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IMPORTANCE Bacteria are ubiquitously found as surface-attached communities and cellular aggregates in nature. Here, we address how bacterial adhesion is coordinated in response to diverse environments using two complementary approaches. First, we examined how Rhodopseudomonas palustris, one of the most metabolically versatile organisms ever described, varies its adhesion to surfaces in response to different environmental conditions. We identified critical genes for the production of a unipolar polysaccharide (UPP) and showed that UPP is important for adhesion when light and organic substrates are used for growth. Looking beyond R. palustris, we performed the most comprehensive survey to date on the conservation of UPP biosynthesis genes among a group of closely related bacteria that occupy diverse niches. Our findings suggest that UPP is important for free-living and plant-associated lifestyles but dispensable for animal pathogens. Additionally, we propose guidelines for classifying the adhesins produced by various Alphaprotebacteria, facilitating future functional and comparative studies.
Diverse bacteria produce cell surface adhesins that facilitate attachment to biotic and abiotic surfaces (1, 2). Some of the earliest observations of bacterial adhesion reported bacterial “stars,” later termed rosettes, in which cells aggregate by attaching to each other at a single pole (3). Similarly, initial observations of bacterial adhesion to abiotic surfaces also noted polar attachment (4). It has since been recognized that the same polar adhesins responsible for rosette formation in many alphaproteobacterial species also mediate irreversible attachment to surfaces and thereby act to initiate the formation of surface-associated communities known as biofilms (1, 5, 6).
Polar surface attachment in Alphaprotebacteria has been most well studied in the freshwater bacterium Caulobacter crescentus (5, 7–10) and more recently in the plant pathogen Agrobacterium tumefaciens (11–13). The polar adhesin of C. crescentus and other members of the order Caulobacterales is called holdfast (1, 14). The polar adhesin of A. tumefaciens has been termed unipolar polysaccharide (UPP) (11). These two unipolar adhesins are distinct but share certain genetic, biochemical, and functional characteristics (1, 11). The synthesis of both adhesins involves a Wzy-dependent polysaccharide synthesis and export pathway. For holdfast, this pathway is encoded by the holdfast synthesis (hfs) gene cluster (8, 15). For UPP, the pathway is partially encoded by the core upp biosynthesis gene cluster, with other components encoded separately in the genome (11). The hfsEFGHCBAD and uppABCDEF gene clusters each have distinct organization and content (i.e., synteny) (Fig. S1). Only hfsD and hfsE have close sequence similarity to uppC and uppE, respectively (Table S1), although other genes likely encode functionally analogous proteins between these two gene clusters. A contrasting feature of these two adhesins is that holdfast-mediated adhesion requires proteins encoded by the holdfast anchor (hfa) operon, which keeps holdfast attached to the cell (16). No apparent homologs of hfa genes are encoded by A. tumefaciens (11) or most other Rhizobiales species (Data Set S1). Holdfast and UPP also exhibit some biochemical similarity, as both contain N-acetylglucosamine (7, 11), allowing the adhesins to be visualized by fluorescence microscopy after staining with the fluorophore-conjugated wheat germ agglutinin (5, 17).
Beyond C. crescentus and A. tumefaciens, polar polysaccharide adhesins are also a common morphological trait across ecologically diverse Alphaprotebacteria (1, 14, 18), especially among Rhizobiales species (19–25). However, the genetic and biochemical diversity of the adhesins across this clade is unclear. Furthermore, the potential environment-specific production and/or function of these adhesins remain largely unexplored. Here, we examine polar adhesin production by the Rhizobiales member Rhodopseudomonas palustris. This purple nonsulfur bacterium was first reported to produce a polar adhesin almost 50 years ago (26), but the genes involved in its biosynthesis were never characterized. Additionally, R. palustris is renowned for its metabolic versatility (27), a feature that allowed us to investigate if adhesin production is coordinated by different metabolic modules. We show that the putative R. palustris uppE (RPA2750) and uppC (RPA4833) orthologs are required for synthesis of a UPP adhesin. UPP is differentially required for R. palustris biofilm formation under various conditions but is particularly influential under photoheterotrophic conditions, in which light energy and organic substrates are used to support growth. Moving beyond R. palustris, we also explored whether UPP is associated with different bacterial lifestyles by performing a comparative genomic analysis across diverse Rhizobiales species. Our results indicate that UPP is a conserved ancestral trait of the Rhizobiales, and that upp genes have been independently lost multiple times during the evolution of the Rhizobiales clade. Based on our analysis, we propose that genetic synteny of adhesion biosynthesis genes is a valid criterion on which to designate the polar adhesins of various Rhizobiales members as UPP.
Genomic organization of the putative R. palustris CGA009 core upp gene cluster.R. palustris has long been known to form rosettes (26, 28); however, the genetic loci responsible for polar adhesin biosynthesis remain uncharacterized. Recently, bioinformatic analysis revealed that R. palustris contains a putative upp gene cluster (23). Such clusters have been shown to function in UPP production in other Rhizobiales species (12, 24). We confirmed that R. palustris CGA009 encodes a putative upp gene cluster using a TBLASTN reciprocal best-hit approach with the A. tumefaciens C58 UppABCDEF proteins as query sequences. We identified four adjacent genes in R. palustris with close identity to A. tumefaciens uppABDE (Fig. 1A and Table S2). The candidate orthologs for both uppC (RPA4833) and uppF (RPA4581) are outside the putative R. palustris uppABDE cluster (RPA2753 to RPA2750) (Fig. 1A). As expected based on species relatedness, the synteny of the putative R. palustris upp gene cluster is more similar to that of the A. tumefaciens upp gene cluster than to the C. crescentus hfs gene cluster (Fig. S1). We did not identify any candidate hfa homologs in R. palustris (Data Set S1), which are required for holdfast anchoring in C. crescentus and which are similarly absent in A. tumefaciens (11).
Synteny of A. tumefaciens C58 and R. palustris CGA009 core upp gene clusters and proposed protein functions. (A) Genes (arrows) are colored based on functional prediction and sequence similarity (>50% query cover, >25% identity, >40% positives, and an E value <1 × 10−20). Double slashes represent large (>100 kb) genomic regions not shown in the figure. (B) Model of the proposed Wzy-dependent synthesis and export pathway for UPP based on references 15, 29, and 30). Rhizobiales core upp gene clusters lack an important Wzx-like flippase (white), which is contained elsewhere in the genome. Gray hexagons represent repeat saccharide units of the UPP. IM, inner membrane; PG, peptidoglycan; OM, outer membrane; PCP, polysaccharide copolymerase; OPX, outer membrane polysaccharide export; PHPT, polyisoprenyl-phosphate hexose-1-phosphate transferase.
The putative R. palustris uppABDE, uppC, and uppF genes are predicted to encode a partial Wzy-dependent polysaccharide export pathway (Fig. 1B). Wzy-dependent pathways are broadly distributed across Gram-negative bacteria (29) and have been most well characterized in lipopolysaccharide and capsular polysaccharide biosynthesis and export in E. coli (30). We propose a Wzy-dependent model for UPP synthesis and export based on the current understanding of Wzy-dependent pathways (Fig. 1B), similar to what has been proposed for Wzy-dependent holdfast production (15). Briefly, an iterative multienzyme process assembles repeat saccharide units on the inner membrane (IM)-associated lipid carrier undecaprenyl phosphate (und-PP). The assembly is then translocated across the IM and into the periplasm, where the repeat saccharide units are transferred from und-PP to add to the growing polysaccharide chain on another und-PP carrier. Ultimately, the polysaccharide chain is exported onto the cell surface (Fig. 1B). It should be noted that for UPP, certain enzymes thought to be required for synthesis are encoded outside the core upp cluster, such as a flippase (Fig. 1B, white), which is responsible for translocation across the IM. This genetic arrangement is distinct from that of C. crescentus and most other Caulobacterales species, which encode putative Wzx-like flippases (HfsF) in their hfs gene clusters (Fig. S1) (15, 17, 31).
Visualization of R. palustris unipolar adhesin.To facilitate genetic and phenotypic characterization of the R. palustris adhesin, we first tested if we could visualize adhesin on wild-type (WT) R. palustris cells using the fluorophore-conjugated lectin WGA-488. Adhesins produced by diverse Alphaprotebacteria have been shown to bind WGA (5, 7, 32), which itself binds N-acetylglucosamine residues. When we stained R. palustris with WGA-488, we observed fluorescence at single poles of some individual cells and at the center of every rosette (Fig. 2A). WGA-488 can potentially stain N-acetylglucosamine present in peptidoglycan if the outer membrane is compromised, but we seldom observed nonpolar cell body staining under standard photoheterotrophic conditions. From this, we conclude that we are indeed staining the unipolar adhesin produced by R. palustris, which contains N-acetylglucosamine, similar to the UPP of other Rhizobiales species (11, 23, 24), as well as Caulobacterales holdfast (7, 17).
uppE and uppC are required for UPP biosynthesis, cell-cell adhesion, and biofilm formation. (A) Epifluorescence microscopy of cells stained with WGA-488 after 2 days of growth under standard photoheterotrophic conditions. Scale bars, 5 μm. (B) Normalized total WGA-488 fluorescence from batch UPP quantification following 3 days of growth under standard photoheterotrophic conditions. Different letters indicate significant differences between strains (P < 0.05; one-way analysis of variance [ANOVA] followed by Tukey's multiple-comparison test; n = 4 to 5). (C) Biofilm formation levels (A570) were quantified by CV staining of adherent biomass following 4 days of growth in microtiter wells under standard photoheterotrophic conditions. All strains grew equivalently, so A570 values were not normalized. Different letters indicate significant differences between strains (P < 0.0001; one-way ANOVA followed by Tukey's multiple-comparison test; n = 10 or 15, pooled from three independent experiments). (B and C) Symbols indicate biological replicates and lines indicate the means. Time (t) of sampling following inoculation is indicated in lower left corner.
UppE and UppC are required for R. palustris UPP biosynthesis, cell-cell adhesion, and biofilm formation.We next addressed the genetic requirements underlying polar adhesin production in R. palustris. In A. tumefaciens, uppE (12, 13) and uppC (C. Fuqua, personal communication) are essential for UPP biosynthesis. Similarly, the uppE ortholog (gmsA) of the root-nodulating symbiont Rhizobium leguminosarum is necessary for root hair attachment (20). In C. crescentus, the putative uppC homolog hfsD is required for holdfast-mediated attachment (8). Thus, we chose the putative uppE and uppC orthologs of R. palustris as targets for in-frame deletions to determine whether they are required for adhesin synthesis.
