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The synthesis of the peptidoglycan cell wall is carefully regulated in time and space. In nature, this essential process occurs in cells that live in fluctuating environments. Here we show that the spatial distributions of specific cell wall proteins in Caulobacter crescentus are sensitive to small external osmotic upshifts. The penicillin-binding protein PBP2, which is commonly branded as an essential cell elongation-specific transpeptidase, switches its localization from a dispersed, patchy pattern to an accumulation at the FtsZ ring location in response to osmotic upshifts as low as 40 mosmol/kg. This osmolality-dependent relocation to the division apparatus is initiated within less than a minute, while restoration to the patchy localization pattern is dependent on cell growth and takes 1 to 2 generations. Cell wall morphogenetic protein RodA and penicillin-binding protein PBP1a also change their spatial distribution by accumulating at the division site in response to external osmotic upshifts. Consistent with its ecological distribution, C. crescentus displays a narrow range of osmotolerance, with an upper limit of 225 mosmol/kg in minimal medium. Collectively, our findings reveal an unsuspected level of environmental regulation of cell wall protein behavior that is likely linked to an ecological adaptation.
Most bacteria possess a peptidoglycan (PG) polymer as a major component of their cell wall. The PG consists of glycan chains that are linked covalently by short peptides, resulting in a strong, elastic meshwork that surrounds the cytoplasmic membrane (36). Under most conditions, this PG meshwork is essential for cell viability by providing a mechanical resistance to the turgor pressure caused by the high concentration of solutes in the cytoplasm relative to the environment. The PG layer also plays a structural role by maintaining cell shape. Growth of the PG requires the coordinated actions of both synthetic and lytic enzymes (22) and is regulated by the cytoskeleton (5) and, at least in some Gram-negative bacteria, by specific outer-membrane lipoproteins (24, 33). During cell elongation, most rod-shaped bacteria insert new PG material into the existing PG along the cylindrical sidewall following a patchy/helical pattern (9, 13). Division and new pole formation proceed by localized PG synthesis directed by the cytokinetic FtsZ ring and associated proteins. In some bacteria, such as Caulobacter crescentus (and possibly Escherichia coli to a lesser extent), a significant amount of cell elongation stems from FtsZ ring-dependent PG growth (1, 13, 34) in a process also known as preseptal growth (36).
Early studies in E. coli (29) suggest that the penicillin-binding proteins (PBPs) PBP2 and PBP3 are specifically involved in cell elongation and division, respectively. Both enzymes are inner-membrane proteins that catalyze the transpeptidation reaction linking adjacent glycan strands of the PG in the periplasm. Consistent with its role in division, PBP3 and homologs have been shown to localize at the FtsZ ring in various bacteria (7, 10, 37). Comparatively, the localization of PBP2 displays more variability among species. In E. coli, PBP2 is spatially distributed throughout the cell according to a reported patchy/helical pattern, with some ring-like accumulation in the midcell region (12, 35). In C. crescentus and Helicobacter pylori, PBP2 exhibits a patchy distribution throughout the cell with no midcell accumulations (14–16, 19). In contrast, in Rhodobacter sphaeroides, PBP2 is mostly concentrated in a midcell ring (28). It is unclear if such localization variability among species reflects differences in PBP2 activity, growth mode, experimental conditions, or adaptation to distinct habitats.
Besides PBP2, many other proteins are involved in cell wall morphogenesis (40). Some of these proteins are enzymes directly involved in peptidoglycan biosynthesis in either the cytoplasm (e.g., MurG) or the periplasm (such as PBPs) (40). Others (e.g., MreB, RodZ, RodA, MreC, and MreD) have important, albeit less well understood, cell shape functions (40). While cell wall morphogenetic proteins have been the focus of many studies, their behaviors have yet to be studied in situations of environmental flux, which occur in nature. In this study, we discovered that in C. crescentus, PBP2, RodA, and PBP1a change their spatial distribution in response to a small increase in external osmolality. Our findings unravel a surprising environmental regulation that may reflect an ecological adaptation.
Strains, plasmids, and growth conditions.The strains and plasmids used in this study are listed in Table 1, and the construction method is provided in the supplemental material. C. crescentus strains were grown in peptone yeast extract (PYE) medium (2 g/liter Bacto peptone, 1 g/liter yeast extract, 1 mM MgSO4, and 0.5 mM CaCl2) or defined minimal (M2G) medium (0.87 g/liter Na2HPO4, 0.54 g/liter KH2PO4, 0.50 g/liter NH4Cl, 0.2% [wt/vol] glucose, 0.5 mM MgSO4, 0.5 mM CaCl2, and 0.01 mM FeSO4) under aerobic conditions at 30°C (17) unless otherwise stated. Antibiotics were added when appropriate. Transformations, conjugation, and transductions were performed as previously described (17). Synchronization of cell populations with respect to the cell cycle was performed as previously described (18). Induction of gene expression from the xylose- or vanillate-inducible promoter was achieved by adding xylose or vanillic acid to the growth medium at the final concentrations of 0.03% and 0.5 mM, respectively, except for the experiments shown in panels D of Fig. 1 and 2, for which 0.3% xylose was used. The presence of 0.03% xylose and 0.5 mM vanillic acid increases the osmolality of the solution by approximately 1.9 and 0.5 mosmol, respectively.
E. coli strains were grown in M9 glucose (M9G; 42 mM Na2HPO4, 22 mM KH2PO4, 8.5 mM NaCl, 1.9 mM NH4Cl, 0.2% glucose, 1 mM MgSO4, 3.3 μM thiamine) or LB (10 g/liter tryptone, 5 g/liter yeast extract, and 10 g/liter NaCl) medium at 37°C with antibiotics when appropriate. Exponentially growing cultures were used in all experiments. Induction of gene expression from the IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible promoter was achieved by adding IPTG to the growth medium at the final concentration of 100 μM (adding ∼0.1 mosmol to the solution).
