text:
Equal contribution.
Surface contact stimulates the just-in-time deployment of bacterial adhesins
Article first published online: 22 NOV 2011
DOI: 10.1111/j.1365-2958.2011.07909.x
© 2011 Blackwell Publishing Ltd
Additional Information
How to Cite
Li, G., Brown, P. J. B., Tang, J. X., Xu, J., Quardokus, E. M., Fuqua, C. and Brun, Y. V. (2012), Surface contact stimulates the just-in-time deployment of bacterial adhesins. Molecular Microbiology, 83: 41–51. doi: 10.1111/j.1365-2958.2011.07909.x
Publication History
- Issue published online: 20 DEC 2011
- Article first published online: 22 NOV 2011
- Accepted manuscript online: 7 NOV 2011 01:51AM EST
- Accepted 27 October, 2011.
- Top of page
- Summary
- Introduction
- Results and discussion
- Experimental procedures
- Acknowledgements
- References
- Supporting Information
The attachment of bacteria to surfaces provides advantages such as increasing nutrient access and resistance to environmental stress. Attachment begins with a reversible phase, often mediated by surface structures such as flagella and pili, followed by a transition to irreversible attachment, typically mediated by polysaccharides. Here we show that the interplay between pili and flagellum rotation stimulates the rapid transition between reversible and polysaccharide-mediated irreversible attachment. We found that reversible attachment of Caulobacter crescentus cells is mediated by motile cells bearing pili and that their contact with a surface results in the rapid pili-dependent arrest of flagellum rotation and concurrent stimulation of polar holdfast adhesive polysaccharide. Similar stimulation of polar adhesin production by surface contact occurs in Asticcacaulis biprosthecum and Agrobacterium tumefaciens. Therefore, single bacterial cells respond to their initial contact with surfaces by triggering just-in-time adhesin production. This mechanism restricts stable attachment to intimate surface interactions, thereby maximizing surface attachment, discouraging non-productive self-adherence, and preventing curing of the adhesive.
- Top of page
- Summary
- Introduction
- Results and discussion
- Experimental procedures
- Acknowledgements
- References
- Supporting Information
Many organisms derive important competitive advantages from their attachment to surfaces and the mechanisms by which they settle on a surface are subject to important regulation. For example, in sessile marine and aquatic invertebrates such as mussels, barnacles and oysters, it is well established that during the transition from the free swimming larval stage to the sessile adult, the larva reversibly contact the surface, testing and responding to the physical and chemical composition of its environment, prior to cementation and an irreversible commitment to stationary life (Morse, 1990; Tamburri et al., 1992). Surface attachment is also advantageous for bacteria because it increases nutrient access and resistance to environmental stress, and they have evolved many strategies that improve the efficiency of the attachment process.
Decades of research have clearly determined that bacterial attachment proceeds in two stages, a reversible stage that involves transient surface interactions and an irreversible stage that depends on adhesins responsible for mediating permanent attachment (van Loosdrecht et al., 1990a,b; Vigeant et al., 2002; Hinsa et al., 2003; Agladze et al., 2005; Beloin et al., 2008; Otto, 2008; Van Dellen et al., 2008). Flagellar motility and thin surface structures such as pili often mediate the reversible adhesion phase and allow cells to overcome the electrostatic repulsion as they approach a surface. In addition, bacteria produce exopolymers, such as adhesive polysaccharides, to mediate the transition from reversible to irreversible binding and increase their strength of adhesion to surfaces. The temporal mechanism by which this transition occurs however has remained mysterious (Karatan and Watnick, 2009).
In this study, Caulobacter crescentus is used as a model bacterium to study the dynamics of cell-to-surface binding. C. crescentus exhibits a dimorphic life cycle in which each cell division produces a motile swarmer cell and a sessile stalked cell (Fig. 1). Swarmer cells (SW) harbour pili and a flagellum at the same pole. These cells are unable to initiate DNA replication and are motile for approximately 25–30 min under the conditions used in these studies, unless they attach to a surface. After the motile period, swarmer cells differentiate into stalked cells (ST) by shedding their flagellum, retracting their pili and synthesizing an adhesive polysaccharide holdfast and a stalk at the same pole. The holdfast is composed at least in part of a polysaccharide of N-acetylglucosamine that is required for irreversible adhesion to surfaces (Merker and Smit, 1988). Stalked cells initiate DNA replication, elongate and eventually synthesize a flagellum at the pole opposite the stalk, forming the pre-divisional cell (PD). Flagellum rotation is initiated just prior to the completion of cell division, which always produces a swarmer cell and a stalked cell. Initial surface adhesion occurs during the swarmer phase (Bodenmiller et al., 2004; Levi and Jenal, 2006) and permanent adhesion is cemented by the holdfast, which binds to surfaces with an impressive strength in the µN range (Tsang et al., 2006). Therefore, the availability of a portion of the life cycle in which the adhesive polysaccharide is absent allows the investigation of factors that regulate its synthesis and its correlation with surface adhesion.
Here, we take advantage of the fact that C. crescentus and several species of alphaproteobacteria synthesize polar adhesive polysaccharides (Laus et al., 2006; Brown et al., 2009; Tomlinson and Fuqua, 2009) to show that surface contact triggers the rapid production of adhesins and to investigate the mechanism that drives the transition between reversible and irreversible attachment. We find that the interplay between pili and the flagellum during initial surface interactions causes the rapid, just-in-time production of the C. crescentus adhesin, the holdfast polysaccharide, thereby driving the transition from reversible to irreversible attachment. We show that the surface contact stimulation of adhesive polysaccharides is a general phenomenon as it also occurs in two other bacterial genera, the stalked bacterium Asticcacaulis biprosthecum and the plant pathogen Agrobacterium tumefaciens.
Results and discussion
- Top of page
- Summary
- Introduction
- Results and discussion
- Experimental procedures
- Acknowledgements
- References
- Supporting Information
Surface contact stimulates the rapid, protein synthesis independent deployment of the holdfast adhesin
In order to determine if surface contact influences the biosynthesis of the holdfast polysaccharide adhesin, C. crescentus swarmer cells, which do not harbour a holdfast, were collected following synchronization and divided into two treatments. To monitor holdfast production in attached cells, an aliquot of swarmer cells was placed on a coverslip for 5 min to allow cells to attach. Unattached cells were washed away and the attached cells were grown, followed by labelling for holdfasts. To observe holdfast production in unattached cells, synchronized swarmer cells were diluted 100-fold in peptone-yeast extract (PYE) medium such that few cells could contact the surfaces until they were labelled for holdfasts. The holdfasts of both populations were labelled at specific time points with fluorescein-conjugated WGA lectin (fluorescein-WGA), which specifically binds to holdfast polysaccharide (Umbreit and Pate, 1978; Merker and Smit, 1988). Fluorescence microscopy analysis indicated that attached cells produce a holdfast much earlier than unattached cells; more than 90% of attached cells had a holdfast within 7.5 min in contrast to only 2% of unattached cells. Unattached cells took ∼37.5 min to reach a level similar to that of attached cells at 2 min (Fig. 1).
To improve the time resolution of the study of holdfast synthesis in single cells, total internal reflection fluorescence (TIRF) microscopy was used to observe and quantify holdfast production in real time by measuring the integrated fluorescence intensity of the labelled holdfast of individual attached cells in the presence of fluorescein-WGA (Fig. 2A and Movie S1). During the first ∼20 min after the synchrony, virtually all cells lacked holdfast prior to their contact with the surface (Figs 2B–D and S1A). Throughout this time interval, the timing of holdfast production depended on the time of attachment to the surface, rather than the age of the cell, and it occurred within 1–2 min of contact (Figs 2D and S1A). Furthermore, stimulation of holdfast production did not require de novo protein synthesis since holdfast synthesis was still triggered by surface contact in the presence of a growth inhibiting concentration of kanamycin (Fig. 3A). This observation is consistent with previous work showing that addition of kanamycin, choloramphenicol or tetracycline had no effect on attachment or holdfast biogenesis during the cell cycle of planktonic C. crescentus cells (Levi and Jenal, 2006). Thus, surface contact stimulates rapid holdfast production by a post-translational mechanism that would involve activating the holdfast polysaccharide biosynthesis or export machinery (Smith et al., 2003; Toh et al., 2008). In contrast, 25 min after synchronization, nearly all C. crescentus cells, including the unattached cells in suspension, had detectable holdfast (Figs 1, 2C and D and S1A). This observation is readily explained by cell cycle-dependent holdfast production, which occurs slightly prior to swarmer-to-stalk cell differentiation (Levi and Jenal, 2006).
The results presented above imply that the initial attachment of newborn swarmer cells to surfaces is not mediated by the holdfast, whose synthesis or export must be stimulated in order to transit from reversible attachment to holdfast-mediated permanent attachment. In order to test this model, we observed attachment of an ΔhfsA mutant, which is deficient in holdfast synthesis (Smith et al., 2003), using a quantitative dark-field microscopy assay. After processing overlays of sequential images (see Experimental procedures) attached cells appear as stationary foci and are readily distinguished from swimming cells, which generate trajectories (Fig. S2). Swarmer cells of the ΔhfsA mutant initially attached to the glass surface nearly as efficiently as wild type (Fig. 4B). However, they did not permanently adhere and progressively detached from the surface (Fig. 4C). In contrast, once attached, the number of attached wild-type cells remained the same for at least 90 min (data not shown). Thus, the holdfast is not required for the initial reversible weak attachment, suggesting that the stimulation of holdfast production drives the transition to the permanent adhesion stage.
Pili-mediated flagellar rotational arrest stimulates holdfast synthesis
How is reversible attachment mediated when cells encounter a surface? We used video microscopy to observe the dynamics of single cell attachment in real time. As C. crescentus wild-type cells attached to the surface, they rapidly became tethered (Movie S2). Immediately after attachment, the cell body continued to rotate due to the rotation of the flagellar motor. The majority of attached cells stopped rotation within a few seconds and formed a stable association with the surface, whereas a low proportion of cells continued rotation for a short period, but then detached and swam away. In order to quantify the efficiency of surface attachment in a population, synchronized cells were placed between two glass surfaces separated by 12 µm and were observed with dark-field microscopy with a 10× objective, allowing cells at any depth in the field of view to be imaged. Surface attachment was extremely rapid; 68% of the swarmer cells had attached to the surface within 1 min, and virtually all the cells had attached within 5 min (Fig. 4A).