Deletion of either the putative uppE or uppC ortholog eliminated both rosette formation and WGA-488 binding (Fig. 2A). Complementation of each mutant from a plasmid restored rosette formation as well as unipolar WGA-488 binding to single cells and at the center of rosettes (Fig. 2A). In addition to microscopic visualization of the adhesin on cells, we also developed an assay to quantify adhesin production at the population level by measuring total WGA-488 fluorescence in batch culture samples. Similar to trends observed by microscopy, total WGA-488 fluorescence was significantly lower in the putative ΔuppE or ΔuppC mutant cultures than in the WT and complemented cultures (Fig. 2B). Overall, these results demonstrate an essential role for both of these orthologs in adhesin production in R. palustris. Based on these results, we henceforth refer to these genes as uppE and uppC and to the R. palustris unipolar adhesin as UPP.
Having established that uppE and uppC are critical for R. palustris UPP synthesis and rosette formation, we next assessed if R. palustris UPP contributes to biofilm formation. After 4 days of standard photoheterotrophic growth, the ΔuppE and ΔuppC mutants showed significantly less biofilm formation than the WT and complemented strains (Fig. 2C). Thus, we conclude that UPP is the primary adhesin facilitating biofilm formation under standard photoheterotrophic conditions.
Survey of UPP-mediated biofilm formation across environmental conditions.R. palustris is metabolically versatile, allowing it to adopt distinct lifestyles to thrive under diverse conditions. When growing anaerobically in light, R. palustris performs anoxygenic photosynthesis to transform energy (27). During phototrophic growth, R. palustris can obtain carbon by consuming organic substrates (photoheterotrophy) or by fixing CO2 (photoautotrophy) (27). It can also grow by aerobic respiration in the dark (chemoheterotrophy). Additionally, R. palustris is a diazotroph, meaning it can grow with N2 gas as the sole nitrogen source by the process of N2 fixation (33). While R. palustris has almost exclusively been studied under freshwater conditions, it was recently noted that an environmental isolate could grow in salt concentrations of up to 4.5% (34).
The metabolic versatility of R. palustris provided an opportunity to assess whether UPP-mediated surface attachment and biofilm formation are favored by some growth conditions over others. To address this, we examined UPP production and biofilm formation under various growth conditions for both WT R. palustris and the ΔuppE mutant. We proceeded with only the ΔuppE mutant because we did not observe any phenotypic differences between the ΔuppE and ΔuppC mutants (Fig. 2). We chose growth conditions that encompass both the metabolic capabilities of R. palustris (e.g., N2 fixation and photoautotrophy) and abiotic conditions it might normally encounter (e.g., low inorganic phosphate [Pi] and high salinity). Total WGA-488 fluorescence values were not compared across conditions, as they were not always reflective of UPP synthesis. For example, some growth conditions, such as low Pi, resulted in occasional staining at both poles and at what appeared to be cell division septa, suggesting that WGA-488 was staining N-acetylglucosamine moieties in peptidoglycan (Fig. S2).
UPP-assisted biofilm formation is favored by R. palustris in adverse photoheterotrophic environments.We first examined if biofilm formation was stimulated or inhibited in response to three adverse photoheterotrophic conditions. These conditions are considered to be less favorable for R. palustris growth due to nutrient limitation (low Pi), less-preferred nutrients (N2 fixation), or osmotic stress (high salinity). Thus, we used these conditions to assess whether biofilm formation might function to increase R. palustris survival under suboptimal conditions or to foster persistence in favorable environments (2, 35). We also examined if UPP is utilized by R. palustris across these growth conditions. Two main trends were observed under all three adverse conditions. First, WT R. palustris formed more biofilm under all adverse conditions than standard photoheterotrophic conditions (Fig. 3A), even though standard conditions supported the highest growth rates and highest cell densities (data not shown). Second, UPP contributed to biofilm formation under all photoheterotrophic conditions, as the WT formed more biofilm than the ΔuppE mutant in each case (Fig. 3A). These biofilm trends were consistent with microscopy results, which showed that WT R. palustris exhibited comparable WGA staining patterns under standard and adverse photoheterotrophic conditions (Fig. 3B). Beyond this, there were also condition-specific phenotypes observed.
UPP is important for biofilm formation across photoheterotrophic conditions. (A) Biofilm formation levels were normalized to final planktonic cell density (A570/OD660) and then made relative to normalized WT standard photoheterotrophic values, which was set to 1. **, P < 0.01; ****, P < 0.0001; ns, not significant; based on multiple unpaired, two-tailed t tests without assuming equal variance and followed by Holm-Šídák correction for multiple comparisons; n = 10, pooled from two independent experiments. Significance is only indicated for pairwise comparisons between WT and the ΔuppE mutant under each condition because the assumption of homogeneity of variances was violated in comparisons across conditions. Results from other statistical analyses comparing across conditions are listed in Table S3. (B) Epifluorescence microscopy of cells stained with WGA-488 after 3 days of photoheterotrophic or chemoheterotrophic growth and after 8 days of photoautotrophic growth. Scale bars, 5 μm. (C) Biofilm formation normalized to final planktonic cell density (A570/OD660) following 4 days of photoheterotrophic growth with 1.5% sea salts or 1.5% NaCl. Different letters indicate significant differences between groups (P < 0.05; two-way ANOVA followed by Tukey's multiple-comparison test; n = 5). (D) Relative biofilm formation (A570/OD660) after 10 days of photoheterotrophic or photoautotrophic growth, with WT values under standard photoheterotrophic conditions set to 1. Different letters indicate statistically significant differences between groups (P < 0.05; two-way ANOVA followed by Tukey's multiple-comparison test; n = 4 to 5). (A, C, and D) Symbols indicate biological replicates, and lines indicate the means. Time (t) of sampling following inoculation is indicated in lower left corner.
Under low-Pi conditions, the ΔuppE mutant formed loosely attached lawns at the bottom of microtiter wells. These lawns were easily disrupted and washed away. Such lawns were not formed by the ΔuppE mutant under standard conditions and were unlike all WT photoheterotrophic biofilms, which were firmly attached to the sides and bottom of the wells. The genetic and biochemical basis for these loose biofilms remains to be determined. Stimulation of biofilm formation in response to Pi limitation has also been observed in A. tumefaciens (12, 36). This common observation raises the possibility that increased biofilm formation is a conserved response to Pi limitation across some Rhizobiales species. It has been speculated that low Pi serves as a signal to A. tumefaciens that plant surfaces are nearby, as plants sequester Pi, locally depleting it from the rhizosphere (36). Given that no symbiotic association between R. palustris and plants has been identified, it is possible that biofilm formation serves a different function in this species, such as increasing survival when essential nutrients, such as Pi, are limiting.
We also observed 2-fold higher biofilm levels by WT under N2-fixing conditions than under standard conditions (Fig. 3A). N2 fixation is energetically expensive compared to using other nitrogen sources, such as NH4+, and is therefore tightly regulated (33, 37). We hypothesize that increased aggregation under N2-fixing conditions might function to help retain costly NH4+, which can passively diffuse out of cells as NH3 (38).
In contrast to all other photoheterotrophic conditions, ΔuppE mutant biofilm levels were 13-fold higher under 1.5% sea salt conditions than WT cells under standard conditions, despite lacking UPP (Fig. 3A). Similar trends were seen with 1.5% NaCl, confirming that the enhanced biofilm formation of both the WT and the ΔuppE mutant was due to high salinity and not another component of the sea salt supplement (Fig. 3C). The high ΔuppE mutant biofilm levels under high-salinity conditions suggest that additional factors besides UPP contribute to this response. Thus, while UPP-mediated surface attachment contributes to robust biofilm formation by R. palustris during photoheterotrophic growth, UPP is less crucial under high-salinity conditions.
UPP-independent biofilm formation is stimulated by nonphotoheterotrophic conditions.We also examined UPP production and biofilm formation under chemoheterotrophic and photoautotrophic conditions. Under chemoheterotrophic conditions, UPP was not necessary for biofilm formation, as WT and the ΔuppE mutant formed similar levels of biofilm. We were surprised by this result, as it suggested that biofilm formation was entirely UPP independent. Aerobically grown bacteria typically adhere near the air-liquid interface (39). However, the adherent biomass of both the WT and the ΔuppE aerobic biofilms was at the bottom of the microtiter well, suggesting that R. palustris might preferentially form biofilms at microaerobic or anaerobic zones. In support of this, the adherent biomass was pigmented, indicating the production of bacteriochlorophyll and carotenoids, which is stimulated in response to low O2 (40). Additionally, chemoheterotrophic conditions seem to favor biofilm formation, as WT and ΔuppE biofilm levels were approximately 12-fold higher than those of the WT under standard photoheterotrophic conditions. (Fig. 3A). Separately, although WGA-488 staining was observed on some single cells, we did not observe any rosettes under chemoheterotrophic conditions (Fig. 3B). It is therefore possible that UPP is produced but is dispensable for chemoheterotrophic biofilm formation.
During photoautotrophy with sodium bicarbonate as the carbon source and thiosulfate as an electron donor, R. palustris has a specific growth rate approximately one-fourth that of during photoheterotrophic growth (41, 42). Because of the slower growth, we extended photoautotrophic incubations from 3 days for epifluorescence microscopy and 4 days for biofilm assay to 8 days and 10 days, respectively, in order to allow cultures to reach final densities similar to those observed after 3 to 4 days of photoheterotrophic growth. After 8 days of photoautotrophic growth, we observed WT rosettes that stained very little or not at all with WGA-488, suggesting that less UPP is produced or that UPP composition is different under these conditions (Fig. 3B). Under photoautotrophic conditions, the WT and the ΔuppE mutant showed similar levels of biofilm formation (Fig. 3D), suggesting that biofilm formation was UPP independent. Similar trends were seen in parallel control cultures for which we allowed for 10 days of photoheterotrophic growth (Fig. 3D), unlike results from the 4-day photoheterotrophic experiments, where the ΔuppE mutant formed less biofilm than WT (Fig. 3A). There are multiple nonmutually exclusive explanations for why the difference in biofilm formation between the WT and the ΔuppE mutant after 4 days was not also observed after 10 days. Because UPP is thought to mediate the initial irreversible surface attachment of cells (5), prolonging the incubation period may have led to some degradation of UPP and/or might have allowed sufficient time for as-of-yet unknown adhesins or other factors, such as DNA release following cell lysis, to facilitate attachment. Such factors could also contribute to the increased biofilm formation observed across the different conditions tested herein.