Light microscopy.Imaging was performed using 100× differential interference contrast (DIC) or phase-contrast objectives on a Nikon E1000 microscope equipped with a Hamamatsu Orca-ER camera, on a Nikon Eclipse 80i microscope equipped with Andor iXonEM+ (DU-897E) EMCCD and Hamamatsu Orca II-ER cameras, or a Nikon Eclipse Ti-U microscope with a Hamamatsu Orca-ER camera. Cells were mounted on 1% agarose pads made with the indicated medium. Images were taken with Metamorph software (Molecular Devices).
Image analysis.Cell identification and analysis were performed using the MATLAB-based, open-source software MicrobeTracker (27). "Demographs" were created by segmenting each cell, integrating the fluorescence in each segment, normalizing the fluorescence by the brightest segment of each cell, sorting cells by length, and plotting fluorescence values as a heat map from 0 (no fluorescence) to 1 (maximum-fluorescence segment in the cell). When appropriate, cells were oriented according to their pole identity prior to sorting. Cell polarity was determined either by using polar markers (for example, FtsZ-yellow fluorescent protein [YFP] accumulation identifies the new pole of swarmer cells) or by identifying the new pole generated during division in time-lapse recordings.
Osmolality measurements.Average osmolalities were calculated from three separate measurements using a Vapro Vapor Pressure 5520 osmometer (Wesco Biomedical Systems).
Growth rate measurements.To determine whether differences in growth rate cause midcell accumulation of PBP2, we first determined the doubling time of cultures growing at different temperatures. For this experiment, 25-ml flasks of culture were grown in a water bath shaker at the indicated temperature and growth medium. Samples were taken every hour for manual optical density reading at 660 nm (OD660) using an Ultrospec 2100 Pro spectrophotometer. For the experiment shown in Fig. 6, cells were grown at 30°C in a Biotek Synergy2 96-well plate reader and optical density measurements (at 660 nm) were taken every 2 min. For each condition, a doubling time was determined by fitting an exponential curve to the interval represented by exponential growth. Averages and standard deviations (SDs) were obtained for three biological replicates for each condition.
Immunoblot analysis.For immunoblot analysis of cell extracts, cultures were grown under indicated conditions to an OD660 of 0.4 at 30°C. After growth, cells were harvested, resuspended in 100 μl of 50 mM sodium phosphate buffer (pH 7.4) containing 50 mM NaCl and 1 mM EDTA, and sonicated. Whole-cell extracts were electrophoretically resolved on 12% SDS-PAGE gels, and the resolved proteins were electrotransferred to polyvinylidene difluoride (PVDF) membranes, which were probed with anti-green fluorescent protein (anti-GFP) antibodies (1:1,000; Clontech).
PBP2 accumulates at the FtsZ ring location.In C. crescentus cells, PBP2 has been reported to have a patchy/helical pattern of localization throughout the cell (14, 15, 19). Surprisingly, we found that when cells (strain JAT878) grown in rich PYE medium were imaged on an agarose pad containing M2G minimal medium (commonly used because M2G generates less fluorescence background than does the PYE medium), a vanillate-inducible, chromosomally expressed N-terminal mCherry fusion of PBP2 displayed a ring-like accumulation near midcell in many cells (Fig. 1A). We observed a similar localization pattern when using a GFP-PBP2 fusion expressed from either the xylose-inducible promoter (Fig. 1B) or the native pbp2 promoter in place of wild-type pbp2 (Fig. 1C), indicating that midcell localization occurs regardless of the type of fluorescent protein fused to PBP2 (GFP or mCherry) or the expression level of the fusion (inducible or native; see Fig. S1 in the supplemental material). Fusion to a fluorescent protein did not appear to affect the function of PBP2, since replacement of wild-type PBP2 by a GFP-PBP2 fusion (expressed from the native promoter on the chromosome in place of wild-type PBP2) supported viability and this strain did not display any apparent growth or morphological defects (data not shown).
We showed that the midcell localization was associated with specific cell cycle stages by examining the localization of both cyan fluorescent protein (CFP)-PBP2 and FtsZ-YFP over time in synchronized cell cycle populations (strain CJW3402) grown in liquid PYE cultures (and spotted on an M2G agarose pad for visualization at 20-min intervals). At early cell cycle stages, GFP-PBP2 showed patchy localization throughout the cells with occasional accumulation at a cell pole (Fig. 1D). After FtsZ-YFP ring formation but before cell constriction became discernible, the intensity of the CFP-PBP2 signal throughout the cells diminished in favor of a ring-like (band) localization coinciding with the FtsZ-YFP ring near midcell (Fig. 1D). CFP-PBP2 remained colocalized with FtsZ-YFP throughout most of the cell constriction process (Fig. 1D).
PBP2 colocalizes with MreB at the FtsZ ring location in an FtsZ-dependent but MreB-independent manner.The FtsZ ring accumulation of PBP2 near midcell was surprising for at least two reasons. First, PBP2 is often referred to as a cell elongation-specific PBP, and second, the presence of MreB at the FtsZ ring has been proposed to prevent PBP2 from being recruited to that location (15). To examine the localization of PBP2 in large, unsynchronized cell populations, we imaged multiple fields of view and identified cells and their outline using the open-source image analysis software MicrobeTracker (27). The normalized fluorescence intensity was measured along the cell length, and then cells were sorted by cell length, to create what we call a demograph. Here, cell length was used as a measure of cell cycle progression. Figure 2A shows demographs of FtsZ-YFP and CFP-PBP2 localization in 1,000 cells producing both fluorescent fusions. The cell-length sorting reflects cell cycle progression, with smaller cells showing an accumulation of FtsZ-YFP at the new pole and longer cells displaying FtsZ-YFP accumulation near midcell (characteristic of the FtsZ ring). The corresponding demograph of CFP-PBP2 confirmed that PBP2 colocalizes with the FtsZ ring during a significant portion of the cell cycle. GFP-PBP2 was unable to form bands in FtsZ-depleted cells (Fig. 2B), indicating that recruitment of GFP-PBP2 near midcell is FtsZ dependent. We verified that GFP-PBP2 levels were unaffected under FtsZ-depletion conditions (see Fig. S2 in the supplemental material).