Caulobacter crescentus pili are colocalized with the flagellum at the future pole of holdfast synthesis and are required for efficient surface attachment (Bodenmiller et al., 2004; Entcheva-Dimitrov and Spormann, 2004; Levi and Jenal, 2006). In order to assess the role of pili in the dynamics of single cell attachment, we synchronized swarmer cells of a mutant lacking pili, ΔpilA, and observed the cells by video microscopy. ΔpilA cells were rarely found attached to the surface. Occasionally, a ΔpilA cell became tethered to the surface by its flagellum, but the motor continued to rotate until the cell detached and swam away (Movie S3). Notably, the flagellum motor of tethered ΔpilA cells rotates considerably slower (60 Hz) than the motor of free-swimming ΔpilA cells (∼330 Hz) (Li and Tang, 2006). Thus, the decrease in flagellum motor rotation upon tethering of ΔpilA cells does not lead to permanent attachment. In contrast, the complete stopping of motor rotation following tethering of wild-type cells results in permanent adhesion (Movie S2). Thus, we infer that pili are required to arrest flagellum rotation when cells are bound to a surface.
Quantitative cell attachment assays using dark-field microscopy indicated that, in stark contrast to wild-type cells, the effective attachment of ΔpilA swarmer cells only began to occur more than 20 min after cell division and occurred at a 24-fold lower rate than wild type (Fig. 4A). The timing of attachment of ΔpilA swarmer cells was consistent with the timing of holdfast synthesis of unattached cells (Figs 1, 2C and 5), suggesting that contact between a preexisting holdfast, whose synthesis was triggered by the developmental pathway, and the surface is responsible for the attachment. This hypothesis is supported by the fact that a mutant synthesizing neither pili nor holdfasts (ΔpilA ΔhfsDAB) does not bind to surfaces since both initial reversible attachment and permanent attachment have been abolished (data not shown). Thus, we conclude that pili-mediated initial attachment is followed by rapid holdfast production that drives the transition from reversible to permanent adhesion.
How does the initial reversible attachment stimulate holdfast production and the transition to permanent attachment? Video microscopy of the ΔpilA mutant binding to surfaces revealed that attachment of the flagellum filament enables transient attachment of the cell to the surface (Movie S3). Since the objective is focused on the surface and the cell pole remains out of focus, we infer that the flagellum-mediated attachment is not sufficient to bring the cell body into close proximity with the surface. Notably, tethering by the filament, even though it slows down flagellum rotation, appears insufficient to stimulate holdfast production since attachment of the ΔpilA mutant only becomes efficient once developmental regulation has triggered holdfast synthesis ∼20 min after cell division (Figs 4 and 5). Like the ΔpilA mutant, cells from a double ΔpilA ΔflgE mutant attached at a slow rate (Fig. 4D). In contrast to wild-type and ΔpilA cells, which swam to the surface and whose behaviour is consistent with tethering (Movies S2 and S3), cells from the ΔpilA ΔflgE mutant simply diffused to the surface. We hypothesize that adhesion by the pili brings the flagellum filament in close proximity to the surface, thereby increasing the probability of filament adhesion. Indeed, the aggregate interaction of the filament and the pili with the surface causes the rapid inhibition of motor rotation (Movie S2). This pure mechanical force may be sufficient to inhibit flagellum rotation, although it is possible that the mechanism involves a clutch or a brake type mechanism (Blair et al., 2008; Paul et al., 2010) regulated at the post-translational level. However, the rapidity by which the cell body is immobilized to the surface by the pili precludes the distinction between these mechanisms by video microscopy. Irrespective of the mechanism, inhibition of flagellum rotation is immediately followed by stimulation of holdfast synthesis, independent of the stage of the cell cycle.
Polymer crowding agents that arrest flagellum rotation stimulate holdfast synthesis
Inhibition of the flagellar motor has been reported to trigger various changes in bacteria (McCarter et al., 1988; Anderson et al., 2010). To test if stopping flagellar rotation could stimulate holdfast production independent of surface attachment, we used the observation that polymer crowding agents can cause bacteria to aggregate in solution (Eboigbodin et al., 2005; Schwarz-Linek et al., 2010), due to osmotic pressure caused by the crowding polymers being excluded from the space between neighbouring cells, an effect called the depletion effect (Tang et al., 1997; Hosek and Tang, 2004). We found that C. crescentus cells formed aggregates in 10% Ficoll within 1 min after the addition of the polymer. Single cells and some aggregates continued to move (Movie S4), propelled by the rotating flagellar filaments in the aggregates. Almost all the cells and aggregates stopped moving at 4 min (Movie S5), indicating that all flagella in the aggregates stopped rotation. The cells at age 17.5 min were labelled with fluorescein-WGA for fluorescence microscopy. The majority of the cells (78%) showed clear holdfasts in contrast to free swimming cells in medium without crowding agent of which only 29% had holdfast (Table 1 and Fig. 6).
Test matrix | Viscosity (cp) | Holdfasts/cells | % with holdfast | Aggregation | Motility at age 17.5 min |
PYE (control) | 1 | 152/529 | 29% | No | Swimming |
Percoll, 67% | 2.2 | 29/95 | 31% | No | Swimming |
PEG (8000 Da), 3.5% | 2.2 | 286/817 | 35% | Yes | Some moving aggregates and cells |
PEG (35 000 Da), 5% | 5.4 | 214/328 | 65% | Yes | No moving aggregates or cells |
Dextran (100 000–200 000 Da), 5% | 2.2 | 228/314 | 73% | Yes | No moving aggregates or cells |
Ficoll (400 000 Da), 5% | 2.2 | 151/179 | 84% | Yes | No moving aggregates or cells |
Ficoll (400 000 Da), 10% | 5.2 | 174/223 | 78% | Yes | No moving aggregates or cells |
The stimulation of holdfast production was also observed with polymers of different chemical composition that stopped flagellar motor rotation (Table 1). In contrast, polymers that did not stop flagellum rotation despite being added to a similar viscosity did not stimulate holdfast production (Table 1). The dependence of holdfast production on the inhibition of flagellum rotation triggered by viscous solution supports the notion that arrest of flagellum rotation is required for contact-dependent holdfast production. However, it is possible that the osmotic shock induced by the introduction of the viscous solution may trigger membrane stress (or another physiological change) responsible for stimulating holdfast production, although not all polymer crowding agents had this effect.
These results were confirmed with TIRF microscopy experiments in the presence of Ficoll. In contrast to what was observed in complex medium, cells in the presence of 10% Ficoll arrived at the surface with a holdfast (Figs 3B and S3A), indicating that holdfast synthesis was stimulated prior to surface contact. Ficoll had a similar effect on ΔpilA cells, which also arrived at the surface with holdfasts already formed and adhered permanently to the surface (Figs 3C and S3B). Therefore, pili are not required for the stimulation of holdfast synthesis by viscous agents as they are for the surface contact stimulation. This result also supports our earlier inference that, prior to the developmental trigger of holdfast synthesis, ΔpilA cells do not adhere productively upon surface contact because they are unable to stimulate holdfast synthesis.
We conclude that the loss of flagellum rotation upon surface contact triggers holdfast synthesis. In addition, our results suggest that on surfaces pili are mediating the arrest of flagellum rotation, but with viscosity agents the flagellum is intrinsically impeded and the need for the pili in stimulating holdfast synthesis is bypassed. Future studies will utilize fla and mot mutants to directly determine the contribution of the flagellum filament and motor in initial binding and the transition to permanent adhesion. At present, these experiments are complicated by the fact that these mutants have a delay in cell separation and cannot be synchronized via either the plate synchrony or density centrifugation methods. Furthermore, these mutants have altered timing of cell cycle-dependent holdfast production (data not shown). We are currently exploring alternative methods of cell synchrony to enable these studies.
Surface-contact stimulation probably activates previously assembled holdfast synthesis machinery since it does not require de novo protein synthesis. Indeed, at least the holdfast anchoring machinery (Hardy et al., 2010) and the outer membrane export protein HfsD (J. Javens, Z. Wan and Y.V. Brun, in preparation) are already localized at the flagellar pole in newborn swarmer cells, in close proximity to the flagellum and the pili.
Surface contact stimulation of adhesin synthesis in other bacterial genera
To determine if surface contact stimulated adhesin synthesis is a general phenomenon, we observed polar polysaccharide formation in A. biprosthecum and A. tumefaciens. A. biprosthecum has a dimorphic life cycle similar to that of C. crescentus; however, this bacterium produces two lateral stalks and the holdfast is made at the pole of the cell body (Pate et al., 1973). The unipolar polysaccharide (UPP) of the plant pathogen A. tumefaciens is rarely detected in single planktonic cells, but is frequently detected in cells attached to each other in rosettes or single cells attached to a surface (Tomlinson and Fuqua, 2009). Stimulation of holdfast and UPP production following contact with a surface was readily observed in both species using TIRF microscopy. For A. biprosthecum, nearly every cell arrived at the surface without a holdfast, but these were detected as early as 2 min after contact, irrespective of cell age (Figs 2E and S1B; Movie S6). For A. tumefaciens, most cells produced UPP within 3 min of attaching to a glass surface (Figs 2F and S1C; Movie S7). We also tested whether the production of UPP was stimulated by contact with Arabidopsis roots, a more natural surface for A. tumefaciens attachment. UPP production occurred preferentially among cells attached to an Arabidopsis root when compared with unattached cells from the same population (Fig. 7A; Movie S8) and UPP production occurred after initial attachment of A. tumefaciens cells to the Arabidopsis root (Fig. 7B; Movie S9). These data indicate that surface contact-dependent stimulation of polar polysaccharide production is conserved in multiple bacterial species.