Overall, our survey of R. palustris biofilm formation across growth conditions can be summarized as follows. UPP mediates biofilm formation under photoheterotrophic conditions, especially those photoheterotrophic conditions that are less favorable to growth. Under certain photoheterotrophic conditions, such as high salinity, biofilm formation involves additional factors that are independent of UPP. Finally, chemoheterotrophic and photoautotrophic conditions also stimulate biofilm formation but in a manner that appears to be independent of UPP.
Conservation of core upp biosynthesis genes across Rhizobiales species.Beyond C. crescentus, R. leguminosarum, A. tumefaciens, and now R. palustris, the characterization of polar adhesins in other Alphaprotebacteria has been cursory. Historically, all polar adhesins were referred to as holdfast. However, designation of alphaproteobacterial adhesins has been complicated by functional differences. For example, the polar glucomannan adhesin of R. leguminosarum plays a unique role in root hair attachment but is not required for attachment to abiotic surfaces (19, 20). The R. leguminosarum glucomannan biosynthesis gene cluster is orthologous to the A. tumefaciens uppABCDEF cluster, which A. tumefaciens uses to attach to both biotic and abiotic surfaces (5, 11, 12). Thus, R. leguminosarum polar glucomannan and A. tumefaciens UPP are homologous adhesins with functional differences. Also contributing to the ambiguity in classifying previously identified Rhizobiales polar adhesins is the compositional diversity (1, 12, 20–22). For example, A. tumefaciens UPP contains N-acetylgalactosamine in addition to N-acetylglucosamine (12), the R. leguminosarum glucomannan adhesin contains primarily glucose and mannose (19), the Bradyrhizobium japonicum polar adhesin contains galactose and lactose (22), and the Hyphomicrobium polar adhesin likely contains galactose and mannose (21). We therefore propose that alphaproteobacterial adhesins be classified according to genetic synteny. Based on the synteny (Fig. 1) and functional requirement of upp orthologs for adhesin production (Fig. 2), we conclude that R. palustris produces UPP.
With the criterion of genetic synteny in mind, we explored the phylogenetic distribution and genomic organization of the core uppABCDEF orthologs across 22 Rhizobiales species, representing the lifestyle diversity of this clade (Fig. 4). The topology of this tree is largely consistent with the alphaproteobacterial phylogeny inferred from a concatenation of 104 protein alignments (43). Our analysis revealed broad conservation of putative upp gene clusters, indicating that UPP is an ancestral trait of the Rhizobiales clade. Almost all of the Rhizobiales plant symbionts, including the plant pathogen A. tumefaciens, the root-nodulating diazotrophs R. leguminosarum, S. meliloti, Mesorhizobium loti, and B. japonicum, the stem-nodulating photosynthetic diazotroph Bradyrhizobium sp. strain BTAi, and the leaf epiphyte Methylobacterium extorquens, contain complete or near-complete upp gene clusters (Fig. 4). The exception to this trend is the root-nodulating diazotroph Azorhizobium caulinodans (44), which does not contain a upp cluster (Fig. 4 and Data Set S2). We were also unable to identify a upp cluster in Xanthobacter autotrophicus, a free-living diazotroph closely related to A. caulinodans (Fig. 4). This absence suggests that the upp cluster was lost before these lineages split. Despite the absence of a upp cluster in A. caulinodans, it still appears to produce a polar adhesin and form rosettes (25). Upon closer examination of the A. caulinodans ORS571 genome, we identified a putative Wzy-like polysaccharide biosynthesis gene cluster with high similarity to the Vibrio fischeri symbiosis polysaccharide (syp) locus (Data Set S3) (45). These putative syp homologs seem to have been acquired horizontally and might have been coopted for polar polysaccharide synthesis in A. caulinodans.
Conservation of core UPP biosynthesis genes among Rhizobiales species. A maximum likelihood phylogeny was inferred based on a concatenated alignment of 6 conserved housekeeping proteins using an LG+G+I substitution model (58) with four discrete gamma categories and invariable sites in MEGA6 (56). The tree with the highest log likelihood is shown. Node values indicate branch support from 100 bootstrap replicates. Scale bar represents the number of substitutions per site along branches. Leaf and mouse symbols indicate known plant and animal symbiotic relationships, respectively.
While UPP is well conserved in plant-associating Rhizobiales species, the opposite is true for animal pathogens. This trend was first noted upon the initial discovery of the upp gene cluster in R. leguminosarum, which noted that this cluster is absent in the Rhizobiales intracellular mammalian pathogen Brucella melitensis (20). Rather than being entirely absent (20), our data corroborate more recent bioinformatic evidence (23) that Brucella spp. contain a cluster of 3 putative upp orthologs (uppBCE) (Fig. 4 and S1, and Data Set S2). It is not known whether this partial upp cluster is involved in the synthesis of a functional UPP. In the closely related intracellular animal pathogens of the genera Bartonella, upp orthologs are completely absent (Fig. 4 and Data Set S2). In contrast, the soil-dwelling opportunistic human pathogen Ochrobactrum anthropi (46), which is more closely related to Brucella than Bartonella, contains a complete uppABCDEF gene cluster (Fig. 4). Ochrobactrum spp. are thought to be rhizosphere community members but are capable of infecting animal hosts (46, 47). We hypothesize that the entire upp cluster was first lost in the Bartonella lineage during adaptation to an intracellular lifestyle after diverging from Brucella/Ochrobactrum. More recently, the Brucella lineage has similarly lost multiple upp orthologs during its transition to becoming intracellular pathogens. The independent loss of upp orthologs in both Bartonella and Brucella would suggest convergent evolution upon adaptation to intracellular niches within animal hosts, supporting the hypothesis that UPP is not important for such lifestyles. Conversely, the conservation of upp orthologs in plant symbionts and free-living species suggests that UPP is beneficial in other diverse environments. Considering this, we hypothesize that Ochrobactrum anthropi has retained the complete upp cluster because it is typically free-living in the soil and thus benefits from producing UPP.
Unipolar adhesins are also used by Alphaprotebacteria outside the Rhizobiales. In the order Caulobacterales, hfs and hfa gene clusters for holdfast synthesis are well conserved (17, 31). Despite the differences in synteny between the upp and hfs gene clusters (Fig. S1), both encode Wzy-dependent pathways for polar polysaccharide synthesis and export, and uppC and uppE show sequence similarity to hfsD and hfsE, respectively (Table S1). Because of these similarities, we hypothesize that holdfast and UPP share an evolutionary origin and that the upp and hfs loci diversified in genomic organization following divergence of the Rhizobiales and Caulobacterales clades.
Other alphaproteobacterial species of the marine Roseobacter clade within the order Rhodobacterales also produce polar adhesins and form rosettes but do not contain either upp or hfs or hfa homologs (18) (Data Sets S1 and S2). The polar polysaccharide adhesin of the Roseobacter species Phaeobacter inhibens contains N-acetylglucosamine based on WGA binding, indicating that the biochemical composition is at least somewhat similar to that of UPP and holdfast (32). In this case, polar adhesion synthesis is encoded on a plasmid, since plasmid curing prevented P. inhibens rosette formation and diminished attachment to abiotic surfaces and algal cells (6). Furthermore, genetic disruption of the plasmid-encoded putative rhamnose operon lowered biofilm formation (48). Plasmids encoding putative rhamnose operons are widely distributed among other Roseobacter species (48), suggesting that polar polysaccharide synthesis and export in this clade are genetically distinct from those of UPP and holdfast. It is not clear whether acquisition of these plasmids led to the loss of gene clusters similar to either upp or hfs loci.
While polar polysaccharide adhesins are a common morphological trait across ecologically diverse Alphaprotebacteria, there is considerable genetic, compositional, and functional variation, which likely reflects adaptation to different niches. We propose here that genetic synteny of biosynthetic loci is a suitable criterion on which to base classification of polar adhesins. This criterion bypasses uncertainty arising from compositional differences while highlighting the shared underlying biosynthetic pathway. As such, holdfast and UPP are distinct adhesins despite facile similarities. Likewise, the A. caulinodans adhesin and the Roseobacter rhamnose adhesins should each receive their own designation, as they are genetically distinct from both holdfast and UPP, as well as from each other. Adoption of a unified classification scheme will facilitate both the comparison of adhesins and the exploration of functional differences within and between adhesin types.
Bacterial strains and growth conditions.All R. palustris strains were derived from CGA009 (27) and are listed in Table 1. Unless otherwise indicated, R. palustris was grown statically in 10 ml of defined photosynthetic medium (PM) (49) in sealed 16-ml anaerobic tubes with argon gas in the headspace. All R. palustris cultures were incubated at 30°C. All phototrophic cultures were illuminated with a 60-W light bulb. For all heterotrophic conditions, PM was supplemented with succinate as the sole carbon source (15 mM in liquid cultures or 10 mM in agar). Incubation in PM with 15 mM succinate and light are here referred to as standard photoheterotrophic conditions. For low-inorganic phosphate (Pi) conditions, PM was modified by replacing Na2HPO4 and KH2PO4 (12.5 mM each) with equimolar concentrations of Na2SO4 and K2SO4. A 1:1 molar mixture of Na2HPO4 and KH2PO4 was added for a final Pi concentration of 30 μM. For N2-fixing conditions, (NH4)2SO4 was omitted from PM, and argon was replaced with N2. For high-salinity conditions, PM was supplemented with 1.5% (wt/vol) sea salts (Sigma) or NaCl. For chemoheterotrophic conditions, cultures were grown in 10 ml of aerobic PM supplemented with 0.05% yeast extract in addition to 15 mM succinate in 50-ml Erlenmeyer flasks shaken at 225 rpm in darkness. For photoautotrophic conditions, anaerobic PM was supplemented with 60 mM NaHCO3 as the inorganic carbon source and 30 mM Na2S2O3 as an inorganic electron donor. Plasmid-harboring R. palustris strains were grown with 50 μg/ml gentamicin in liquid culture and 100 μg/ml gentamicin on agar plates. The Escherichia coli strains used for cloning (DH5-α and S17-1) were grown aerobically in Luria-Bertani medium (BD) supplemented with 15 μg/ml gentamicin when necessary.