In C. crescentus, MreB localization has been reported to change during the cell cycle from a patchy pattern throughout the cell in the beginning of the cell cycle (stage 1) to an FtsZ ring-dependent midcell localization in the stalked and predivisional stages (stage 2) and back to a patchy pattern in the late predivisional stage (stage 3) (19, 20). Demographs of CJW3397 cells coproducing YFP-MreB and CFP-PBP2 confirm this previously noted three-stage cell cycle localization pattern for MreB (Fig. 2C). Interestingly, the patchy signal of YFP-MreB in the beginning of the cell cycle (stage 1) was most concentrated in the central region of the cells, as evident from the demograph (Fig. 2C). Similarly, in long, deeply constricted cells corresponding to the late predivisional stage (stage 3, Fig. 2C), the MreB-YFP patchy signal was most concentrated between the one-fourth and three-fourths regions, which correspond to the central regions of the future daughter cells. The biological significance of this pattern is unclear. However, more relevant to our PBP2 study, the corresponding demograph clearly shows that CFP-PBP2 colocalizes with MreB-YFP near midcell during stage 2 (Fig. 2C), indicating that MreB does not prevent PBP2 accumulation at the FtsZ ring under these conditions.
PBP2 localization at the FtsZ ring was independent of MreB. GFP-PBP2 bands near midcell were still discernible in cells treated with 50 μM A22 (Fig. 2D), a drug that inhibits MreB polymerization (4, 21). Since A22-treated cells have a cell shape defect and are rounder than untreated cells (21), we also examined GFP-PBP2 localization in mreBQ26P mutant cells, which have a normal shape but produce an MreB mutant, MreBQ26P, that largely fails to condense into a ring near midcell (1). Since GFP-PBP2 also retained its midcell localization in these mreBQ26P cells (Fig. 2E), we conclude that MreB has no apparent positive or inhibitory effect on PBP2 accumulation at the FtsZ ring location.
Transition under environmental conditions causes the accumulation of PBP2 at the FtsZ ring location.Our observation of PBP2 recruitment to the FtsZ ring was inconsistent with previous reports (14, 15), raising the possibility that a difference in experimental conditions may be responsible for the discrepancy. We noted that in the previous studies, M2G cultures were used, whereas we used PYE cultures. Indeed, the midcell accumulation of GFP-PBP2 observed when using PYE cultures (Fig. 3A) was largely abrogated when cells were grown in M2G medium (Fig. 3B) instead of PYE medium, as evident from the relative uniformity of fluorescence seen in the demograph. In both cases, M2G agarose pads were used for visualization.
Since C. crescentus grows faster in the more nutrient-rich PYE medium, we examined whether PBP2 accumulation near midcell was dependent on the growth rate by varying the temperature. At 20 and 23°C, PYE cultures displayed doubling times of 235 ± 10 and 162 ± 4 min (mean ± standard deviation [SD], n = 3 independent experiments), respectively, which are longer than the doubling time of M2G cultures at 30°C (115 ± 2 min, n = 3). Yet, GFP-PBP2 still accumulated near midcell when cells were pregrown in liquid PYE culture medium at 20 or 23°C (see Fig. S3 in the supplemental material), indicating that it is not the growth rate but rather some aspect of the medium that affects PBP2 localization.
Surprisingly, the PYE culture medium per se did not cause the difference in PBP2 localization. Instead, it was the transition from the PYE culture to the M2G pad that caused midcell accumulation of PBP2, as spotting cells from a PYE culture to a PYE pad (i.e., an agarose pad containing PYE instead of M2G) for visualization did not result in midcell accumulation of mCherry-PBP2 (Fig. 3C). PBP2 localization was, on the other hand, insensitive to the transition from the M2G culture to the PYE pad (Fig. 3D). Midcell accumulation was observed not only when cells grown in PYE were visualized on an M2G pad (Fig. 3A) but also when cells grown in PYE were washed in M2G and visualized in M2G liquid (instead of on an M2G agarose pad; data not shown).
Relocation of PBP2 to midcell happens fast upon medium change whereas the return of PBP2 to its patchy localization is slow and growth dependent.Our data so far indicated that a switch from PYE to M2G medium results in PBP2 relocation to the FtsZ ring location. The PBP2 relocation event is not controlled by changes in gene expression, as midcell localization of GFP-PBP2 still occurred when the cells were pretreated with the transcription inhibitor rifampin or the translation inhibitor chloramphenicol (see Fig. S4 in the supplemental material).
The relocation is remarkably quick, since it was already initiated within the 40 s that it took to spot cells from a PYE culture onto an M2G pad and image them on the microscope (Fig. 4A). The relocalization process was complete within 2 min, as evident from tight midcell accumulation that remained unchanged over the following 3 min (Fig. 4A). Thus, relocation of mCherry-PBP2 to midcell is likely too fast to involve changes in peptidoglycan composition through remodeling. Even a transient exposure to M2G was sufficient to cause midcell relocation. This was shown by growing cells in PYE and then washing the cells in M2G prior to imaging them on a PYE agarose pad (Fig. 4B). In this assay, the cells were exposed to M2G medium for only approximately 3 min during the wash. No relocation to midcell was observed for PBP2 if the wash was a PYE solution (Fig. 4B) instead of M2G.