Advantages of surface contact stimulation of adhesin synthesis
We have shown that the ability to rapidly produce a permanent adhesin is shared among at least three members of the Alphaproteobacteria, in which motile cells differentiate to become sessile cells. For many motile bacteria, initial attachment is reversible, allowing cells to detach and sample other surfaces (Korber et al., 1994; Hinsa et al., 2003). The ability to rapidly deploy a permanent adhesin may be advantageous for swimming cells that have encountered a favourable environment, enabling colonization.
Another advantage of just-in-time adhesin deployment upon surface contact is that it reduces the chances that the adhesive will be inactivated. Once exported from the cell, two main factors can reduce the adhesiveness of an adhesin: curing, as observed in the mussel byssal thread (Kamino, 2008) and also with man-made glues, and occlusion of the adhesin with microscopic particles after random collision events. Either mechanism would be expected to decrease adhesiveness over time, limiting productive interactions with a surface. Indeed, previous experiments showed that permanent surface adhesion is highest during the swarmer phase of the C. crescentus developmental cycle and drops dramatically during the stalked cell stage despite the presence of a holdfast on stalked cells (Bodenmiller et al., 2004; Levi and Jenal, 2006). These results support the hypothesis that holdfast adhesiveness decreases with time after export and suggest that the ability to rapidly stimulate adhesin formation upon initial contact with a surface dramatically improves the efficiency of surface interaction and colonization.
Polar attachment of single bacterial cells as a prelude to the transition from reversible to irreversible attachment has also been seen in other proteobacteria such as Escherichia coli (Agladze et al., 2005) and Pseudomonas aeruginosa (Caiazza and O'Toole, 2004). Therefore, contact-dependent adhesin deployment may be a commonly utilized strategy to improve adhesion. As surface-associated adhesins may also act to stimulate host defence responses, this timing may also come into play during pathogenesis to ensure that potential antigens are not prematurely exposed to the host immune system.
Experimental procedures
- Top of page
- Summary
- Introduction
- Results and discussion
- Experimental procedures
- Acknowledgements
- References
- Supporting Information
Bacterial strains and synchronization
Caulobacter crescentus strains were grown at 30°C in PYE medium (Poindexter, 1964) with 10 µg ml−1 kanamycin when indicated. The following C. crescentus strains were used in this study: YB135 (CB15 wild type), YB4038 (CB15 ΔpilA), YB5086 (CB15 ΔpilA ΔflgE) and YB2833 (CB15 ΔhfsA). A. biprosthecum strain JH2 (YB1113) was grown at 26°C in PYE. A. tumefaciens strain C58 (YB2951) was grown at 26°C in LB. Synchronized newborn cells were obtained using the plate release technique (Degnen and Newton, 1972). Briefly, cultures were grown for ∼10 h with shaking at 100 r.p.m. Three millilitres of the culture was added to 20 ml of fresh growth medium in a 9 cm diameter plastic Petri dish and grown at room temperature overnight (or 48 h for A. tumefaciens with medium changes every 12 h) with shaking at 60 r.p.m. After growth, a monolayer of cells was attached to the surface of the Petri dish. The Petri dish was washed thoroughly with water to remove loosely attached and unattached cells. Twenty millilitres of fresh medium was added to the Petri dish and the cells were grown further at room temperature for ∼4 h with shaking at 60 r.p.m. The Petri dish was then grown for 1 more hour at 30°C for C. crescentus and 26°C for A. biprosthecum and A. tumefaciens. The Petri dish was washed repeatedly with oxygenated fresh medium to remove unattached cells and 1 ml fresh medium was added to the plate, which was incubated for 5 min without shaking. The synchronized cells were harvested from the 1 ml of fresh medium. The resulting newborn cells differ in age by a maximum 5 min after cell division.
Fluorescence labelling and fluorescence microscopy
Labelling holdfasts of attached cells. The holdfast is composed of a polysaccharide containing N-acetylglucosamine (GlcNac), which can be labelled with fluorescein-conjugated wheat germ agglutinin lectin (fluorescein-WGA) (Merker and Smit, 1988; Ong et al., 1990). A drop of culture containing synchronized swarmer cells was placed on a coverslip for 5 min to allow some cells to attach to the glass surface. The unattached cells were rinsed with PYE. The cells attached to the coverslips were then grown in oxygenated PYE at 30°C for various amounts of time. After growth, the coverslips were rinsed with water and the cells were labelled with 0.02 mg ml−1 fluorescein-WGA (Molecular Probes) and 0.05% w/v of the bacteriocide sodium azide on ice for 15 min. After labelling, the coverslips were rinsed with 0.05% w/v sodium azide three times and antiphotobleaching solution (20 µg ml−1 catalase, 0.5 mg ml−1 glucose, 0.1 mg ml−1 glucose oxidase and 0.25% v/v mercaptoethanol) was added to the slide for fluorescence microscopy.
Labelling holdfasts of unattached cells. One millilitre of synchronized swarmer cells was added to 100 ml of oxygenated PYE and grown at 30°C without agitation for various amounts of time. In this large volume, very few cells get sufficiently close to the surface to attach. After growth, the unattached cells were supplemented with 0.05% w/v sodium azide and transferred to a pre-cooled glass beaker. Fluorescein-WGA was added to a final concentration of 20 µg ml−1 and the cells were labelled for 15 min on ice. The solution was then passed through an Isopore membrane of 0.4 µm pore size (Millipore) to collect the cells. The collected cells on the membrane were further washed with cold 0.05% w/v sodium azide solution to remove excess dyes. Antiphotobleaching solution was added for fluorescence microscopy.
Attachment of C. crescentus on a glass surface
Immediately after synchronization, an aliquot of synchronized cells was placed between a coverslip and a glass slide that had been cleaned with a solution of 6.5 g of Nochromix (Godax Laboratories) in 100 ml of sulphuric acid for 30 min, rinsed thoroughly with water and dried in air before use. The slide sample was quickly transferred to a warm stage kept at 30°C and observed with dark-field microscopy with a 10× objective, allowing cells at any depth in the field of view to be imaged. To monitor the attachment process, a 1-s-long video was taken at 10 frames per second every 2–3 min. To identify attached cells, an average image was obtained by overlaying the 10 frames of a video and dividing the intensity of the overlay by 10. Since the swimming cells are moving, the brightness of swimming cells is 10 times weaker than that of attached cells in the average image, and therefore the attached cells can be identified by appropriately scaling the image to show only the bright spots (Fig. S2A and B). The attached cells were counted from such images. Attached cells can be distinguished from unattached non-swimming cells because the later drift randomly due to Brownian motion, while attached cells do not. To image the swimming cells, we removed the attached cell from the video by subtracting the average image from each frame. Three consecutive frames were then overlaid and the number of trajectories was counted as the number of swimming cells (Fig. S2C and D). The thickness of the sample was measured using phase-contrast microscopy with a 100× objective by focusing on the top and bottom surfaces respectively (Li and Tang, 2004).
The strain YB2833 (ΔhfsA) lacks a holdfast, is unable to form a stable monolayer and therefore cannot be synchronized by the plate release method. To quantify its attachment, YB2833 cells were grown to exponential phase in a mixed culture, centrifuged and resuspended in fresh PYE. An aliquot was confined between a coverslip and a glass slide and the sample was observed by dark-field microscopy at 30°C. The culture at mid-exponential phase is a mixture of swarmer cells, stalked cells and pre-divisional cells. We measured the decrease in the number of swimming cells as a measure of attachment by counting the number of cell trajectories as described above. The attached cells could easily be washed off the surface, unlike similarly treated wild-type cells, which are tightly attached through their holdfast. Although this strain is able to attach to glass surface through pili, the attachment is not permanent. To quantify the detachment, we added a drop of cell culture at mid-exponential phase to a coverslip, allowed it to sit for 2 min and rinsed gently to remove unattached cells. The number of attached cells was recorded under the light microscope over time.
Labelling holdfast in viscous media
The test viscosity agents used included Percoll (GE), PEGs of molecular weight 8000 Da and 35 000 Da (Fluka), dextran of molecular weight 100 000–200 000 Da (Sigma) and Ficoll of molecular weight 400 000 Da (Fluka). The viscosity was measured with the falling ball method (Tang et al., 1999), except for PEG 8000 (Gunduz, 1996) and Ficoll (Chen and Berg, 2000), which are obtained from the literature. Synchronized C. crescentus wild-type cells were mixed with these agents at the designated concentration and 10 µg ml−1 fluorescein-WGA in PYE in a 1 ml tube. A portion of the mixture was taken out to make slide samples at different times to observe cell swimming and aggregation. To observe holdfast labelling 15 min after mixing, the mixture was passed through a 0.44 µm microfilter (Millipore), followed by passing 60 ml of water to wash the cells trapped by the filter. About 100 µl of water was left in the filter to collect the cells, which became dispersed by the procedure. The cells were then dried on a coverslip for microscopic observation.
Fluorescence microscopy
A Nikon Eclipse E800 epifluorescence microscope with a 100× oil immersion objective lens (Plan Apo) was used to image the fluorescently labelled holdfast. A highly sensitive and linear CoolSnap camera controlled by MetaMorph software (Universal Imaging, PA) was used to record labelled holdfast fluorescence. The fluorescence images were taken at 0.1 s exposure time. To keep the same photobleaching time for each sample, we focused on the holdfast by taking successive pictures under fluorescence microscopy after readjusting the sample stage height between pictures. If the holdfast was in focus prior to the 11th exposure, subsequent exposures were taken and the picture taken at the 11th exposure was used to measure the fluorescence intensity.
TIRF microscopy
TIRF microscopy was performed with a Nikon E2000 inverted microscope with a TIRF objective (NA = 1.49). Two hundred microlitres of synchronized swarmer cells (2.5 ± 2.5 min old) in 20 µg ml−1 fluorescein-WGA lectin solution in PYE was added to a cleaned coverslip sitting on top of the TIRF objective. Some of the dye adsorbed to the coverslip, facilitating focusing of the microscope. After focusing, the TIRF illumination was kept on for 2 min to photobleach the adsorbed dye. Since the TIRF setting varies from experiment to experiment, it is hard to control the light intensity on the holdfast and inconsistent levels of photobleaching are observed. Light microscopy images and TIRF images were taken alternately with 30 s interval to get a time series. The integrated intensity of the TIRF image of individual holdfasts was measured over time. For experiments in the presence of polymers, cells were mixed with the polymers as indicated and 20 µg ml−1 fluorescein-WGA lectin prior to TIRF observation. All the experiments were performed at approximately 22°C.