R. palustris strain construction.All plasmids and primers are listed in Tables 1 and 2, respectively. Deletion vectors for uppC (RPA4833) and uppE (RPA2750) were generated by PCR amplification of the genomic regions flanking the gene to be deleted, as described previously (41). PCR product pairs were fused by overlap extension PCR and cloned into pJQ200SK (50). Vectors were introduced into R. palustris by conjugation with E. coli S17-1 (51) or by electroporation (52). Complementation vectors for uppC and uppE were generated by PCR amplification of each gene along with the putative ribosomal binding site. PCR products were cloned into pBBPgdh (53), and complementation and empty pBBPgdh vectors were introduced into R. palustris by conjugation with E. coli S17-1.
Epifluorescence microscopy and image analysis.Unless stated otherwise, R. palustris cultures used for microscopy were grown in liquid without agitation for 2 to 3 days, except for photoautotrophic cultures, which were grown for 8 days. Culture samples were centrifuged, and the cell pellet was resuspended in 0.1 mM phosphate-buffered saline (PBS) (Fisher Scientific) to an optical density at 660 nm (OD660) between 0.6 and 0.9. Wheat germ agglutinin Alexa Fluor 488 conjugate (WGA-488) (Molecular Probes) was added to cells suspended in PBS at a final concentration of 2 μg/ml and incubated in darkness at room temperature for 15 min. Cells were washed with PBS three times to remove unbound dye and then resuspended in PBS. Cells were imaged on agarose pads using a Nikon Eclipse 90i light microscope equipped with a 100× oil immersion objective and a Photometrics Cascade 1K electron-multiplying charge-coupled-device (EMCCD) camera and processed using the Nikon NIS-Elements software. Images were subsequently analyzed using the ImageJ distribution Fiji (54).
Batch UPP quantification via total WGA fluorescence.R. palustris cultures were grown under standard photoheterotrophic conditions for 3 days to early stationary phase. A 400-μl sample of each culture was centrifuged, and the cell pellet was resuspended in 400 μl of PBS. A 100-μl aliquot of each cell suspension was set aside for use as the unstained control. WGA-488 was added to the remaining 300 μl of resuspended cells to a final concentration of 1.5 μg/ml and incubated in darkness at room temperature for 15 min. WGA-488-stained cells were washed three times with PBS and then resuspended in 120 μl of PBS to account for cells lost during washes. A 100-μl aliquot of the stained cells and the reserved unstained samples were each transferred to empty wells of a black polystyrene 96-well μClear flat-bottom microtiter plate (Greiner Bio-One). Fluorescence (top-120, excitation 485/20; emission 528/20) and the OD660 were measured using a Synergy H1 microplate reader (BioTek). Fluorescence readings were normalized to cell densities (relative fluorescent units [RFU]/OD660), and background fluorescence was removed by subtracting RFU/OD660 values of unstained samples from the WGA-488-stained samples.
Crystal violet microtiter plate biofilm assay.Biofilm formation was quantified using a modified version a crystal violet microtiter plate assay (39). Briefly, starter cultures were grown under standard photoheterotrophic conditions supplemented with 0.1% yeast extract. A 1.5-μl volume of stationary-phase culture was used to inoculate the wells of a lidded untreated polystyrene 24-well plate (Corning) containing 1.5 ml of the specified sterile medium. All plates were incubated statically at 30°C. For anaerobic phototrophic growth conditions, plates were incubated in a BD GasPak EZ container with two EZ anaerobe container system sachets (BD) and illuminated by two 60-W light bulbs, one on either side of the container. For chemoheterotrophic growth, plates were incubated aerobically in darkness. For all heterotrophic growth conditions, plates were incubated for 4 days. For photoautotrophic growth conditions (and paired heterotrophic controls), plates were incubated for 10 days. After incubation, plates were shaken at 150 rpm for 3 min on a flat-bed rotary shaker to disrupt loosely attached cells. A 400-μl aliquot of culture was removed for quantifying cell density (OD660) for normalization. Then, 400 μl of 0.1% (wt/vol) crystal violet (CV) was added to each well, and plates were incubated statically at room temperature for 15 min. Wells were then washed 3 times with 2 ml of deionized water to remove unbound CV. Next, 750 μl of 10% (vol/vol) acetic acid was added to each well, followed by shaking at 150 rpm for 3 min to solubilize bound CV. A 150-μl sample of solubilized CV was transferred to a 96-well plate, and absorbance at 570 nm (A570) was measured. Uninoculated wells containing sterile medium were treated the same way as described above to determine background A570, which was subsequently subtracted from all A570 measurements.
Identification of orthologous core upp gene clusters and phylogenetic analysis.The putative orthologs of the core UPP biosynthesis genes in R. palustris CGA009 (GenBank accession no. BX571963.1) were initially identified by reciprocal best-hit analysis using the UppABCDEF proteins of A. tumefaciens C58 (GenBank accession no. AE007869.2) as the query sequences for a TBLASTN search against the translated nucleotide database of R. palustris CGA009. The best hits in R. palustris CGA009 were subsequently used as query sequences for a BLASTX search against the proteome of A. tumefaciens C58. All putative R. palustris orthologs showed >50% query cover and an E value of <1 × 10−20 (Table S2). Previous studies noted that the core uppABCDEF biosynthesis gene cluster is conserved among several Rhizobiales species (20, 23), which was confirmed by using BLASTP with A. tumefaciens C58 UppABCDEF proteins as query sequences (Data Set S2). Several additional species that contain complete or near-complete upp gene clusters were also identified using BLASTP (minimum threshold for homology of query cover, >50%; E value, <1 × 10−10) (Data Set S2).
For phylogenetic analysis, amino acid sequences for 6 conserved housekeeping proteins, GyrA, GyrB, RpoA, RpoB, FusA, and RecA, from 26 alphaproteobacterial species were individually aligned using MUSCLE (55) with default settings in MEGA6 (56). Gaps and ambiguous sites were removed from alignments using Gblocks (57), with a minimum block length of 10 positions, and gaps were allowed at a position for no more than half of the sequences. The sequences were subsequently concatenated in the order shown above. The final concatenated alignment contained 4,379 amino acid positions (92% of the original positions). Phylogeny was inferred for the concatenated amino acid sequence using the maximum likelihood method based on the Le and Gascuel (LG) model (58), with 4 discrete gamma categories, which allowed for some sites to be evolutionarily invariable, implemented in MEGA6 (56). The LG+G+I model was selected because it was the best-fitting substitution model based on having the lowest Bayesian information criterion score. Initial tree(s) for the heuristic search were obtained by applying the neighbor-joining method to a matrix of pairwise distances estimated using a Jones-Taylor-Thornton model.
Statistical analysis.All statistical analyses were performed using GraphPad Prism version 6.07. Additional information about statistical analyses is in the figure legends. For Fig. 3A, multiple statistical analyses were performed to reach a consensus for comparisons across growth conditions, which are summarized in Table S3.
We thank Yves Brun for use of microscopy facilities and reagents, Clay Fuqua for sharing unpublished data, and members of the Brun and Fuqua labs for thoughtful discussions.
This work was supported in part by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, under award DE-SC0008131, and the Indiana University College of Arts & Sciences.
Supplemental material for this article may be found at https://doi.org/10.1128/AEM.03035-16.
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. 2015. Plasmid curing and the loss of grip–the 65-kb replicon of Phaeobacter inhibens DSM 17395 is required for biofilm formation, motility and the colonization of marine algae. Syst Appl Microbiol 38:120–127. doi:10.1016/j.syapm.2014.12.001.
. 2009. Surface colonization by marine roseobacters: integrating genotype and phenotype. Appl Environ Microbiol 75:6027–6037. doi:10.1128/AEM.01508-09.
. 2006. A novel polar surface polysaccharide from Rhizobium leguminosarum binds host plant lectin. Mol Microbiol 59:1704–1713. doi:10.1111/j.1365-2958.2006.05057.x.
. 2008. Glucomannan-mediated attachment of Rhizobium leguminosarum to pea root hairs is required for competitive nodule infection. J Bacteriol 190:4706–4715. doi:10.1128/JB.01694-07.
. 1981. Attachment and rosette formation by hyphomicrobia. Appl Environ Microbiol 42:751–757.
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. 2016. Short-stalked Prosthecomicrobium hirschii cells have a Caulobacter-like cell cycle. J Bacteriol 198:1149–1159. doi:10.1128/JB.00896-15.
. 2016. Cyclic di-GMP regulates multiple cellular functions in the symbiotic alphaproteobacterium Sinorhizobium meliloti. J Bacteriol 198:521–535. doi:10.1128/JB.00795-15.
. 2011. Involvement of the azorhizobial chromosome partition gene (parA) in the onset of bacteroid differentiation during Sesbania rostrata stem nodule development. Appl Environ Microbiol 77:4371–4382. doi:10.1128/AEM.02327-10.
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. 2004. Phosphorus limitation enhances biofilm formation of the plant pathogen Agrobacterium tumefaciens through the PhoR-PhoB regulatory system. J Bacteriol 186:4492–4501. doi:10.1128/JB.186.14.4492-4501.2004.
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text:
Welcome to Heat Tech Innovations Plumbing & Heating. We do everything from ‘A’ rated boiler changes with 12 year warranties available to full central heating systems, underfloor heating with smart controls, solid fuel stoves including pressurised wood burners, running at 93% efficiency and full bathrooms / wet rooms.
Offering a wide range of services, specialising in boiler breakdowns in Plymouth. Bathroom refurbishments, landlord certification and central heating are also part of our service. Take a look at all the services we provide by clicking the link below.
Helping keep homes and water warmer for customers in Plymouth and surrounding areas, 365 days a year.
SIT BACK, WE CAN DO IT ALL!
Offering a wide range of services, specialising in boiler breakdowns in Plymouth. Bathroom refurbishments, landlord certification and central heating are also part of our service.
We also provide a full plumber service in Plymouth to incorporate wet rooms, boiler changes, full kitchen installs and lots more so if you’re unsure just give us a call and we will be more than happy to give advice or guidance based on your requirements and personal tastes.
The only thing we don’t do is weddings!
See our latest work on facebook and instagram!
Thank you Ross for your efficiency with the installation of our new boiler, especially as you did it all in one day. You & your mate were very professional & we will not hesitate to recommend you. We just had our first gas bill since the new boiler & what a difference it has made. Thanks again.
Many thanks to Ross for sorting out the downstairs WC, some highly professional plumbing and very personable service… highly recommended.
Do you need help with any kind of plumbing and heating issue? Fill out this form to get in touch.