Next, we performed time-lapse experiments on cells that were grown on a PYE culture but imaged on an M2G pad and showed that PBP2 returns to a patchy localization over time (Fig. 4C). This redistribution was, however, slow and took approximately 2 h, which corresponds to about 1 to 2 generations. Surprisingly, the redistribution of mCherry-PBP2 appeared progressive, with the mCherry-PBP2 band at midcell getting wider and wider over time (Fig. 4C), as if PBP2 localization were expanding concomitantly with peptidoglycan growth (1). This redistribution required cell growth, as it was inhibited when cells were spotted on an M2 pad that lacked glucose (carbon source) for growth (Fig. 4D).
We concluded from these experiments that in response to a switch from PYE to M2G, PBP2 relocation to midcell is very rapid (≤40 s) but temporary because PBP2 gradually returns to a patchy localization; this return to patchy localization is, however, slow (∼1 to 2 generations) and dependent on cell growth.
An upshift in medium osmolality is sufficient to cause PBP2 recruitment to midcell.Since PBP2 relocation to the FtsZ ring location occurs when the extracellular environment changes from PYE to M2G, we investigated what physical or chemical differences between these two media might cause PBP2 relocation. PYE and M2G media notably differ in chemical composition and pH. However, despite intensive efforts, we were unable to link PBP2 relocation to the presence of a specific chemical ingredient or to a difference in pH (data not shown). Interestingly, measurements with a vapor pressure osmometer revealed that PYE and M2G media also differ in osmolality (Fig. 5A). (Note that osmolality, which is the measure of osmoles of solute per kilogram of solvent, often is the preferred measure as it is insensitive to changes in pressure and temperature, unlike osmolarity, which is the measure of osmoles per liter of solution.) Perhaps counterintuitively, the osmolality of the nutrient-rich PYE medium (21.7 ± 1.5 mosmol/kg, mean ± SD, n = 3 measurements) was lower than that of the minimal M2G medium (52.6 ± 1.2 mosmol/kg, n = 3) (Fig. 5A). While richer in nutrients because of the presence of yeast extract and peptone (0.1 and 0.2%, respectively), PYE has fewer osmolytes than does M2G, which contains M2 salts (6.1 mM Na2HPO4, 3.9 mM KH2PO4, and 5.6 mM NH4Cl), 11.1 mM glucose, and other, less-abundant components (0.5 mM MgSO4, 0.5 mM CaCl2, and 0.01 mM FeSO4).
Using a PYE culture/wash/PYE pad assay in which the only variable is the composition of the wash solution, we discovered that any solutions with an osmolality higher than that of PYE resulted in mCherry-PBP2 localization near midcell. For example, phosphate-buffered saline (PBS), which is commonly used as a wash solution in biological research, resulted in relocation of PBP2 to midcell (Fig. 5B), similarly to an M2G wash and unlike a PYE wash (Fig. 4B). The osmolality of PBS was 280 ± 2.6 mosmol/kg (n = 3), over 10-fold higher than that of PYE (Fig. 5A). The type of components used to increase osmolality (inorganic or organic) did not seem to matter. For instance, brief exposure to 1× M2 salts or PYE medium containing 20 mM NaCl or 2.5% sucrose (73 mM) all resulted in mCherry-PBP2 relocation to midcell (Fig. 5B). All of these solutions have higher osmolality than that of PYE (Fig. 5A). Conversely, purified water, which has a lower osmolality than that of PYE (Fig. 5A), had no effect on mCherry-PBP2 localization (Fig. 5B). We also noted small differences in PBP2 concentrations at the pole, but they tended to vary from experiment to experiment, suggesting that they may be caused by minute differences in procedures (e.g., in the hydration of the pad or in the time between washing and imaging). However, in all cases, an upshift in osmolality was reproducibly accompanied with a relocation of mCherry-PBP2 to the FtsZ ring location.
We obtained similar results when using an M2G culture/wash/M2G pad assay (instead of PYE culture/wash/PYE pad) in which we started with M2G cultures, washed them with solutions of various osmolalities, and imaged them on an M2G pad. Again, when the osmolality of the wash solutions was lower than or equal to that of M2G, such as with PYE, M2G, M2 salts, and purified H2O (Fig. 5C), mCherry-PBP2 retained its dispersed patchy distribution. In contrast, wash solutions of higher osmolality than M2G, such as 3× M2 salts or M2G medium containing 20 mM NaCl or 2.5% sucrose (Fig. 5A), caused mCherry-PBP2 relocation to midcell (Fig. 5C). Thus, whether cells are initially present in low (PYE)- or higher (M2G)-osmolality medium does not seem to matter; what seems important for PBP2 relocation to occur is an increase in external osmolality.
We also noted that the quality of the agarose pads used for microscopy can matter, as any significant drying increases the concentration of solutes in the pad, creating an osmotic upshift (data not shown).
C. crescentus displays low osmotolerance.We wondered if this osmotic response relates to the ecological niche of C. crescentus. C. crescentus is an oligotroph because of its ability to thrive in environments containing very low levels of nutrients, such as lakes and streams. These environments are also characterized by a very low osmolality. The rich (complex) medium of choice for C. crescentus growth in the laboratory is PYE, which is not only poorer in nutrients but also ∼20-fold lower in osmolality than LB (Fig. 6A). M2G is the most common minimal medium used by Caulobacter researchers; it has the same amount of carbon as does the minimal medium M9 glucose commonly used for E. coli but has an osmolality ∼5-fold lower than that of M9 glucose (Fig. 6A), primarily because of a much lower concentration of phosphate buffer (3.98 mM versus 25 mM). We found that C. crescentus is unable to grow in LB or M9 glucose after overnight culture (data not shown).
