m so excited that after this I will be done!


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TAGS. Dakine Hip Pack. Dakine is located at 408 Columbia St, Ste 300, Hood River, OR 97031. View Details. Dakine can be contacted at. In a town of 7,200 residents, the fate of a company as big as Dakine is a pretty big deal. View Details. Hood River outdoor sports and lifestyle brand Dakine has a new private equity owner. Women’s Olukai ‘Ohana. “As far as backcountry legends go, the Dakine Heli Pack is as prestigious as some of the top athletes” A few years after Dakine was founded, the company headquarters were moved from Hawaii to the mainland: Hood River, Oregon. Hood River outdoor gear maker Dakine is closing its office there, laying off 39 and moving its headquarters to Southern California. Founded in Hawaii in 1979, Dakine moved to Hood River in 1986 where it became one of the state's signature brands for outdoor gear selling backpacks, luggage, ski and snowboarding gear. The Torrance, Calif., based sports equipment company will vacate its Portway offices and move its … Doug's Hood River. Dakine, headquartered in Hood River, is a leading backpack and accessory brand within the action sports market. Společnost se … Dakine is ending its 34-year presence in Hood River. Dakine's annual revenues are $10-$50 million (see exact revenue data). Browse Dakine, Inc. has 85 total employees across all of its locations and generates $14.14 million in sales (USD). Dakine moved to Hood River in 1986 where it became one of Oregon’s signature brands for outdoor gear selling backpacks, luggage, ski and snowboarding gear. Founded in Hawaii and located in Hood River, Oregon. Founded in Hawaii in 1979 and located at the base of Mt. Dakine moved to Hood River in 1986 after being founded in Hawaii seven years earlier and became one of the Oregon’s signature brands for outdoor gear selling backpacks, luggage, and ski and snowboarding gear. DaKine byla založena v roce 1979 v Maui County na Havaji Robem Kaplanem.V roce 1986 došlo k přesunu sídla společnosti do Hood River v Oregonu ve Spojené státech amerických, zde firma stále sídlí ().V srpnu 2009 DaKine získala Billabong International Limited za přibližně 100 miliónů amerických dolarů. HOOD RIVER, Ore. — Outdoor gear maker Dakine is closing its Hood River office and laying off 39 employees, officials said “The current health crisis, and the corresponding Oregon stay at home order were unforeseen business circumstances, and have made it financially unfeasible for the company to continue operating,” Dakine … Get directions, reviews and information for Dakine in Hood River, OR. The Oregon Live reported this morning that Dakine is laying off 39 people, closing the brand’s Hood River headquarters and moving to Southern California. Hood and the Columbia River, the new DAKINE home is adjacent to a world-class kiteboarding and windsurfing … There are 54 companies in the Dakine, Inc. corporate family. It is classified as operating in the Sporting & Recreational Goods & Supplies Merchant Wholesalers industry. Find 2 listings related to Dakine in Hood River on YP.com. Dakine Equipment LLC will be closing its Hood River headquarters and reportedly moving to Torrence, CA. 2005: The DIY Mountain biking scene explodes around Hood River and Dakine answers with rider-driven bike packs focused on the need for hydration. The closing will result in the layoffs of 39 workers, according to a letter filed with state workforce officials on May 1 attained by The Portland Business Journal. Coronavirus Dakine Hood River. Is this an introspective on Dakine or Hood River? Share This! Either way, I'm in both every day! $24* *Shipping and Taxes may be added during checkout. This was the perfect location for a brand that was now starting to gain … V roce 1989 jako první vstoupil na snowboardový trh s příslušenstvím a batohy na snowboard. Customer Service Representative (Former Employee) - Hood River, OR - March 5, 2013 American outdoor apparel/sport goods specializing in sportswear and sports equipment for alternative sports such as wind surf, … Hood River, Ore. (June 17, 2013) – After two decades in downtown Hood River, Ore., DAKINE is proud to announce the move of its headquarters to a new waterfront development at the city’s port. Reviews (541) 386-3166 Website. That's a tune Hood River residents are happy to hear Dakine executives humming. Menu & Reservations Make Reservations . $65. See reviews, photos, directions, phone numbers and more for Dakine locations in Hood River, OR. Marquee Brands LLC has announced that it has acquired the Dakine brand. Dakine, Inc. is located in Hood River, OR, United States and is part of the Sporting Goods Stores Industry. Hood River, OR 97031 dakine.com: Note: Revenues for privately held companies are statistical evaluations. $65. A free inside look at company reviews and salaries posted anonymously by employees. DAKINE, Hood River, OR. This is the Dakine company profile. DAKINE Opens New Waterfront Headquarters in Hood River, Ore. Press Release from Dakine. David Orr, the GM of Dakine Equipment and longtime Oregon resident, is departing the company as it prepares to move from Hood River, Oregon, to Torrance, Calif. Dakine Equipment is owned by Dakine licensee JR286 and comprises the part of Dakine’s business that the industry is familiar with – … Buy Now. Hood in Hood River, Oregon, Dakine builds backpacks, travel bags, accessories, outerwear and clothing for people who love to surf, snowboard, skateboard, mountain bike, ski, windsurf, kiteboard and travel. Olukai ‘Ohana. COVID-19 continues to literally take the wind out of the Gorge economy. Doug's Hood River. Doug's Hood River. After two decades in downtown Hood River, Ore., DAKINE is proud to announce the move of its headquarters to a new waterfront development at the city’s port. Doug's Hood River Dakine Hip Pack. Dakine makes equipment to get you from the water, to the mountain and every journey in between. Email a Purchase Request. V průběhu 90. let rozšířil svou nabídku o první modely zimních rukavic. Glassdoor gives you an inside look at what it's like to work at Dakine, including salaries, reviews, office photos, and more. Dakine 603 Portway Ave Hood River OR 97031. Get reviews, hours, directions, coupons and more for Dakine at 603 Portway Ave # 101, Hood River, OR 97031. 205K likes. Other products by Doug's Hood River. V roce 1986 DaKine otevřel svoji první severoamerickou pobočku (včetně výroby a skladu) v Hood River v Oregonu, kde má dodnes svoji základnu. Search for other Sporting Goods in Hood River on The Real Yellow Pages®. Founded on Maui’s North Shore in 1979 and currently based in Hood River, Oregon, Dakine is an outdoor sports and lifestyle brand offering snow, surf and biking bags, backpacks, gear and accessories to outdoor enthusiasts. Conveniently located between the base of Mt. All content is posted anonymously by employees working at Dakine. Adblock Detected. Get ratings, reviews, hours, phone numbers, and directions. 5 Dakine reviews in Hood River, OR.
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domain: geography