A. tumefaciens UPP production during attachment to an Arabidopsis root
Sterilized Arabidopis thaliana seeds were placed on ½ MS salts plates with 1% agar and 1% sucrose and allowed to germinate and grow until the roots were approximately 3 cm long (Ramey et al., 2004). Wild-type A. tumefaciens cells were grown in LB medium at 26°C until reaching exponential phase. Two hundred microlitres of cell culture was washed and resuspended in a solution containing 1 mM calcium chloride and 0.4% sucrose. The bacterial cell suspension was spotted into a Petri dish and a root segment was floated in the bacterial cell suspension. The root segments were incubated in the bacterial culture overnight in the dark at room temperature. The root segments were rinsed in 1 mM calcium chloride and 0.4% sucrose to remove unattached cells and placed on an agarose pad containing Alexa Fluor 488 conjugated-WGA. The attachment of A. tumefaciens cells to the root segments was observed using time-lapse microscopy. A. tumefaciens attachment to the root occurs prior to the formation of UPP and this structure is rarely observed in the unattached cells (Fig. 7).
- Top of page
- Summary
- Introduction
- Results and discussion
- Experimental procedures
- Acknowledgements
- References
- Supporting Information
Initial stages of this work were supported by National Institutes of Health grant GM51986 and an Indiana University Faculty Research Support Program grant to Y.V.B., and by a grant from the Indiana METACyt Initiative of Indiana University (funded in part through a major grant from the Lilly Endowment) to C.F. and Y.V.B. The major portion of this work was supported by National Institutes of Health Grant GM077648 to Y.V.B. and J.X.T. and GM080546 to C.F. P.J.B.B. was supported by a postdoctoral National Institutes of Health National Research Service Award number F32AI072992 from the National Institute of Allergy and Infectious Diseases. The authors thank Peter Merritt for his assistance in growing Arabidopsis thaliana, Daniel Kearns, and members of their laboratories for critical reading of the manuscript.
- Top of page
- Summary
- Introduction
- Results and discussion
- Experimental procedures
- Acknowledgements
- References
- Supporting Information
- 2005) Spatial periodicity of Escherichia coli K-12 biofilm microstructure initiates during a reversible, polar attachment phase of development and requires the polysaccharide adhesin PGA. J Bacteriol 187: 8237–8246. , , and (
- 2010) Sense and sensibility: flagellum-mediated gene regulation. Trends Microbiol 18: 30–37. , , and (
- 2008) A short-time scale colloidal system reveals early bacterial adhesion dynamics. PLoS Biol 6: e167. , , , , and (
- 2008) A molecular clutch disables flagella in the Bacillus subtilis biofilm. Science 320: 1636–1638. , , , , and (
- 2004) Development of surface adhesion in Caulobacter crescentus. J Bacteriol 186: 1438–1447. , , and (
- 2009) Complex regulatory pathways coordinate cell-cycle progression and development in Caulobacter crescentus. Adv Microb Physiol 54: 1–101. , , , and (
- 2004) SadB is required for the transition from reversible to irreversible attachment during biofilm formation by Pseudomonas aeruginosa PA14. J Bacteriol 186: 4476–4485. , and (
- 2000) Torque-speed relationship of the flagellar rotary motor of Escherichia coli. Biophys J 78: 1036–1041. , and (
- 1972) Chromosome replication during development in Caulobacter crescentus. J Mol Biol 129: 671–680. , and (
- 2005) Role of nonadsorbing polymers in bacterial aggregation. Langmuir 21: 12315–12319. , , , and (
- 2004) Dynamics and control of biofilms of the oligotrophic bacterium Caulobacter crescentus. J Bacteriol 186: 8254–8266. , and (
- 1996) Evaluation of viscosities of polymer-water solutions used in aqueous two-phase systems. J Chromatogr B 680: 263–266. (
- 2010) A localized multimeric anchor attaches the Caulobacter holdfast to the cell pole. Mol Microbiol 76: 409–427. , , , , , , and (
- 2003) Transition from reversible to irreversible attachment during biofilm formation by Pseudomonas fluorescens WCS365 requires an ABC transporter and a large secreted protein. Mol Microbiol 49: 905–918. , , , and (
- 2004) Polymer-induced bundling of F-actin and the depletion force. Phys Rev E Stat Nonlin Soft Matter Phys 69: 0519071–0519079. , and (
- 2008) Underwater adhesive of marine organisms as the vital link between biological science and material science. Mar Biotechnol (NY) 10: 111–121. (
- 2009) Signals, regulatory networks, and materials that build and break bacterial biofilms. Microbiol Mol Biol Rev 73: 310–347. , and (
- 1994) Effect of motility on surface colonization and reproductive success of Pseudomonas fluorescens in dual-dilution continuous culture and batch culture systems. Appl Environ Microbiol 60: 1421–1429. , , and (
- 2006) A novel polar surface polysaccharide from Rhizobium leguminosarum binds host plant lectin. Mol Microbiol 59: 1704–1713. , , , , , and (
- 2006) Holdfast formation in motile swarmer cells optimizes surface attachment during Caulobacter crescentus development. J Bacteriol 188: 5315–5318. , and (
- 2004) Diffusion of actin filaments within a thin layer between two walls. Phys Rev E Stat Nonlin Soft Matter Phys 69: 061921. , and (
- 2006) Low flagellar motor torque and high swimming efficiency of Caulobacter crescentus swarmer cells. Biophys J 91: 2726–2734. , and (
- 1990a) Influence of interfaces on microbial activity. Microbiol Rev 54: 75–87. , , , and (
- 1990b) Physical chemical description of bacterial adhesion. J Biomater Appl 5: 91–106. , , and (
- 1988) Flagellar dynamometer controls swarmer cell differentiation of V. parahaemolyticus. Cell 54: 345–351. , , and (
- 1988) Characterization of the adhesive holdfast of marine and freshwater Caulobacters. Appl Environ Microbiol 54: 2078–2085. , and (
- 1990) Recent progress in larval settlement and metamorphosis: closing the gaps between molecular biology and ecology. Bull Mar Sci 46: 465–483. (
- 1990) Attachment of the adhesive holdfast organelle to the cellular stalk of Caulobacter crescentus. J Bacteriol 172: 1448–1456. , , and (
- 2008) Considering the first steps toward a stable and orderly way of bacterial life. PLoS Biol 6: e180. (
- 1973) Asticcacaulis biprosthecum sp.nov.: life cycle, morphology, and cultural characteristics. Antonie Van Leeuwenhoek 39: 569–583. , , and (
- 2010) The c-di-GMP binding protein YcgR controls flagellar motor direction and speed to affect chemotaxis by a 'backstop brake' mechanism. Mol Cell 38: 128–139. , , , , and (
- 1964) Biological properties and classification of the Caulobacter crescentus group. Bacteriol Rev 28: 231–295. (
- 2004) The FNR-type transcriptional regulator SinR controls maturation of Agrobacterium tumefaciens biofilms. Mol Microbiol 52: 1495–1511. , , and (
- 2010) Polymer-induced phase separation in suspensions of bacteria. Europhys Lett 89: 68003. , , , , , , et al. (
- 2003) Identification of genes required for synthesis of the adhesive holdfast in Caulobacter crescentus. J Bacteriol 185: 1342–1442. , , , , and (
- 1992) Natural sources and properties of chemical inducers mediating settlement of oyster larvae: a re-examination. Biol Bull 183: 327–338. , , and (
- 1997) Opposite effects of electrostatics and steric exclusion on bundle formation by F-actin and other filamentous polyelectrolytes. Biochemistry 36: 12600–12607. , , , , and (
- 1999) Thiol oxidation of actin produces dimers that enhance the elasticity of the F-actin network. Biophys J 76: 2208–2215. , , , and (
- 2008) Characterization of the Caulobacter crescentus holdfast polysaccharide biosynthesis pathway reveals significant redundancy in the initiating glycosyltransferase and polymerase steps. J Bacteriol 190: 7219–7231. , , and (
- 2009) Mechanisms and regulation of polar surface attachment in Agrobacterium tumefaciens. Curr Opin Microbiol 12: 708–714. , and (
- 2006) Adhesion of single bacterial cells in the micronewton range. Proc Natl Acad Sci USA 103: 5764–5768. , , , , and (
- 1978) Characterization of the holdfast region of wild-type cells and holdfast mutants of asticcacaulis biprosthecum. Arch Microbiol 118: 157–168. , and (
- 2008) Genetic analysis of Vibrio cholerae monolayer formation reveals a key role for DeltaPsi in the transition to permanent attachment. J Bacteriol 190: 8185–8196. , , and (
- 2002) Reversible and irreversible adhesion of motile Escherichia coli cells analyzed by total internal reflection aqueous fluorescence microscopy. Appl Environ Microbiol 68: 2794–2801. , , , and (
Supporting Information
- Top of page
- Summary
- Introduction
- Results and discussion
- Experimental procedures
- Acknowledgements
- References
- Supporting Information
Filename | Format | Size | Description |
MMI_7909_sm_FS1-3.pdf | 3680K | Supporting info item | |
MMI_7909_sm_MovieS1.mov | 149K | Supporting info item | |
MMI_7909_sm_MovieS2.mov | 548K | Supporting info item | |
MMI_7909_sm_MovieS3.mov | 100K | Supporting info item | |
MMI_7909_sm_MovieS4.mov | 174K | Supporting info item | |
MMI_7909_sm_MovieS5.mov | 137K | Supporting info item | |
MMI_7909_sm_MovieS6.mov | 190K | Supporting info item | |
MMI_7909_sm_MovieS7.mov | 33K | Supporting info item | |
MMI_7909_sm_MovieS8.mov | 630K | Supporting info item | |
MMI_7909_sm_MovieS9.mov | 5K | Supporting info item |
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.