Increasing the osmolality of M2G by adding increasing concentrations of NaCl (from 10 mM to 80 mM) gradually decreased the growth of C. crescentus as shown by the increase in doubling time (Fig. 6B). Growth was completely inhibited in M2G containing NaCl at concentrations of 85 mM or more (data not shown). C. crescentus was, however, able to grow in the presence of higher concentrations of NaCl when the osmolality of the medium was decreased to compensate. For example, exponential growth was observed in the presence of up to 95 mM NaCl when the concentrations of glucose, phosphate buffer, and nitrate in M2G were decreased by half (referred to as "M2Ghalf"; Fig. 6B). The trade-off was that these cultures reached stationary phase sooner (data not shown), as these osmolytes, which also are nutrients, become more rapidly depleted from the medium. Exponential growth was also faster in M2Ghalf than in M2G for the tested NaCl concentrations of 40 mM and above, as shown by a decrease in doubling time (Fig. 6B, top). For example, cells growing in M2Ghalf plus 60 mM NaCl had a doubling time of 141.8 ± 1.5 min (mean ± SD, n = 3 biological replicates) compared to 197.2 ± 6.4 min (n = 3) for cells growing in M2G plus 60 mM NaCl. When the doubling times for the cultures grown in M2G or M2Ghalf containing various concentrations of NaCl were plotted as a function of osmolality (Fig. 6B, bottom) instead of NaCl concentrations (Fig. 6B, top), the two curves overlapped, indicating that the growth inhibition is due to the increased external osmolality, rather than salt intolerance.
These observations suggest that in minimal medium (which lacks osmoprotectants), C. crescentus has a low osmotolerance with an upper limit of approximately 225 mosmol/kg (Fig. 6B). In contrast, E. coli grows in media with osmolalities as high as 3,000 mosmol/kg (25), which is consistent with its broad ecology (ranging from streams to the human gut).
The localization of E. coli PBP2 is largely unaffected by osmotic upshifts.We wondered whether the relocalization of PBP2 in response to osmotic upshifts was somehow connected to the ecological niches of C. crescentus. If true, we expected bacteria such as E. coli to display no or little response. In E. coli, PBP2 has been reported to adopt a patchy localization throughout the cell membrane with some ring-like accumulations in the midcell region (11, 35). We observed a similar localization pattern for a GFP-PBP2 fusion in E. coli cells grown in LB or M9G medium, as shown in demographs (Fig. 7A and B), and this pattern was maintained following an osmotic upshift with the addition of 100 mM NaCl. Even when the cells were grown in low-osmolality PYE medium and spotted on PYE pads containing increasing concentrations of NaCl (20 to 60 mM), there were no significant differences in the GFP-PBP2 localization profile (Fig. 7C). Under these conditions, PBP2 relocates to the division site in C. crescentus. These results support the notion that the sensitivity of PBP2 to osmotic stress might reflect ecological differences between C. crescentus and E. coli.
The localization of PBP1a and RodA is also affected by osmotic upshifts in C. crescentus.There are a large number of proteins involved in the biosynthesis and regulation of the cell wall in C. crescentus. Some of them, such as the morphogenetic protein MreB (19, 20), the precursor PG synthesis enzyme MurG (1), and the morphogenetic protein RodZ (3), have been shown to localize to the FtsZ ring. We found that these midcell accumulations occurred whether or not the cells were subjected to an osmotic upshift (see Fig. S5 in the supplemental material). We also examined the localization of MreC, which has been reported to have a patchy localization near the cell membrane in C. crescentus (14). This pattern remained dispersed and patchy even when the cells experienced an osmotic upshift (see Fig. S5).
We did, however, observe a difference in localization pattern for fluorescent fusions of RodA and PBP1a in response to an osmotic upshift (Fig. 8A and B), although not to the same extent as with PBP2. RodA, a polytopic membrane protein of the SEDS family of PG precursor flippases, has been proposed to act in concert with PBP2 in PG synthesis during the elongation of E. coli (23, 30). Since RodA localization has not been reported in C. crescentus, we created a strain (CJW3207) in which a YFP-RodA fusion is produced under the xylose-inducible promoter from the chromosome. When cells were grown in PYE liquid medium and spotted on an M2G pad (resulting in an osmotic upshift), YFP-RodA displayed dispersed/patchy membrane localization in nondividing cells and a weak but discernible midcell accumulation that was most apparent in deeply constricted cells (Fig. 8A). Such accumulation was less prevalent in cells grown in M2G medium and spotted onto an M2G pad (i.e., no osmotic shift) (Fig. 8A), suggesting a response to an increase in external osmolality.
Similarly, a GFP fusion to a PBP1a homolog (CC1516), which contains transglycosylase and transpeptidase domains, displayed a discernible accumulation at the site of division in many constricted cells following osmotic upshift; such midcell accumulation was much less apparent without osmotic upshift (Fig. 8B).
Since RodA and PBP1a have been proposed to be part of a multienzyme complex with PBP2 (for a recent review, see reference 32), we considered the possibility that the midcell accumulation of YFP-RodA and GFP-PBP1a under osmotic-upshift conditions might be weak because the higher level of these xylose-inducible fusions results in saturation of the natively expressed PBP2 at midcell. If true, we would expect that a concomitant overproduction of PBP2 would result in a more prominent midcell accumulation of YFP-RodA and GFP-PBP1a under osmotic-upshift conditions. This was indeed what we observed. When mCherry-PBP2 and YFP-RodA were coexpressed from the vanillic acid- and xylose-inducible promoters, respectively, the midcell accumulation of YFP-RodA was considerably enhanced following the hyperosmotic shock (Fig. 8C). The same happened for GFP-PBP1a (Fig. 8D).