industry_corpus2: Real Estate & Construction


text:
Digital marketers are familiar with in-feed advertising. Facebook and Instagram have ‘sponsored and boosted’ posts where brand messaging can appear in a fan’s Facebook or Instagram news feed. Twitter has a similar model with ‘promoted tweets’. More recently, LinkedIn and Pinterest launched similar advertising formats too.
Back in 2015, Tencent’s trailblazing app WeChat launched WeChat Moments Advertising — but with limited functionality and huge minimum spends for Western advertisers and brands. However, in April of 2019, this all changed and WeChat Moments Advertising is now available for all brands with no minimum spend requirement.
This product has now launched globally. For specifics on how you can advertise on WeChat, download our WeChat Advertising Guide and check out our blog post on “Why Brands Should Care about WeChat Advertising“.
WeChat has achieved tremendous growth since its launch in early 2011. As of November 2014, WeChat’s parent company, Tencent, announced that its user base has grown to 468 million monthly active users. But despite the drastic growth, Tencent has struggled to monetize that user base. A big part of its success was due to their commitment to user experience, in an intimate space tailored to people and not marketers — which has been a strategic move to not inundate their audience with advertising.
From the beginning, WeChat has been cautious with monetization. In July 2014, it launched an advertising service in China that allowed verified WeChat accounts to place ads on other companies’ official pages with more than 100,000 followers. These ads, however, did not appear in users’ newsfeeds.
WeChat has a measured rollout plan in place to minimize the risk of turning off users. The first set of test ads will only be shown to a group of people who WeChat determines to be extremely active in Moments or those who frequently engage with any advertising. Only when these people interact with the test ads, will the ads show up in their friends’ Moments, and so on and so forth. In addition, if users don’t interact with the ads at all, the ad will disappear from Moments within 6 hours. WeChat also has an impression cap on the number of times users would see an ad — a typical user would see one ad every other day.
In the past, brands had limited options when it came to utilizing WeChat as a marketing channel. Other than the advertising feature that launched in July 2014 mentioned above (which was open to a very limited number of accounts), the only other option was to create business pages. However, it was difficult to build a following and have any engagement on the business pages without an amplification strategy. WeChat’s newsfeed ads point out a direction for advertisers to see it as a marketing channel, which they can incorporate as a part of their media plan. So far, brands invited to take part in the test include BMW, Coca-Cola, and Cadillac.
Although Chinese Internet companies were initially regarded as copies of Google, Twitter, and Facebook, they have since evolved into innovative companies with unique business models. WeChat is not simply a Chinese version of WhatsApp; it is an aggregate of several social platforms, with the functionalities of Facebook, Twitter, and chat, and even acts as a walkie-talkie. The app holds a dominant position in the world’s largest smartphone market. WeChat Moments Advertising offering comes as it works to create a platform where users not only send messages to friends, but can also shop, make purchases, play games, ride-sharing, and more without having to leave the app.
With WeChat’s cautious move towards monetization via advertising and its continued emphasis on user experience, high standards for advertisers are expected. Now, the question for advertisers is, what is the best way to make an impression on a platform built on private conversations?
Read the updated WeChat advertising blog post, where we dive into specifics such as the anatomy of WeChat advertising, bidding strategies, and pricing.