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Phoronix Meet-Up - New York City - 21 December
Published on 06 December 2012 10:48 AM EST
Written by
Michael Larabel
Comment On This Article
For Phoronix readers in the New York City area, on 21 December there are tentative plans being made for a usual
Phoronix meet-up
for those interested in Linux, open-source software, benchmarking, and computer hardware enthusiasts.
21 December 2012 might be the day that
the world ends
according to the Mayans or
the day that Enlightenment E17
is finally released, so what better way to celebrate than enjoy a few beverages while discussing Linux hardware and open-source software? (Better yet, as usual for Phoronix meet-ups, those that buy your's truly a beer receive
a subscription to Phoronix Premium
The location within Manhattan and time for the Phoronix meet-up are still to be determined based upon interest level. If you're from the New York City area and would be interested in the Phoronix meet-up that day,
reply to this thread
within the forums or
let me know via Twitter
(@MichaelLarabel). For any Linux professionals or organizations within NYC that would like to discuss the
Phoronix Test Suite
or other matters,
contact me
if you would like to meet separately. More details forthcoming.
Other recent Phoronix meet-ups have been
the annual Phoronix at Oktoberfest
the Munich summer event
, and
the great Oslo event
, among others.
Comment On This Article
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Michael Larabel is the principal author of Phoronix.com and founded the web-site in 2004 with a focus on enriching the Linux hardware experience and being the largest web-site devoted to Linux hardware reviews, particularly for products relevant to Linux gamers and enthusiasts but also commonly reviewing servers/workstations and embedded Linux devices. Michael has written more than 10,000 articles covering the state of Linux hardware support, Linux performance, graphics hardware drivers, and other topics. Michael is also the lead developer of the
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Google Programming Contest Winner 229
from the check-out-the-big-brain-on-brad dept.
Please create an account to participate in the Slashdot moderation system
This is impressive bit of database manipulation. Somehow I didn't think that all of the datatypes, etc would be so easily parsed.
Although I do recall telephone directories that used to give you results for a specified radius for certain types of businesses
$10,000 in cash
VIP visit to Google Inc. in Mountain View, California
Potentially run your prize-winning code on Google's multi-billion document repository (circumstances permitting)"
A Markov process is basically a series of random variables where the value of random variable X^(i+1) only depends on X^i. The idea is that if you want to predict the value of X^(i+1), all of the information you could possibly use is in the value of X^i.
Lots of processes are Markovian- for instance, a random walk. If you're at point x at time t, then you know that there's a fifty-fifty chance you will be at x-1 or x+1 at time t+1. Knowing all of the previous points along the random walk won't help you predict the next point any better than that.
Nothing succeeds like the appearance of success. -- Christopher Lascl


text:
Jack Frost may still be nipping at their nose a little bit, but that's not stopping the Mississippi State Bulldogs from hitting the softball field for competition this weekend.
The Bulldogs start the 2014 campaign today with a pair of games.
MSU plays host to Mississippi Valley State and Northern Kentucky in the Bulldog Kickoff Classic with first pitches at 3 and 5:30 p.m. respectively.
To subscribe to the E-edition, please click here [3].
[1] http://www.starkvilledailynews.com/sites/default/files/Vann px 7.jpg
[2] mailto:email@example.com
[3] http://www.starkvilledailynews.com/eedition


text:
RF PMEL Tech Position in GA (478)926-3959 - RF Cafe Forums
Because of the high maintenance needed to monitor and filter spammers from the RF Cafe Forums, I decided that it would
be best to just archive the pages to make all the good information posted in the past available for review. It is unfortunate
that the scumbags of the world ruin an otherwise useful venue for people wanting to exchanged useful ideas and views.
It seems that the more formal social media like Facebook pretty much dominate this kind of venue anymore anyway, so if
you would like to post something on RF Cafe's
Facebook page, please do.
Below are all of the forum threads, including all the responses to the original posts. Post subject: RF PMEL Tech Position in GA (478)926-3959 Unread postPosted: Sun Aug 29, 2004 11:50 pm Immediate position in PMEL for RF Tech taht repairs and calibrates Spectrum Analyzers, RF Signal Generators, and other Radar Test Sets. Email resume to email@example.com. Posted 11/12/2012
Custom Search
More than 10,000 searchable pages indexed. Your RF Cafe
single-handedly redefining what an engineering website should be. |


text:
Demo Derby: Official PlayStation Magazine: Issue 58
Become Premium to Watch
The chaos and fun of Saints Row is fully intact in the underworld, but a new setting can only go so far.
Dimensions recaptures enough of Geometry Wars' thrills to stand as the best new dual-joystick shooter in quite a while.
The little mushroom man finally gets his name on the marquee, and the result is a charming, unique, and puzzle-heavy adventure.
Character »
The Sly Collection is a re-mastering of the Sly Cooper trilogy made available on a single Blu-ray disc. Along with HD graphics and trophies, the collection includes 3D support and new minigames with PlayStation Move support.
Sly 2: Band of Thieves follows the Cooper Gang as they attempt to recover the dismantled parts of Clockwerk from an international criminal organization known as the Klaww Gang.
This edit will also create new pages on Giant Bomb for:
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Because you're new to wiki editing, we sent your submission off to our moderators to check it over. Most changes are approved within a few hours. We'll send an email when it is.
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January 10, 2015
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Got Eras campaign or battle reports, stories and AARs? Share them here.
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Posts by Author
Saints look literally to rebound
That's the headline of today's story previewing Siena's game at Radford (Va.) tonight. It was written before the news broke that Siena reserve sophomore guard Patrick Cole has been suspended indefinitely. Dressing only eight scholarship players, the Saints figure to have a tough task tonight against Radford, a team picked to finish third in the […]
[Read More]
Siena men's basketball suspends Cole indefinitely (updated)
Siena men's basketball player Patrick Cole is suspended indefinitely from the team, the school's athletic communications department announced today, confirming a Times Union report. The reason for the suspension wasn't immediately available. Siena is expected to make the announcement shortly. Cole, a 6-foot-5 reserve guard, didn't make the trip with the team to tonight's game […]
[Read More]
Siena's Long sprains ankle, out 2-3 weeks (updated)
The Siena men's basketball team took another injury hit today when sophomore forward Lavon Long went down in practice with a high left ankle sprain, according to head coach Jimmy Patsos. Long will be out for two to three weeks and won't return until Metro Atlantic Athletic Conference play resumes next month, said Patsos, who […]
[Read More]
Former Saint Rose player appears in Foot Locker commercial
Former Saint Rose men's basketball player Jin Hong is appearing in a national television commercial with Portland Trail Blazers point guard Damian Lillard and former NBA All-Star Tracy McGrady that debuted last week. For that, Hong can thank an unusual request that a man named Clifton Malone made on Facebook on Nov. 20. "Does anyone […]
[Read More]
Siena's Poole says players aren't taking accountability
LOUDONVILLE – Siena head coach Jimmy Patsos, who usually appears alone at his weekly media luncheon, brought company with him this afternoon in the form of senior guard Rob Poole. Poole gave some interesting insight after Patsos talked about how the team is not rebounding well enough in the absence of junior forward Brett Bisping […]
[Read More]
Coach: UAlbany must be firm
That's the headline of today's story, written by UAlbany beat writer Tim Wilkin, on the latest in the negotiations between Siena and UAlbany about extending the Albany Cup rivalry.