In nature, bacterial species are found in vastly different osmotic environments. While some species display broad osmotolerance, many, if not most, do not, and yet they remain susceptible to osmotic fluctuations within their habitats. In this study, we show that C. crescentus displays a narrow dynamic range of osmotolerance, with an upper limit of growth osmolality of about 225 mosmol/kg (in a minimal medium lacking osmoprotectants). This is consistent with the ecological distribution of this organism in the wild.
We also show that in C. crescentus, PBP2 and at least two other cell wall proteins, RodA and PBP1a, are recruited to the divisome following osmotic upshifts. This effect occurs even in response to an increase in external osmolality as low as 40 mosmol/kg (for example, by adding ∼20 mM NaCl to the growth medium, which corresponds to an addition of only 1.2 g of NaCl per liter of solution). This result has important implications, as many laboratory procedures routinely include steps in which cells are exposed to an environment with a different osmolality (e.g., washing in buffered solutions). Osmotic upshifts as low as 40 mosmol/kg are also likely to occur in natural environments, which often are unstable. Furthermore, many bacteria, including Caulobacter species, can form high-cell-density biofilms where the osmolality is expected to fluctuate, for example, with the release of cellular components from cell death occurring within the biofilm.
While very small in absolute terms, an osmotic upshift of 40 mosmol/kg is considerable in relative terms when it occurs in low-osmolality environments. For instance, the PYE medium has an osmolality of ∼22 mosmol. An increase in 40 mosmol generates an approximately 3-fold increase in the osmolality of the growth medium. Such a relative increase causes water to flow out of the cytoplasm. Cells respond to an increase in external osmolality by transporting and synthesizing compatible solutes to increase their internal osmotic strength and return water to the cytoplasm (8, 38). Long-term adaptation in bacteria, including C. crescentus (2, 41), involves gene expression and cellular remodeling.
Efflux of water due to an osmotic upshift results in loss of turgor pressure and shrinkage of the cytoplasmic space (6). Concomitantly, the PG becomes less stretched. The relocation of PBP2 to the division site does not require gene expression, and the observation that it occurs remarkably fast (≤40 s) suggests that this event happens in response to the initial phase of osmotic stress. The specific event triggering PBP2 relocation is unclear. Perhaps PBP2 senses the relaxation of the PG layer or local contractions of the cytoplasmic membrane, which may affect the interaction of the inner-membrane-anchored PBP2 with its PG substrate. It is possible that the division plane is less sensitive to such perturbations because of the accumulation of trans-envelope complexes (e.g., the divisome and the essential Tol/Pal complex [39]) that connect the outer membrane, the PG, and the inner membrane.
Interestingly, after osmotic upshift, dispersion of PBP2 from midcell is very slow and requires cell growth. The observation that PBP2 is recruited to the divisome for one to two generations raises the possibility that PBP2 may participate in the adaptation to higher-osmolality environments. It is theoretically possible that the accumulation of PBP2 at the FtsZ ring location may increase the cross-linking of the septal PG and thereby help strengthen the septal PG during the recovery period when the cell remains vulnerable. Addressing these hypotheses will require a better understanding of cell wall morphogenesis and osmotic stress.
While a role for PBP2 in osmotic adaption in C. crescentus remains speculative, our study clearly uncovers a previously unsuspected link between important cell wall proteins (PBP2, RodA, and PBP1a) and osmotic stress in C. crescentus. PBP2 and RodA are often referred to as cell elongation-specific proteins essential for lateral PG growth. Our study shows that in C. crescentus, these proteins can be recruited to the divisome upon osmotic upshifts. Under these conditions, PBP3 remains colocalized with the FtsZ ring (data not shown), indicating that both PBP2 and PBP3 transpeptidases coexist at the division plane. This suggests that the notions of phase specificity (cell elongation for PBP2 and cell division for PBP3) and mutual exclusivity of these transpeptidases are likely oversimplifications, a view also supported by studies in other bacteria (11, 28, 35). Do PBP2 and PBP3 use different PG substrates near midcell, or do they compete for the same substrates? Are these two essential transpeptidases part of the same PG-synthesizing complexes? C. crescentus can offer a valuable model system for addressing these questions in the future.
In conclusion, our findings uncover an unsuspected environmental regulation of the localization of proteins involved in cell wall morphogenesis and stress the need for a better understanding of the fluctuating physicochemical environments in which bacterial cells live.
We thank Tanneke Den Blaauwen for the LMC1840 strain and Janet Wood and the members of the Jacobs-Wagner laboratory for helpful discussion. We are also grateful to Michael Higley for sharing his osmometer.
This work was supported by the National Institutes of Health (GM076698 and GM065835 to C.J.-W.). C.J.-W. is an Investigator of the Howard Hughes Medical Institute. R.P. was supported in part by a Yale Anderson fellowship. C.N.T. was in part supported by a Yale College Dean's Office Science, Technology and Research Scholars (STARS II) fellowship. T.C. was in part supported by a Yale Anderson fellowship and by a postdoctoral fellowship (SFRH/BPD/26232/2006) from the Fundação para a Ciência e Tecnologia.
- Received 21 February 2012.
- Accepted 5 April 2012.
- Accepted manuscript posted online 13 April 2012.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.00260-12.
- Copyright © 2012, American Society for Microbiology. All Rights Reserved.