industry_corpus2: Computing & Telecommunications


text:
Members of Winnipeg’s gay community are mourning after two people died in a fire at a local bathhouse.
Oct. 13, 2009 1:28 p.m.
WINNIPEG — Members of Winnipeg’s gay community are mourning after two people died in a fire at a local bathhouse.
Steven Yablonski, 23, was asleep when fire broke at the Aquarius Men’s Bath around 7 a.m. Sunday, says his father, Robert.
Winnipeg police are trying to confirm the other man’s identity, said spokeswoman Const. Jacqueline Chaput.
Yablonski’s father said investigators told him the blaze appears accidental and may have been caused by careless smoking.
The blaze broke out on the top floor of the two-storey building. Several patrons and employees escaped as the floor filled with smoke.
Customers said it isn’t uncommon for people to go against the rules — and law — by smoking in rooms at Aquarius, a 24-hour co-ed bathhouse that is exclusive to men three nights a week.
Yablonski, who was openly gay, found an outlet for expressing himself and entertaining at Gio’s Club & Bar and Club 200, where he was a drag queen performer.
“It’s quite devastating for our community,” said Bob Rogers, president of Gio’s.
Yablonski’s stage name, Kerri O’Kee, was a play on the word karaoke, an activity he loved, his friends and family said.
On Oct. 17, Yablonski was to take part in a drag queen competition at Gio’s, but the event has been cancelled and a memorial service will be held.
“He was a great kid. He loved performing and he loved being in front of the camera and on stage,” said his dad.
Yablonski was also a board member of the charity Gio’s Cares, which provides support for people with HIV and AIDS.
As a volunteer, he delivered supplies to HIV/AIDS service organizations in Winnipeg, the charity said in written a statement.

industry_corpus2: Hospitality & Catering


text:
FinTechnology was established by founder and Managing Director, David Williams to work with financial services firms to provide both in house managed technology and an income stream through offering managed services to their clients through them.
Dissatisfied with the disconnect between external advice and internal business goals, David started FinTehcnology with a small team. Their mission: bridge the gap between IT and business, and ensure that business growth and success is the priority in every partnership.
We align everything we do, from the smallest projects to the biggest partnerships, with our core values. We believe that by measuring up to them, you, as our partner, will always receive solutions and advice tailored specifically to your business with guaranteed success.
"We have found FinTechnology to be very beneficial to our business. The staff are very prompt to attend to any issues or concerns. I would happily recommend their service to anyone."
David is the Managing Director of FinTechnology. Along with leading the company, he assists clients with astute business and strategic advice. An IT veteran with over 20 years commercial experience, he served in a number of senior leadership roles, including Group CIO for a manufacturing company, before founding FinTechnology. An expert in applying technology to solve business problems, David takes the time to understand the needs of your business and only suggests solutions that best fit your requirements.
David not only heads FinTechnology, but also is seen working with clients to adapt their technology strategy through the vCIO service to best support their business goals and ensure their ongoing success. David is a current PRINCE2 Practitioner, holds ITIL certification and continues to develop his skills and exposure to new technologies and business methodologies and processes.

industry_corpus2: Finance & Economics


text: ACDelco can help keep your vehicle in optimal running condition. Bring your vehicle to your dealership or trusted independent workshop, where your technician can maintain and repair critical vehicle components and systems. Or, if you prefer to do the work yourself, make sure you use the quality ACDelco auto parts you know and trust.

industry_corpus2: Automobiles


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Sunday, Jan 3, 2016 marks the end of the holiday weekend and the 15/16 holiday season. If you’re like a lot of people you’re feeling a little puffy. Here are easy ways to get rid of that Post Holiday Puffy feeling.
Drink water. Drink lots of water. Much of the holiday puff is from water retention from eating a lot of holiday carbs. All those extra calories coming from sugary treats causes your body to store glycogen in your muscles and liver. Glycogen holds onto water. Oddly, the way to get rid of retained water is to flush it away by drinking more water!
Avoid alcoholic drinks. They do the opposite of water. Your goal is to hydrate, not dehydrate!
Focus on lean protein. Protein is the macronutrient that doesn’t make you retain water or feel puffy. It does the opposite. Lean meat, poultry, and fish are good sources of protein, but if you don’t eat flesh you can also get high-quality protein from legumes.
Snack on fruit and vegetables. Skip the cookies, crackers and cheese.
Get moving. You don’t have to go to the gym if it isn’t your thing, but do get moving doing something you enjoy, preferably, because that helps you move more. Moving stimulates body functions that help reduce puff. In other words, moving helps you poop.
That’s it! Do these 5 steps and in no time you’ll be feeling and looking less puffy!

industry_corpus2: Health & Medicine


text:
A range of jagged mountains thrusting into the splendid desolation of the southern Atlantic, the Cape Peninsula is one of the world's most beautiful places.
Pristine beaches, sheltered bays and secluded coves encircle the majestic might of Table Mountain, a World Heritage Site and home to some 8,500 species of flower. Between the mountain's looming crags and Table Bay's deep blue sweep lies South Africa's Mother City and the cosmopolitan hub of the Western Cape Province - Cape Town.
The best way to revel in the Peninsula's unrivalled beauty is to travel to the Cape of Good Hope Nature Reserve along one of two coastal drives, each dramatically different. To the west, the famous Chapman's Peak route traverses Sea Point's cosmopolitan seaboard and the natural beauty of Clifton's famed beaches, leading one from the quiet rusticity of Hout Bay over the awe-inspring, cliff-hugging Chapman's Peak Drive to the endless, white expanse of Noordhoek's Long Beach. The False Bay road heads north, hugging the mountains, snaking past Fish Hoek, a popular family beach, Kalk Bay, a fishing village imbued with character and colour, and Muizenberg, renowned for its safe swimming and excellent surfing.
Life in the City Bowl centres around the Victoria & Alfred Waterfront, a pulsating waterfront Mecca, historical site and working harbour offering everything from upmarket shopping malls, arts and crafts markets, theatres and live music to entertainment showpieces including the Telkom Exploratorium and the awe-inspiring Two Oceans Aquarium.
From the Waterfront, it's a short boat ride to Robben Island, one of the most significant historical sites in South Africa. First used by the Dutch East India Company (VOC) as a victualling station, it became an asylum and leper colony. Later, it fell under the Army and Navy and, in 1960, under the Department of Correctional Services. It gained notoriety as a prison housing , for more than two decades, former President Nelson Mandela.
Museums and art galleries abound and South Africa's oldest building, the Castle of Good Hope, is in the city centre. The cobbled streets of Bo-Kaap with their delightful old buildings and the first mosque in South Africa stand in cultural contrast to the buildings of the oldest homestead and wine farm in the Cape, Groot Constantia. On the Constantia wine route, Groot Constantia is tied by history and the vine to estates in the Helderberg and Durbanville.
A short drive from the theme parks, malls, cineplexes and entertainment centres of Century City, the energetic, and those wanting to explore the abundance of the Cape Floral Kingdom's fauna and flora, can take to the hills, choosing from myriad walks and climbs: up Table Mountain or Lion's Head, through the Silvermine or Tygerberg Nature Reserves, Newlands Forest, the Cape of Good Hope or Helderberg Nature Reserves, or one of several other inner-city wildlife areas.

industry_corpus2: Travel & Geography


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Selected Productions from Eric Whiptastic below – check out more recent projects HERE!
– Dj faLAYsheOH rayDIO MUMBERS EP – debut ep from drug induced rapper Dj faLAYsheOH rayDIO. This album is multiple minutes of largely improvised raps and Auto Tuned beats (really). The album has multiple appearances of characters from The Mike and Eric Show.
– WEMZ* – Expanded acoustic melodies, songs by Mike Wilson – Recorded and Mixed.
– Ogden* – A time traveler or crazy man, disoriented in the present trying to make sense of his situation. Official Selection – 2011 Royal Flush Film Fest!
– Rumanian Buck – Blindness of the Bat – Music video cut from footage from the band’s debut show and singer Aaron Lazar’s Punk Rock Pillow Fight match.
– Baby Steps McCarthy – Remixed and Remastered album from the unknown legend and true father of rock Anna McCarthy.
– Gay Bar Gone Too Far – A goth man is haunted by the song Gay Bar by Electric Six.
– Chunky Monkey (remix) – Improved visual effects and updated soundtrack.
– Captain Whiptastic Owns You* – going solo – 7 tracks of oddities and awesomeness all performed, mixed and arranged by yours truly (out-of-print).
– Inanimate Girl (band)* – mixed and arranged self titled EP (out-of-print).
– Chunky Monkey* – Acting Awesomeness – A story about 2 teenagers attacked by a stranger. Karate Matrix fighting ensues.
We’ve also filmed live performances for The Giraffes, gONNA gET gOT, Evil Cowards, Consider the Source, Lorrie Doriza, Midnight Foolishness, When Distance Fails and more. Check it out on YouTube.