[Read More]
Patsos says Siena's "lost" right now
ALBANY – Siena head coach Jimmy Patsos pointed to a pair of snapshots that he felt told the story of his team's 77-68 loss to UAlbany on Saturday night. The first came when freshman Jimmy Paige checked into the game and didn't know who he was guarding. He was supposed to have one of the […]
[Read More]
Siena's Wright: "Scared" tweet motivated UAlbany
Did a tweet help beat the Siena men's basketball team on Saturday night? Sophomore guard Marquis Wright thought so after the 77-68 loss to the University at Albany. Wright mentioned the tweet that teammate Patrick Cole sent out in September: "UAlbany scared to play us.! Don't matter when or where we play them. That cup […]
[Read More]
Great Danes win dog fight
That's the headline of today's story recapping UAlbany's 77-68 victory over Siena on Saturday night. Also, the postgame notebook: Singletary gets best of Baltimore buds
[Read More]
Highlights from UAlbany's win over Siena
Here are the video highlights of UAlbany's 77-68 victory over Siena in the Albany Cup, courtesy of Siena athletic communications:
[Read More]
Show More


text: 000 FXUS63 KEAX 011144 AFDEAX AREA FORECAST DISCUSSION NATIONAL WEATHER SERVICE KANSAS CITY/PLEASANT HILL MO 544 AM CST Sun Feb 1 2015 .SHORT TERM...(Today through Monday) Issued at 353 AM CST SUN FEB 1 2015 Short-term focus remains on the next 12-18 hours with ongoing winter storm. While precipitation type is varied across the forecast area due to the variables of a near-surface warm layer, dynamic cooling and precip rate, the general theme has been mainly rain well south of Interstate 70, a rain-snow mix along the Interstate 70 corridor, and all snow along and north of Highway 36 where notable snowfall amounts of 5-7 inches have already been reported in some areas. Precipitation will continue to overspread the area through mid- morning before some breaks in coverage and decrease in rate intensity are expected along and south of I-70. However, occasional precipitation in the form of snow is anticipated along and north of Highway 36 through much of the day. As a secondary vorticity maximum, currently seen on water vapor over the central High Plains, drops into region, precipitation rates may increase. At the same time, snow ratios are expected to increase later in the day. Confidence exists that an additional 2-4 inches of snow will be possible generally along and north of Highway 36 from sunrise into early evening with precip wrapping around the backside of the low. This will bring storm total snowfall during the entire event of 5-10 inches in the warning area. Elsewhere, accumulating snowfall is possible, but with decreasing amounts with southward extent. This includes the north side of the KC metro area, where 0.5-1.5 inches of snowfall have already been reported, with another inch of snow possible. As for headlines, adjusted the warning and advisory slightly south to best reflect current observations/reports and anticipated trends. In addition to the snow, a strengthening pressure gradient will result in increasing wind speeds, with northerly winds on the order of 15-25 mph with gusts to 30 mph late this morning into the early evening. These winds, in combination with the already fallen snow and anticipated more powdery snow, will result in blowing and some drifting of snowfall, reducing visibility and making travel treacherous. Wind gusts with the wet snow accumulated in tree branches may also cause some stress and breaking of tree limbs. Lastly, cold air will quickly advect into the area this afternoon, with temperatures rapidly falling. Wet or slushy roadways may have the potential to quickly refreeze, resulting in additional travel troubles. The snow is expected to end by midnight with bitterly cold temperatures by Monday morning, with lows in the single digits to teens. Much quieter weather is expected on Monday, with much of the area remaining precipitation free despite a quick-moving upper wave passing to the north. .LONG TERM...(Tuesday through Saturday) Issued at 353 AM CST SUN FEB 1 2015 A cool northwest flow will be in place across the nations mid-section by Tuesday. However, despite the northwest flow, temperatures Tuesday will be on a bit of a rebound as a southerly flow at the surface lifts a weak warm front into northern Missouri. This will bring a quick bout of above normal temperatures for areas along and south of the Missouri River, though areas farther north will likely remain near seasonally normal --mid to upper 30s--. The warm weather wont last long though as a trough swinging through the Canadian Plains will send another surge of cold air south for the later half of the work week. Next surge of cold air is advertised as a 1036mb high; so it will be cold. Continued to keep slight chance to chance POPs for snow Wednesday and Wednesday night. ECMWF is now lining up a bit more with the GFS in advertising some light precipitation in that time period. This looks reasonable given the strength of the frontogenic forcing expected to occur on the leading edge of the surface high. Confidence on accumulations is rather low due to concerns about available moisture, but flurries will likely be a minimum. Cold temperatures will return to the region behind the cold front and light snow Wednesday. This will likely allow overnight lows to drop into the single digits in areas north of the Missouri River early Thursday morning. However, there is hope for next weekend as a modest shortwave ridge helps push warmer temperatures back into the Central Plains by Saturday and Sunday. && .AVIATION...(For the 12Z TAFS through 12Z Monday Morning) Issued at 536 AM CST SUN FEB 1 2015 Outside of temporary fluctuations in ceilings and visibility, expect prevailing IFR conditions through late morning. Periods of snow or a rain-snow mix are expected at terminals, with a gradual decrease in coverage/intensity during the late morning and afternoon. MVFR conditions will return this afternoon, as well as increasing northerly winds with gusts up to 30kts. Wind speeds will relax this evening, with a return to VFR just after the valid TAF period. && .EAX WATCHES/WARNINGS/ADVISORIES... KS...WINTER WEATHER ADVISORY until 9 PM CST this evening FOR KSZ025- 102. MO...WINTER WEATHER ADVISORY until 9 PM CST this evening FOR MOZ001- 011>013-020>023-028>033. WINTER STORM WARNING until 9 PM CST this evening FOR MOZ002>008- 014>017-024-025. && $$ SHORT TERM...Blair LONG TERM...Cutter AVIATION...Blair


text:
Tell America's largest chocolate company that slave labor and Valentine's Day chocolate don't mix.
The sweet smell and healing properties of bay, eucalyptus and ginger will make your sweetheart's Val...
A recent study found that 63 percent of all couples plan to stay in for Valentine's Day this year. C...
Forgo store-bought goods this year in favor of a homemade gift. These homemade Valentine's Day gift ...
Valentine's Day is right around the corner. This year, why not show some love to the earth as well as your sweetheart? You can have a romantic Valentine's Day without putting any extra strain on the earth.
1. Cook a meal at home.
Not only will cooking at home save money, but you'll have more control over the quality of your food. Substitute organic produce, naturally raised meat and local ingredients. For a romantic meal, try one of these recipes: • Lamb Ravioli • Roast Chicken and Root Vegetables • Flatiron Steak with Natural Jus and Root Vegetable Mash
2. Drink organic wine.
No romantic celebration is complete without wine. This year splurge a little and get a bottle of organic wine. Organic wine doesn't cost that much more. Many organic wines are grown and bottled in the U.S., meaning your wine won't have to travel as far to get to you.
3. Eat fair-trade, organic chocolate.
While you're thinking of your beloved, consider the earth, too—and her people. Give the gift of organic, fair trade and even vegan chocolate. Indulge in a bar of Yachana Jungle Chocolate or sip on a cup of Dagoba organic drinking chocolate.
4. Give homemade gifts.
Nothing says "I love you" like a homemade gift. Valentine's Day gifts don't have to be elaborate. Create a picture album, decorate a vase, or make homemade candy.
5. Buy fair trade gifts.
If you're not the crafty type, buy your significant other a gift that gives back. Fair trade gifts support artisans in developing countries, and because each one is handmade, fair-trade gifts come with a personal touch.
6. Wear green lingerie.
Just because (almost) no one will see it doesn't mean you should ignore your intimates! Underwear and lingerie come in a variety of eco-friendly fabrics these days, including organic cotton, organic silk and hemp.
7. Get local, organic flowers.
Conventional flowers harbor pesticide residue, and many of them are imported from South America. If flowers are part of your Valentine's Day routine, buy organic flowers from a local florist. Fresher is better!
8. Set the mood with soy candles.
Create an atmosphere of romance with soy candles. Conventional candles are made with paraffin wax, a petroleum byproduct, and some wicks contain traces of heavy metals. Choose soy wax candles as a natural option.
9. Take a green, romantic weekend.
With Valentine's Day on a Sunday, this year might be the perfect opportunity to make a romantic weekend out of it. Get away to a local bed and breakfast, or check into a green hotel in your area. Want to save money on a romantic weekend? Take a staycation. Set aside the weekend for romantic pursuits: bubble baths, watching movies, cooking or whatever you like to do together.
10. Send a recycled card.
Many Valentine's Day cards are made from virgin paper. Give a card that uses recycled paper, or make your own card out of scrap paper you already have.
Subscribe to Mother Earth Living!
Welcome to Mother Earth Living, the authority on green lifestyle and design. Each issue of Mother Earth Living features advice to create naturally healthy and nontoxic homes for yourself and your loved ones. With Mother Earth Living by your side, you'll discover all the best and latest information you want on choosing natural remedies and practicing preventive medicine; cooking with a nutritious and whole-food focus; creating a nontoxic home; and gardening for food, wellness and enjoyment. Subscribe to Mother Earth Living today to get inspired on the art of living wisely and living well.
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text:
- OS: Windows ×
Presentation Software
Showing page 1 of 39.
Jaspersoft Studio The Eclipse-based report development tool for JasperReports
27,297 weekly downloads -
JasperReports Library JasperReports Library, the world's most popular open source business intelligence and reporting engine and JasperReports Server, the most popular open source interactive report server built-on JasperReports Library
4,388 weekly downloads -
PhotoFilmStrip PhotoFilmStrip creates movies out of your pictures in just 3 steps. First select your photos, customize the motion path and render the video. There are several output possibilities for VCD, SVCD, DVD up to FULL-HD. Creates animated slideshows.
2,921 weekly downloads -
DxWnd Window hooker to run fullscreen programs in window and much more...
2,719 weekly downloads -
OpenSong Lyric projection and chord charts for church musicians
1,507 weekly downloads -
Klavaro Touch Typing Tutor A very efficient tutor.
1,346 weekly downloads -
CasparCG: Pro Video & Graphics Play-Out Pro Video & Graphics Play-Out SW, proven in 24/7 broadcasts since 2006
1,463 weekly downloads -
ZedGraph ZedGraph is a class library, user control, and web control for .net, written in C#, for drawing 2D Line, Bar, and Pie Charts. It features full, detailed customization capabilities, but most options have defaults for ease of use.
717 weekly downloads -
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Nurse Jackie: Season 4, Episode 9Are Those Feathers? (10 Jun. 2012) 7.8
The firing of her colleagues, Grace's refusal to go to school, and an overflowing emergency room full of patients redirected from another hospital push Jackie to finally seize control of the E.R. and run it her way. Director:Randall Einhorn |
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Once an alpine skier, Stephen doesn't fear going fast, something she has proved with podium finishes in some of the world's most prestigious cross country races. (Getty Images/AFP/Pierre-Philippe Marcou)
The USSA named 16 athletes to the 2015 Cross Country World Championships Team that will compete in Falun, Sweden.
Jessie Diggins was fifth, Liz Stephen seventh as the U.S. Ski Team closed out a strong weekend in Rybinsk.
Liz Stephen finished second on Friday in the 10k freestyle race in Rybinsk, Russia—the top World Cup distance result by an American woman.
USSA columnist Tom Kelly takes a look at Liz Stephen's phenomenal result at the Tour de Ski in this week's Behind the Gold.
Liz Stephen rewrote the history books again, posting the fourth fastest time up Alpe Cermis to move up to fifth in the final Tour de Ski standings - the best American finish ever.
Liz Stephen skied a strong race to finish fifth in the 10k classic, advancing to sixth in the Tour de Ski.
Liz Stephen (East Montpelier, VT) was the only U.S. racer to compete in stage five of the Tour de Ski in Toblach, Italy, finishing 14th.
Sadie Bjornsen and Liz Stephen continued to pace the U.S. women, standing top 15 after the stage two 10k classic.
Liz Stephen led the U.S. Ski Team with a strong 12th place finish in a 10k freestyle race in Davos.
The U.S. women closed the gap in a 10k classic race in Davos as the World Cup tour left Scandinavia for the higher elevation Swiss Alps.
Sadie Bjornsen skied to 18th overall in the three day World Cup mini-tour in Lillehammer.
The U.S. Cross Country Ski Team will take a strong summer and fall preparation period into this weekend's FIS World Cup opener in Ruka.
On June 29th, U.S. Ski and Snowboard Association (USSA) athletes and staff will compete in the third annual Sufferfest.
Noah Hoffman and Sadie Bjornsen (seen here at 2013 U.S. Championships in Utah) both capped their respective seasons Friday with U.S. distance titles to wrap up the 2014 U.S. Cross Country Championships.