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Obituary: Mary Etta Dailey, 71
Mar 19, 2015 12:44PM
By Kieran Gilman
TEWKSBURY - Mary Etta (Davis) Dailey, age 71, died peacefully on Monday evening, March 16, at the Lahey Clinic in Burlington following a brief illness. She was the beloved wife of John J. Dailey, with whom she had celebrated a 46th wedding anniversary this past October 5th.
Mary Etta was born in Westbrook, Maine, on December 8, 1943, one of four daughters of the late Paul and Nellie (Robinson) Davis. She was raised in Westbrook and graduated from Westbrook High School. She went on to graduate from the Pierce Secretarial School in Boston.
She worked at CMGI in Wilmington as a data entry specialist until her retirement ten years ago.
She was a member of the Daughters of the American Revolution, enjoyed flower gardening, and time with her family. She and her family enjoyed seasonal camping at their trailer in Denmark Maine.
Besides her husband she leaves a daughter, Anne Marie Morgan and her husband Tim of Merrimack, NH; two sons, John P. Dailey of Hyannis, and Matthew T. Dailey of Brighton; two beloved granddaughters, Lauren Morgan and Sarah Morgan; many nieces and nephews; and was predeceased by her infant son, James F. Dailey; and three sisters, Pauline Lorfano, Priscilla Waters, and Sylvia MacDonald.
Visiting hours are Friday, March 20, from 4:00 until 7:00 p.m. in the Tewksbury Funeral Home, corner of 1 Dewey and 975 Main Sts. (Rte. 38) TEWKSBURY CENTER, Ph. (800 in MA or 978) 851 2950. Her Funeral Service will be held Saturday, March 20, at 11:00 a.m. in the funeral home, followed by burial in Tewksbury Cemetery. In lieu of flowers, memorials to the American Liver Foundation, 88 Winchester St., Newton, MA 02461 or www.liverfoundation.org, will be appreciated. tewksburyfuneralhome.com

subset: commoncrawl


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Many states in the U.S., including North Carolina, have strict laws against motorcyclists riding without a helmet. Authorities have tried to emphasize how important it is for motorcyclists to wear helmets because the helmet serves as effective protection in the event of a motorcycle accident. A helmet helps to reduce head injuries, leading to fewer fatalities from accidents.
Points to remember about the revocation of a driver's license
Drivers of commercial vehicles in North Carolina should be aware of the rules regarding revocation of a driver's license. The Motor Vehicles Division has the authority to revoke the driver's license of a person if that person has been convicted of a certain listed offense. If a driver has been convicted of manslaughter while operating a motor vehicle, the same offense may lead to the revocation of the driver's license. Apart from that, if the driver is found to be driving under the influence of alcohol or any other drug, the division has the right to revoke that person's the driver's license.
North Carolina drivers often break the "move over" law
Many North Carolina residents may be aware that in 2002 our state passed a law that required vehicles to move over or reduce their speed if there is an emergency vehicle near them. That law was meant to safeguard accident victims, emergency crews and law enforcement officers who might be present at the scene of an accident. Sadly, as recent reports state, many drivers in the state are unaware of this law.
North Carolina truckers can face higher truck accidents penalties
Commercial vehicles, such as trucks and other large commercial vehicles, which carry large loads in North Carolina, are subject to more stringent laws than other personal vehicles. Commercial vehicles, like trucks, offer their services for a price. With that in mind, in the event of an accident, the trucking company may find itself in the position of being indicted.
What is the statute of limitations for personal injury cases?
The victim of a car accident often not only suffers serious physical injuries but the person can also face extreme financial challenges because of the car accident. Medical expenses to treat injuries are often very high; those injuries include head injuries as well as internal and external bleeding. In many cases, the victim of a car accident suffers permanent disabilities as well.


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A POIGNANT ceremony marking Holocaust Memorial Day was hosted by Cheshire East Council on Friday.
Knutsford Academy Choir provided an emotional soundtrack to the service of remembrance at Sandbach Town Hall, held the day before the international Memorial Day.
Attendees heard personal testaments of two survivors – sisters Sonja Sternberg, 91, and Gisela Feldman, 94 – who were among 1,000 refugees aboard the infamous SS St Louis when it was rejected by Cuba and the USA.
More than a quarter of the ship's passengers died at the hands of the Nazis in concentration camps.
Mark Palethorpe, CEC's acting executive director of people, was among those present, along with CEC mayor Cllr Arthur Moran and mayor of Sandbach Cllr Martin Forster.
Mr Palethorpe said: "It is important that we continue to remember the Holocaust and subsequent genocides and never forget the devastation visited upon so many innocent lives
"I am particularly pleased to see so many young people here today, as it is vital that the lessons learned from the Holocaust are passed from generation to generation.
"This solemn ceremony is an occasion when the people of east Cheshire join together, as a community, to remember, bear witness and say 'never again'."
The Holocaust resulted in the death of an estimated six million Jews, two million Gypsies, 15,000 homosexual people and millions of others by the Nazi regime and its collaborators.
Since 1945 there have been several other attempted genocides across the world – including Rwanda, Cambodia and Bosnia – and these are also commemorated on Holocaust Memorial Day.


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Research interests
- Chemical communication
- Social evolution
- Behavioral Ecology
- Social polymorphism
Scientific projects
Evolution of fertility signals in halictine bees (Hymenoptera: Halictidae)
It is thought that in insects chemical communication is one major precondition for the evolution of sociality. Queen recognition by the means of so-called fertility signals has been shown to occur in many groups of eusocial insects. However, until now the chemical structure of these fertility signals was only characterized in a few species, though insights into the structural identity of fertility signals are crucial to understand the evolution of eusociality and colonial living in insects. Studying criteria accompanying with the evolution of fertility signals in highly eusocial insect taxa like termites, honeybees or ants, which exhibit a highly developed and complex form of eusociality, are complicated as their eusocial living is mainly obligate. Hence it seems to be more suitable to conduct comparative studies with closely related species which exhibit different social level. Halictine or sweat bees (Hymenoptera: Halictidae) are particularly suitable model organisms to study the evolution of sociality in insects as they show a high variability of social behavior ranging from primitive eusocial species to solitary ones including communal as well as socially polymorphic ones.