industry_corpus2: Film & Entertainment


text:
Shopping to buy the latest USB 3.0 port for the computer? The Usb 3. 0 External a great solution! I certainly loved that it had the feature of usb 3. 0 external 2-port, usb 3. 0 19-pin header and molex power connector. Other highlights include low profile bracket included. UPC Number 810154016747. The USB 3.0 port card dimensions are 2"H x 6.75"L x 5.75"W. Have you been considering buying the Usb 3. 0 External and you are simply looking for the very best value for this for it? Or perhaps you need to know the opinions of some other clients before you purchase a USB 3.0 port card? If that's so then you are at the right spot. How to buy a Usb 3. 0 External. I would like for you to get the best price when choosing a USB 3.0 expansion card.
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text:
Friday night at Common Ground, Halsey took the stage and treated the crowd to an amazing performance! And it got personal. Real personal. During her performance of "Sorry" she appeared to have gotten overwhelmed and caught up in the moment. Speculation has led folks to believe she was in her feelings about her "ex" G-Eazy, fresh off the break up that she took to social media about on July 3rd.
On Tuesday, she confirmed the pair were on a break.
And in a story and performance (caught on video by several fans) that has gone viral, you can see that the breakup is still fresh and affecting the singer.


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It’s been nearly a week since IPCPR 2013 ended, and I could help but reflect on the events of the show, good, bad and otherwise.
It makes sense to start at the beginning. After answering questions about it all show, I started to regret making it public, but there was too much comic value in Jerry and I getting kicked off the floor. The embarrassing high-speed security scooter ride, the attempt to confiscate ALL my cigars (including the ones I brought from home to smoke!), it was too good not to tweet about. Not to mention this all happened while the Social Media Seminar was in progress just down the hall. And, let’s face it, we were a little steamed about it.
Some thought we didn’t belong on the floor, others were surprised. The facts of the debacle are these: Press has always had set-up day access to the show, and we use it. (Doc StogieFresh was kind enough to confirm this going back as far as 2003.) There’s no better time to do your still photography, and get quick video booth tours than the afternoon before the show opens. Cases are polished, product is untouched, and booths are nearly empty. Sometimes you get lucky and get a few interviews in as well.
Look at me, I’m getting a little steamed again, and that’s not my intent. The end of the story is Jerry spoke with the folks at IPCPR and was assured this wouldn’t be a problem in the future. I was happy to hear that. I support the IPCPR’s mission and love the event. More than anything else, I value the relationships we make and strengthen each year. Covering the show for Stogie Review is a lot of work (I limped my way onto the flight home, no joke), but it’s also immensely satisfying. Our fingers are crossed for next year.
The questions we hear repeatedly at the trade show are roughly “What’s the best thing you smoked at the show?” and “What’s got the buzz this year?” The first question is a tough one to answer. Believe it or not, we generally don’t smoke may cigars while on the floor. The ones we do light up we often don’t get to finish. I try to avoid smoking someone else’s cigar while interviewing a manufacturer, and in the hustle partially smoked cigars wind up forgotten in an ashtray. Other times we hear “put that down, you gotta try this.” But here are a few cigars I enjoyed and some general observations.
Speaking of big, a number of booths grew as well, and there was some noteworthy consolidation. I didn’t measure it, but I heard the Drew Estate booth was 30% larger this year, making it seem like a city of cigars and art within the trade show. Tatuaje and My Father Cigars teamed up for a large booth in the center of the floor, and similarly EP Carrillo and Crowned Heads joined forces for a larger space this year.
Fire-cured tobacco was definitely something that was talked about on the floor, and I had a chance to smoke an example or two while I was there. My concerns about it being too overpowering seem to have been put to rest. I plan to spend more time burning it soon.
Drew Estate’s Nica Rustica samples were highly sought after at the show, and by the second day, they were long gone. I heard great things, and from my perspective, it seems like buzz for the Nica Rustica may have eclipsed their Kentucky Fire Cured MUWAT offerings. Retailers and manufacturers alike spoke well of the new cigar.
Sam Leccia was back, occupying a large part of the Torano booth, and man was he busy. We finally caught up with him the last day to talk about his new lines, and I asked him about the fate of the Debut line that never made it to market. It’s something I’ve been wondering about. He confirmed that the cigars are still around, and may appear on the market at some point in the future.
Alec Bradley shot their new Mundial cigars into space, nearly. Working with a company that sends up weather balloons, they launched their cigars into “near orbit” and then collected them after they parachuted back down into the desert 2 hours outside of Las Vegas. The plans are to auction off and donate the proceeds of the first cigars in space to charity.
One of my local retailers clued me into something interesting at the Esteban Carrera booth, a cigar called the Bloodline. the cigar employs a process I’ve they’re calling “The Soft Crush”. It originates with the perfume industry and involves pressing the individual tobacco leaves to bring out the oils. I can’t wait to try one.
Another fun find was a French puro cigar named Le Navarre, tucked away in a corner of the trade show (almost the exact same location as the Panamanian puro I came across a few years ago). All the tobacco is grown, and the cigars rolled in Southwest France. Most importantly, it’s a solid cigar. I lit one up just this morning and found it to be an enjoyable experience. They may be a little hard to find right now, but I noticed Smoke Inn has them in stock if you’re interested.
I spoke with another local retailer on the plane back, and he couldn’t say enough good things about the CAO Flathead. He lit it up and loved it. I haven’t smoked it yet, but is a pretty interesting concept, and a box-pressed lancero has me intrigued. Who doesn’t love cars, cigars and retro pin-up girls?
Patrick Vivalo, a sales executive for Rocky Patel blended a cigar for his trips to the factory, and through a series of fortunate events, it wound up in the hands of a couple retailers. Not only did they like it, they asked to have it made for them as a house blend named Vivalo. Jerry and I smoked a sample and dug it. Stories like this are one of the fun parts of the show, we made a point to talk to him about it on camera.
Another couple cigars I did have the chance to light up were RoMa Craft’s CroMagnon Mode-5 and Craft La Campana De Panama Sobrerana. The latter I smoked on evening at the Venetian’s infamous Circle Bar, and it surprised me how much I enjoyed it. Not because I expected it to be bad, but because it lit up after a few drinks, a number of cigars and a full day on the trade show floor and it really shined through. Also it’s a great looking smoke.
I don’t smoke many Macanudos, but I found the new Estate Reserve very enjoyable first thing one morning as we waited for the show to open. The Cru Royale remains my favorite Macanudo, but this may be in second place.
As with every year, there are a few booths that we just don’t get to, or people we have trouble tracking down. Usually it means they’re hard at work selling cigars, but it’s shame to miss a chance to talk. My apologies to anyone who feels slighted that we didn’t stop by, it wasn’t intentional. You have an open invitation to drop me a line when you swing through Atlanta, or perhaps we can catch up soon in Chattanooga.
And finally, a big thanks to Drew Estate for their sponsorship of our work at IPCPR this year. It would have been tough to make it happen without them.
We don’t get to see enough of you and Jerry throughout the year, so it is always great to catch up at the show.
Great coverage as always, guys. It was great to see both of you, even if only for a few moments here and there. With each year, we spend a few more hours at the show. Maybe in a couple years I will be there on day three or four and we can all sit down for a nice smoke and drink. Anytime you guy guys are in Chicago let me know.
First I love what you guys do, I appreciate all the efforts you (and all the bloggers out there) give to get information about our industry to the public. This isn’t a money making gig for you guys, and I certainly am very grateful. I didn’t realize you were removed from the IPCPR, I don’t like hearing that. I am a huge advocate of what you guys do, and believe that The Stogie Review (and most other bloggers) should always have the freedom to walk the trade show floor. You are the independent voices of our industry. Keep up the great work.
Thanks Sam, we appreciate your support.
Can’t thank you enough for your oustanding coverage of IPCPR. You make guys like me feel like we are right there in the midst of it all. Keep up the great, great work, I love what you do.
Great write up Brian and IPCPR is always an adventure hahaha. Great coverage, great controversies great cigars! Also, your not a reporter until you’ve been ejected so welcome to graduating from a blogger!!!!!
Thanks again for doing all the vids and giving others a good feel of the show and what’s to come out from the interviews you do!