Liz Stephen Quick Facts
Nordic Athletes » Liz Stephen |
Additional Info
Liz Stephen WebsitesAthlete Photo Gallery |
Liz Stephen started her young career as an alpine skier at Vermont's Burke Mountain Academy, but later found her calling on the cross country course. The U.S. Team can count their blessings on that decision, as Stephen has quickly become a key distance asset to the women's side, competing on three World Championship teams and reaching her second Olympics in 2014. Stephen's Sochi push was an impressive one, earning the best U.S. finish in the 30k and bolstering the U.S. relay squad in the 4x5k relay. The Vermont native also recorded her highest individual result on the World Cup, snagging sixth in the Szklarska Poreba, Poland 10k classic. She also posted six more scoring results in the World Cup, bringing her career total to 22. In 2013 Stephen was part of the fourth place World Championships 4x5 relay team and took fifth in the 10k freestyle at the same event. Just two years earlier, a young Stephen made her Olympic debut in Vancouver, and two years before that in 2008, she stood on the podium at the U23 World Championships with a bronze medal finish. Stephen will spend her offseason in Park City training at the USSA Center of Excellence as she prepares for another stellar season in 2015. LIZ SAYS FIRST TRACKS OFF THE SNOW |
HighlightsOLYMPICS WORLD CHAMPIONSHIPS WORLD CUP U.S. CHAMPIONSHIPS Other |


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Call to lift the game
PUBLISHED: 28 Feb 2011 00:01:00 | UPDATED: 24 May 2011 09:41:20PUBLISHED: 28 Feb 2011 PRINT EDITION: 28 Feb 2011Proposed new rules governing financial advisers should be extended to others in the sector and ASIC should become more proactive, speakers have told a conference of do-it-yourself super fund advisers.
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People | Deborah Ralston, Mathias Cormann, Bernie Ripoll |
Topics | Financial Services Industry /Accounting , Personal Investment /Superannuation , Politics /Federal Politics |


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Not all the action went down on the court during last night's NBA All-Stars Game in L.A. -- because there was some pretty interesting stuff going on in the stands thanks to appearances by Justin Bieber, Rihanna, Beyonce and more!Check out the gallery to see who randomly was seated next to Lil Wayne, which stars mingled with each other in their floor seats and what Steven Tyler was wearing!
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The Zimbabwe Revenue Authority (ZIMRA) hosted commemorations to mark International Customs Day at a colourful event held on 26 January 2014 in Harare. The event ran under the theme, "Coordinated Border Management: An Inclusive Approach for Connecting Stakeholders".
Attempts to smuggle four illegal immigrants from Malawi to South Africa after hiding them in a false compartment created within the chassis of a commercial truck trailer were thwarted by the Zimbabwe Revenue Authority (ZIMRA) at Beitbridge Border Post recently.
The top hierarchy of ZIMRA management converged in Beitbridge for the annual Strategic Planning meeting which ran from the 12th to the 17th of January 2015. The meeting which was described by the attendees as highly successful zeroed in on a number of issues that will see things turning for the better in the Zimbabwe Revenue Authority and the Beitbridge Border Post in particular through the use of a Results Based Management appraisal system.
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The storyline of the 1913 U.S. Open is simple: Francis Ouimet, a 20-year-old amateur and former caddie who lived across the street from The Country Club in Brookline, Mass., where the Open was being played, tied British pros Harry Vardon and Ted Ray after 72 holes, then beat them in an 18-hole playoff.
It's the tournament's myriad subplots that make it fascinating, even 100 years later. We'll set up the circumstances, then use the accounts of the participants and observers to help tell the story.
Remembering Ouimet | |
• | Baggs: Who was Francis? |
• | Baggs: Search for Ouimet |
• | Tays: Turning point in U.S. |
• | Mosier: Eddie Lowery story |
• | Timeline | Trivia | Bag | Photos |
• | Why Vardon and Ray? |
• | The Country Club |
• | Vardon and the Titanic |
• | Inspiring other writers |
• | Acknowledgeents |
• | Full Coverage |
The preview: Harry Vardon from the Isle of Jersey, a British Crown Dependency located just off the coast of Normandy, France, was the biggest name in the field. He had won five British Opens and one U.S. Open. He was on a tour of the U.S. with fellow Jerseyman Ted Ray, and the USGA had agreed to postpone the Open until September so the two stars could play. It's the only time the USGA has ever made such an accommodation.
Other notables in the field included pros John J. McDermott of Philadelphia, the two-time defending champion (and the first American to win the U.S. Open), Walter Hagen, Wilfrid Reid and Jerome Travers.
What about Ouimet? In Massachusetts, he was well known. In 1910, '11 and '12, he had missed qualifying for the U.S. Amateur by just one shot. In June 1913, Ouimet won the Massachusetts Amateur, then finally qualified for the U.S. Amateur but lost in the second round to eventual champion Travers. USGA president Robert Watson, who had played with Ouimet in a U.S. Amateur qualifying round in 1910, was looking for a local amateur to add to the Open field, and he settled on Ouimet, even entering him without Ouimet's knowledge.
The course: The Country Club course played to a "bogey score" of 80, as par was a concept that was just beginning to take hold. Here are the yardages of each hole:
The Country Club course
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | Out |
430 | 300 | 435 | 300 | 420 | 275 | 185 | 380 | 520 | 3245 |
10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | In |
140 | 390 | 415 | 320 | 470 | 370 | 125 | 360 | 410 | 3000 |
Sept. 16, qualifying, first day
Vardon Beats Boston Boy. Briton's Final Putt Does It.
Ex-President Taft Is Among Spectators.
(Boston Daily Globe, Sept. 17, 1913)
For the first time in the history of the U.S. Open, the size of the field necessitated two 36-hole qualifying rounds, with the low 64 players and ties advancing to the Open proper. Playing on the first day of qualifying, Ouimet shot 74-78–152, which looked like it was going to top the standings, until Vardon (75-76–151) sank a long putt on his final hole.
Writer D.J. McGuiness referred to Ouimet as "a player considered by experts as the golfing sensation of the year."
"Vardon had a gallery of about 500 on his morning round," McGuiness noted, "but after the spectators heard at the conclusion of the first 18 holes that Francis Ouimet, the only amateur who had made any kind of a showing, was leading the field by a stroke, the Woodland player (Ouimet was a junior member of nearby Woodland Golf Club) was followed by at least 700 persons in his afternoon's play, a large majority leaving Vardon."
John G. Anderson also covered the first-day qualifier for the Globe. (He was serving double duty as a player and newspaper correspondent.) He knew Ouimet well. A high school English teacher and golf coach in the Boston area, Anderson had inserted himself into his undermanned team's roster for a match against Ouimet's Brookline High team four years earlier. But Anderson was no ordinary high school coach – at the time he already had won two Massachusetts Amateurs, and in 1913 he would reach the final of a third, losing to Ouimet, and the final of the U.S. Amateur, losing to Jerome Travers, who had taken down Ouimet in the second round.
"I thought a couple of clubs would be enough for any boy I might happen to compete against," Anderson recalled in his Globe article, "and the boy happened to be Ouimet. The first thing I knew, my young opponent was leading me three holes, and with my two clubs, I could not catch him. I'm not saying I could if I had my entire kit."
"Within my knowledge of golf in the United States," Anderson continued, "I know no performance by an amateur in medal play which stands out with quite such brilliancy as that of Mr. Ouimet yesterday."
1. | Harry Vardon | Jersey | 75-76–151 |
2. | Mr. Francis Ouimet | U.S. | 74-78–152 |
3. | Macdonald Smith | Scotland | 77-77–154 |
4. | Alec Ross | Scotland | 76-81–157 |
5. | Tom McNamara | U.S. | 78-80–158 |
5. | Jim Barnes | England | 82-76–158 |
Sept. 17, qualifying, second day
Ted Ray led all qualifiers with a 36-hole course-record 74-74–148, and Ouimet wound up with the fifth-best qualifying score overall.
Henry Leach, an Englishman writing for The American Golfer, wrote that Ray's scores "were the result of sound careful play in every part of the game … but it struck me that he could have driven a little further if he had wanted to do so. When he lets out, however, the rough has a great attraction for him, and at Brookline, he had developed a holy horror of the trees that line the fairway and told me that his greatest fear in regard to the championship was the dire trouble that he might get into through crooked driving."
1. | Ted Ray | Jersey | 74-74–148 |
2. | Wilfrid Reid | England | 72-79–151 |
2. | Robert McDonald | U.S. | 72-79–151 |
4. | George Sargent | U.S. | 79-75–154 |
5. | Walter Hagen | U.S. | 78-89–157 |
5. | Joseph Sylvester | U.S. | 78-79–157 |
1. | Ted Ray | Jersey | 74-74–148 |
2. | Harry Vardon | Jersey | 75-76–151 |
2. | Wilfrid Reid | England | 72-79–151 |
2. | Robert McDonald | U.S. | 72-79–151 |
5. | Mr. Francis Ouimet | U.S. | 74-78–152 |
Sept. 18, Rounds 1 and 2
British Golfers' Showing Is Most Formidable.
Small Chance of Heading Them Off Now.
(Boston Daily Globe, Sept. 19, 1913)
Ouimet shot 77-74–151, tied for seventh, four shots behind co-leader Vardon and two shots behind Ray. Ray, after opening with a disappointing 79, broke the course record with a 70 in his second round. After Ray's first round, Leach wrote, "he uttered a declaration that he would do a 70 in the afternoon to make up for it. And he did it!" It could have been a 69, "had he not missed a two-foot putt on the home green," the Globe's McGuiness noted.
Ouimet got off to a double bogey-bogey start and turned in 41 in his opening round, but came home in 36.
With Vardon and fellow Englishman Wilfrid Reid leading after identical totals of 75-72–147, "Nothing seemed more certain then that the championship title would go abroad," Leach wrote.
1. | Wilfrid Reid | England | 75-72–147 |
1. | Harry Vardon | Jersey | 75-72–147 |
3. | Ted Ray | Jersey | 79-70–149 |
3. | Herbert Strong | England | 75-74–149 |
5. | Macdonald Smith | Scotland | 71-79–150 |
5. | Jim Barnes | England | 74-76–150 |
7. | Mr. Francis Ouimet | U.S. | 77-74–151 |
7. | Alec Ross | Scotland | 71-80–151 |
7. | George Sargent | U.S. | 75-76–151 |
7. | Walter Hagen | U.S. | 73-78–151 |
Sept. 19, Rounds 3 and 4
Brookline Boy to Fore.