industry_corpus2: Others


text:
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industry_corpus2: Transportation


text:
June 13, 2014, 11:10 a.m.
June 9, 2014, 11:13 a.m.
What does the CLS difference mean for professional success?
June 11, 2014, 9:15 a.m.
Pamela Ballard’s degrees offer her constant inspiration.
It is no accident my degrees from the University of Oklahoma are prominently displayed on the office wall directly across from my desk. That’s where I have all my favorite mementos—pictures of my children, grandchildren, various plaques and a couple of photos of George Clooney. On days when decisions are difficult and foresight is blurred, those framed diplomas serve as reminders of what brought me to serve as the executive director of United Way of Enid and Northwest Oklahoma.
While much of my professional career was in banking, I have long been an active community volunteer, serving on numerous nonprofit boards, the Oklahoma Community Service Commission and the National board of America’s Service Commissions.
I thought I had experienced some level of success with my volunteer efforts until I witnessed the post-CLS graduation difference. There really is no other explanation.
Even with 90 percent of my degree completed online, there were lessons learned, relationships established and collaborations forged that have since been the catalyst for programs created in the nonprofit community, whose rewards are more far-reaching than anything I could have ever imagined.
After receiving my degree, I began a new job search. My degree enabled me to apply for jobs that were previously unobtainable. I was hired as the executive director of a nonprofit youth development agency.
I look at my framed undergraduate and graduate degrees not as merely diplomas, but rather as divine encounters—leading me to a place where I was meant to be, doing the work I was meant to do.
There are many more testimonials just like that. Today, two national affiliates are looking to partner and have asked permission to use the Camp C.A.N.O.E. brand to make this concept available to children with autism across the United States. I would never have been in a position to say, “Nothing would thrill me more…” if not for my experience with the College of Liberal Studies.
Though I had experience working with United Way organizations across the state, moving to Enid as the executive director of a regional United Way was a bit daunting. Again, my experiences from CLS were the perfect preparation.
On two different occasions, I had the privilege of participating in Feaver-MacMinn seminars. I spent weekends with scholars who have consulted with Gandhi, Netanyahu and the United Nations. The man who wrote the cover article for Newsweek’s December 2012 issue–“I know him!” Because of the College of Liberal Studies, I know these people!
Whether you are sitting in your living room or a classroom or propped up on pillows in your bed with your laptop—my personal favorite—you simply cannot leave these experiences of working with others, even if it is through discussion boards and group projects, unchanged.
These learned methods of communicating, sharing ideas and teamwork translated into community alliances and collaborations that surpassed our executive committee and board’s expectations for our first few months together in Northwest Oklahoma.
Breaking the traditional model of fundraising, the entire Enid Fire Department—all 81 firefighters—were named as our 2013-14 campaign co-chairs. From the fire chief, the fire marshal and through the ranks, they took complete charge of our community kick-off, distributed packets and have been present and assisted at every event.
Again, our organization stepped outside its traditional funding mechanism when we realized a need in our community for more pre-K classrooms. Working directly with superintendent Shawn Hime (recently named executive director of the Oklahoma State School Board Association), we created an impact initiative, “Little Kids, Big Impact,” and as a result, this fall United Way of Enid and Northwest Oklahoma will host a reception for its donors and the Inasmuch Foundation (who provided a 50 percent match) for a newly renovated Early Childhood Education Center.
These great programs—and I highlight only a few—are possible only because of the wisdom, the insight, the talents, and gifts of many.
I could never have put together Camp C.A.N.O.E. without Pam Newby, who founded Special Care, and an expert staff who knew how to execute a camp.
I certainly couldn’t move to a new town where I knew NO ONE and raise money for an early childhood center without people like Dianne Juhnke, Dr. Bob and Betty Shuttee and Stan and Gail Brownlee—people who knew the community and were willing to help.
I mentioned earlier it would be impossible to be in a classroom with professors of the caliber we have at CLS and graduate the same person you were before. The conversations you have with instructors, and fellow students, who have travelled extensively, consulted with world leaders and advised diplomats on matters of ethics and public policy elevate the way you think and approach life. Indeed, it gives you the confidence to facilitate and lead conversations and public forums within your own community because you have participated in the highest level of civic discourse and global engagement.
I look at my framed undergraduate and graduate degrees not as merely diplomas, but rather as divine encounters—leading me to a place where I was meant to be, doing the work I was meant to do. Seeing them often is a source of continual inspiration—and an occasional smile from George doesn’t hurt, either.
The United Way of America, based in Alexandria, Virginia, is a nonprofit organization that works with more than 1,200 local United Way offices throughout the country in a coalition of charitable organizations. The focus of the United Way is the promotion of education, income stability and healthy living through partnerships with local organizations that are serving critical needs in communities.

industry_corpus2: Subject Education


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Once we arrived at the Library of Birmingham, we walked into a theatre. We watched loads of presentations from different schools: Wheelers Lane, TNT Ladywood and us (Paganel). Our presentations were about Catseyes(a filming group), and we also done a presentation about our Archive room. After we had a break, we ate some of my favourite biscuits. One school girl from Wheelers Lane had 5 biscuits; she must of been hungry. Then there was loads more presentations, there was so many different authors mentioned: Roald Dahl, Jacqueline Wilson and J.K Rowling.
After we had our lunch, and we went into two different groups. The group I was in went onto the 9th floor(William Shakespeare Memorial). There was millions of amazing artwork: books, picture and sculptures. Also there was loads of comfortable chairs, and I went on a massive computer, which had shown a map. The view was extremely beautiful, and you could see everything.
Lilymai and I went down the travelator, along with Mr Philp (my teacher). Me, Lilymai and Mr Philp were carrying heavy boxes, filled with loads of stuff. Lilymai had the box with the pencils in, and we were going down the travelator. All of a sudden pencils started to roll down the travelator. Mr Philp was on his hands and knees trying to pick them all up, some were starting to get stuck. Finally we picked up all the pencils and went back into the theatre.


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Lobster is with out doubt one of many most delectable and prized shellfish chanced on in this day's seafood markets. They are chanced on in saltwater in all ingredients of the globe and they differ in dimension from the stunning, clawed North American lobster to about a of the smaller spiny lobsters which discontinuance no longer relish claws. All lobsters relish 5 pairs of legs and a aggravating outer shell which they shed, or molt, time and over again someday of their life which allow them to grow. The beneficial American lobster known topped the scales at about forty Four pounds. They are slack-increasing and it’s far believed they’ve a lifespan of over a hundred years. Lobsters shed their shell about 20 cases in their first 5-Eight years and as soon as or twice a year after that. It’s far as a outcome of this that it’s far aggravating to resolve the true age of a lobster. They exhaust most of their life strolling however relish the skill to propel backwards with a swift curl of the tail. They are able to discontinuance this at a flee of about eleven miles per hour. There are two fashioned groups of lobsters, clawed and spiny lobsters. The American lobster which will most probably be is named northern or Maine lobster is a correct lobster and the spiny lobster which is sold as lobster is in actuality a varied species. The female lobster is even handed by many to be the most productive drinking. They would maybe well maybe be vital by the leathery fins on the underside where the physique meats the tail. The male has the related fins however they’re very bony.
The fashioned lobster is the most fashioned and the most tantalizing correct lobster chanced on in the US waters. Additionally called northern, American or Maine lobster, the fashioned lobster are chanced on in the chilly waters of the North Atlantic from Canada to North Carolina. It averages about 2 -Four pounds and 9-15 inches in dimension however it completely has been known to succeed in three fe toes and forty Four pounds. It ranges from yellow or reddish-brown to bluish-inexperienced when alive and turns brilliant crimson with white meat when cooked. The fashioned lobster has 5 pair of legs with the entrance three pair having claws. The entrance pair of claws is terribly stunning and are faded for crushing and cutting its meals where the second and third pair is terribly itsy-bitsy. The fashioned lobster is even handed by many to be the most productive tasting of the more than a couple of species. The lobsterette and the langostino are related in shape and taste to the fashioned lobster however they’re vital smaller, about three inches prolonged, and most tantalizing the tails are eaten.
The spiny lobster, also is named warm water or rock lobster, has prolonged spines that they allege for defense and lack the stunning claws attribute of the fashioned lobster. The spiny lobster is prized for its tail meat which compresses about 1/three of its total weight. The majority of frozen lobster tails are taken from spiny lobster. They are marketed under 2 varied kinds, warm water and chilly water. Warm water tails come largely from Brazil and the Caribbean. The Caribbean spiny lobster is the most fashioned warm water tail chanced on in the US. The chilly water tails come largely from Australia, New Zealand and South Africa with Australia being the greatest producer. The spiny lobster has a steady shell that is on the full brownish-inexperienced with stunning yellow or white spots on the first section of the tail. Cool water tails is no longer going to relish these spots. If from the Pacific, they’ve a steady shell and lack the spots and differ in colour from darkish crimson to orange and brown. Esteem the fashioned lobster, the shell turns brilliant crimson when cooked and the meat turns opaque white.

industry_corpus2: Agriculture & Fisheries


text: This Thursday, February 7, 2019 is National Black HIV/AIDS Awareness Day, coordinated by HIV.Gov, a website of the Office of HIV/AIDS and Infectious Disease Policy. If you would like to arrange a program in your community, check out the Event Planning Guide. You may also participate virtually by using the Centers for Disease Control and Prevention’s social media kit, using #NBHAAD, and following HIV.Gov across social media.