Ties Both Ray and Vardon.
(Boston Daily Globe, Sept. 20, 1913)
On a dreary, rainy Friday, Ouimet opened with the best round of the day, a 74, to tie Vardon (78) and Ray (76) for the lead. All three shot 79 in the final round, setting up an 18-hole playoff the following day.
Vardon and Ray both failed to break 40 in the opening nine of their fourth round, "Vardon's putting and Ray's short game being mainly responsible," Leach wrote.
"Vardon very rarely smokes when playing in public, though he is as fond of his pipe as any man alive. It was an indication of his anxiety, therefore, when he lighted up at the fourteenth hole and he played better afterwards. He told me he wished he had begun to smoke earlier!"
Tied for the clubhouse lead at 304, neither Vardon nor Ray felt that score would hold up. Vardon told Leach, "There are three or four still out who will beat us. I am very sorry, but it is my putting that has let me down again. I feared it would."
"I played rotten," Leach quoted Ray, "and to make matters worse Harry went and did the same thing."
The list of contenders that Vardon and Ray feared would surpass them eventually dwindled to one – Ouimet. He had turned in a disappointing 43 and started the final nine badly with a double-bogey 5 at the 10th.
The rules of the era did not allow a player to pick up a ball and clean it or loosen it from a plugged lie, even on the green. In his first-person account of the tournament for The American Golfer, Ouimet explained his strategy at the 10th: "Fearing that a high pitch might mean that the ball would bury itself on the green, as had happened a number of times during the tournament, I tried the experiment of playing the shot with a midiron, trusting to the soft turf to keep the ball from over-running, while at the same time expecting the running shot to prevent any accumulation of mud on the ball. Like a novice, I was too eager to see what happened on the shot, looked up to watch the ball and, consequently, made a bad flub." He reached the green in two, then three-putted for his 5.
"After that wretched five, walking to the eleventh tee between a lane of spectators," Ouimet wrote in "A Game of Golf," "I heard one man say, 'It's too bad, he has blown up.' I knew he meant me, and it made me angry. It put me in the proper frame of mind to carry on."
When he reached the 13th tee, Ouimet knew he needed to play the remaining six holes in 2 under par to tie Vardon and Ray. He got the first birdie by chipping in at the 13th, the report of which caused Vardon and Ray to venture out to the 14th hole to watch the young amateur finish.
Ouimet had targeted the 125-yard 16th hole as the most likely source of a second birdie, but he could manage only a par. But at the 360-yard 17th, he holed what Leach called a "twelve yards putt for a 3. That was a great putt for America and a great 3." Ouimet wrote, "my putt felt good from the moment I hit it."
Needing a par 4 at the 410-yard 18th, Ouimet was just short of the green in two, chipped up and holed "a yard putt at the finish with no more hesitation than if he were practicing," Leach wrote.
The immediate press reports were breathless. Anderson called the reaction of the crowd, estimated at 10,000, the largest for a U.S. tournament to that date, "the most enthusiastic demonstration ever accorded a golfer in this country or probably in the world."
Citing examples of heroes in other sports "hoisted upon the shoulders of his admirers, for a jerky ride of fame," Anderson noted that "such enthusiasm has been generally foreign to the golf links until the modest Woodland boy, Francis Ouimet, carried the gallery completely off its feet at the Country Club yesterday afternoon."
1. | Mr. Francis Ouimet | U.S. | 77-74-74-79–304 |
1. | Harry Vardon | Jersey | 75-72-78-79–304 |
1. | Ted Ray | Jersey | 79-70-76-79–304 |
4. | Walter Hagen | U.S. | 73-78-76-80–307 |
4. | Macdonald Smith | Scotland | 71-79-80-77–307 |
4. | Jim Barnes | England | 74-76-78-79–307 |
4. | Louis Tellier | France | 76-76-79-76–307 |
Sept. 20, 18-hole playoff
First Amateur to Win the American Open Golf Title.
Little Woman Holds Her Ground Until She Congratulates Her Son.
(The New York Times, Sept. 21, 1913)
Cards of the playoff
Hole | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | Out |
Yards | 430 | 300 | 435 | 300 | 420 | 275 | 185 | 380 | 520 | 3245 |
Ouimet | 5 | 4 | 4 | 4 | 5 | 4 | 4 | 3 | 5 | 38 |
Vardon | 5 | 4 | 4 | 4 | 5 | 3 | 4 | 4 | 5 | 38 |
Ray | 5 | 4 | 5 | 4 | 5 | 4 | 3 | 3 | 5 | 38 |
Hole | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | Out |
Yards | 140 | 390 | 415 | 320 | 470 | 370 | 125 | 360 | 410 | 3000 |
Ouimet | 3 | 4 | 4 | 4 | 5 | 4 | 3 | 3 | 4 | 34–72 |
Vardon | 4 | 4 | 5 | 3 | 5 | 4 | 3 | 5 | 6 | 39–77 |
Ray | 4 | 4 | 5 | 4 | 5 | 6 | 4 | 5 | 3 | 40–78 |
Ouimet became the first amateur to win the U.S. Open, shooting 72 to Vardon's 77 and Ray's 78.
"While I did not feel nervous" going into the playoff, Ouimet wrote for The American Golfer, "I did realize the formidable task in hand." Two things and one person "helped me amazingly. The person was my little caddie, Eddie Lowrey (sic); one of the two things was the appeal which he made to my patriotism; the other thing was my determination that Vardon and Ray should not be able to say that my tying them for the championship was a fluke, which I felt they could say if I 'went to pieces' in the playoff."
All three players parred the first hole in 5, but Ouimet cited it as a key to his round. "After playing four shots I was about a yard from the cup and I never felt more pleased over a putt in the entire championship than when that three-footer dropped out of sight, unless it was the putt on the home green the day before which tied me for the championship title," he wrote.
All three players made the turn in 38, and when Vardon and Ray both three-putted the 10th, Ouimet had a lead he would never relinquish. He picked up another stroke at the 12th, with a 4 to the Englishmen's 5s. "About this time it dawned upon me that even against two such wonderful players there might be a chance of landing the title, with which thought I resolved that if they beat me it would be only by playing better than par golf," Ouimet wrote.
Vardon closed within a stroke with a birdie at the 13th, but both Englishmen failed to capitalize on a topped brassie shot by Ouimet at the 14th, where all made 5s.
Ray fell out of contention with a double-bogey 6 at the 15th, where he needed two strokes to escape a greenside bunker. Now it was down to Ouimet and Vardon.
Both parred the par-3 16th, but when Vardon tried to cut the corner on the dogleg of the par-4 17th, he landed in a bunker, had to pitch out sideways and made 5. Ouimet took the safer straight route, reached the green in two and sank his birdie putt for a three-shot lead going to the last. A closing 4 by Ouimet, a 6 by Vardon and a too-little, too-late 3 by Ray, and the U.S. Open had its first amateur champion. Again Ouimet was hoisted on the shoulders of the crowd. A collection was taken up for Lowery.
At the awards ceremony, Vardon and Ray paid tribute to their unlikely conqueror. "I have no hesitation in saying that he played better golf the whole four days than any of us," Ray said. "His was the best golf I have ever seen in my time in America. It has been an honor to play with him and no dishonor to lose to him."
"We have no excuses to make today," Vardon followed, "for we were both defeated by the highest class of golf. … America should be nothing but proud of her new champion. He has proved himself to be a superior golfer and a courageous fighter."
The New York Times blanketed the playoff with 11 different articles. Also reported was a message of congratulations of former President William Howard Taft, who had watched Ouimet during the qualifying rounds. "I am very glad that an American won," Taft said. "It was wonderful for a boy of only 20 years to be able to win a championship in a match of that length, a match that required strength, endurance, and fortitude to so great a degree. I congratulate Ouimet on his victory."
Ouimet closed his American Golfer piece with praise for Lowery, Vardon and Ray: Lowery "was a veritable inspiration all around." As for Vardon and Ray, "I can say only that I admire them as fine golfers and good types of sportsmen, and am only sorry that all three of us could not win."


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'Timber' Goes Vintage! [Video]
Ke$ha and Pitbull got together for 'Timber' in 2014, but what if this was 1950?
Ke$ha and Pitbull got together for 'Timber' in 2014, but what if this was 1950?
That awkward moment when a stranger asks to borrow your phone...
Happy lazy Sunday! Ever wonder why you love your favorite song so much?
Do you put together a family Christmas card? Or maybe a family Christmas letter? This family did just that but with a Miley Twist!
"Are you answering what I'm saying?"
"Because I said I would" is hosting a YouTube video posted by Matthew Cordle, who says he drove drunk and killed a man over the summer.
Recently, Megan and Liz stopped by the 95-7 studios to perform for you live on the air. They are very talented girls who made it big on YouTube and now have a hit song that we play called "Are You Happy Now?"
Whether you loved it or hated it, chances are you viewed Rebecca Black's 'Friday' video at some point during the past year. Probably several times. The infamous clip was the most watched YouTube video in the world in 2011, the video site recently revealed.
I know we have a ton of music fans here in West Michigan. Obviously because thousands come to downtown Grand Rapids every time there's a concert. In the summer time, there are two big music festivals in the U.S. that many aren't able to make it to, but this year YouTube is taking you there.
The website announced just the other day that it will live stream Chicago's Lollapalooza, and Austin City Limits in (obviously) Austin, TX.
What's cooler than having Andy Grammer live in the Channel 957 studio?
Probably having him tweet aCheck out the video after the jump!nd Facebook about how awesome of a job you did covering his song "Fine By Me" in sign language! Check out the video after the jump!
One of the downsides to YouTube is that it provides a steady stream of stark evidence that you are not a very good dancer.
The latest set of humbling dance moves comes from what we believe to be two brothers, one big and one small.
As you can see, the smaller of the duo can match any move the bigger fellow throws at him - and then some. Read on to see their amazing moves.