In this context my PhD thesis focuses on the evolution of fertility signals within different halictine bee species including socially polymorphic as well as primitively eusocial ones. By combining behavioral assays with chemical and genetic analyses, I intend to identify the chemical composition of fertility signals and the key genes which are involved in their biosynthetic pathways.
Steitz I, Ayasse M (2020) Macrocyclic Lactones Act as a Queen Pheromone in a Primitively Eusocial Sweat Bee. Current Biology 30,1-6 doi.org/10.1016/j.cub.2020.01.026
Steitz I, Brandt K, Biefel F, Minat Ä, Ayasse M (2019) Queen Recognition Signals in Two Primitively Eusocial Halictid Bees: Evolutionary Conservation andd Cast-Specific Perception. Insects 2019, 10, 416 . doi: 10.3390/insects10120416
Vogt J, Klaus VH, Both S, Fürstenau C, Gockel S, Gossner MM, Heinze J, Hemp A, Hölzel N, Jung K, Kleinebecker T, Lauterbach R, Lorenzen K, Ostrowski A, Otto N, Prati D, Renner S, Schumacher U, Seibold S, Simons N, Steitz I, Teuscher M, Thiele J, Weithmann S, Wells K, Wiesner K, Ayasse M, Blüthgen N, Fischer M, Weisser WW (2019) Eleven years' data of grassland management in Germany. Biodiversity Data Journal 7: e36387. doi.org/10.3897/BDJ.7.e36387
Steitz I, Kingwell C, Paxton R J, Ayasse M (2018) Evolution of Caste-Specific Chemical Profiles in Halictid Bees. Journal of Chemical Ecology 44: 827-837
Conference contributions
Albrecht I, Ayasse M (2016) Always at your service, Your Majesty: Macrocyclic lactones elicit subordinate behavior in Lasioglossum malachurum workers. Chemoökologentreffen, Stuttgart, Germany (Talk)
Albrecht I, Ayasse M (2016) Always at your service, Your Majesty: Macrocyclic lactones elicit subordinate behavior in Lasioglossum malachurum workers. European Meeting of IUSSI, Helsinki, Finland (Talk)
Albrecht I, Ayasse M (2016) Always at your service, Your Majesty: Macrocyclic lactones elicit subordinate behavior in Lasioglossum malachurum workers. 12. Hymenopterologentagung, Stuttgart, Germany (Talk; award for best student talk)
Albrecht I, Paxton RJ, Ayasse M (2015) Function- and population-specific odor bouquets of the halictine bee Halictus rubicundus (Hymenoptera: Halictidae) and the evolution of fertility signals. IV Central European Meeting of IUSSI 2015, Lichtenfels, Germany (Talk)
Albrecht I, Paxton RJ, Ayasse M (2015) Caste-and function-specific odor bouquets and the evolution of fertility signals in sweat bees (Hymenoptera: Halictidae). 108th annual meeting of the German Zoological Society, Graz, Austria. (Talk)
Albrecht I, Paxton RJ, Ayasse M (2014) Funktions- und populationsspezifische Duftbouquets der Furchenbienen Halictus rubicundus und Lasioglossum malachurum und die Evolution von Fertilitätssignalen. 11. Hymenopterologentagung, Stuttgart, Germany (Talk)
M.Sc. Iris Steitz
Institute of Evolutionary Ecology and Conservation Genomics
Ulm University
Albert-Einstein-Allee 11
D-89081 Ulm, Germany
Tel. +49 (0)731 50 22668
Fax +49 (0)731 50 22683
Office: M25 4115


text:
You are currently browsing comments. If you would like to return to the full story, you can read the full entry here: "Bathroom Hardware".
Hi guys,
I'm a senior editor at Clarkson Potter, the illustrated book division of Random House, and also a fanatical bungalow person……Any chance we can spin your tale into an actual BOOK?
Feel free to call me or email me.


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- Real Carbon Fiber Remote Key FOB Case Cover for 2017-up Mercedes Benz
Real Carbon Fiber Remote Key FOB Case Cover for 2017-up Mercedes Benz E-Class E43 W213 E300 E400 Sedan
Type: Universal Product
Vendor: Xotic Tech
Description:This listing is for one set of carbon fiber glossy finished hard shell remote key FOB cover, specially for Mercedes-Benz keyless entry remote key.
• 2017 Mercedes-Benz E-Class E300 E400 Sedan
This stylish remote key cover is a perfect complement for your luxury Newer Mercedes. It is extremely durable and will protect your smart key from everyday wear and tear.
Product Details
• One set of carbon fiber glossy remote key cover
• 2 pieces snap on shells for easy installation
• Good for 2017 and up smart remote key
• Premium quality hard shell plastic
• Protect your smart key from scratches
• Key is not included
• Please make sure your smart key same as picture shown before purchasing
Other fine products


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chickpea salad with feta
Member recipe by wiels000
ServingsServes 4
mediteranian feast
This recipe has been submitted by the Good Food community. Sign in or create a My Good Food account to upload your own recipe creations. Please note that all recipes will be moderated but they are not tested in the Good Food kitchen.
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- 3 tbsp extra vergine oliveoil
- 1 red onion
- 3 gloves of garlic, crushed
- 1 red chilli
- 300 gr chickpeas
- 100 gr feta crumbled
- some coriander and parsley
- 2 tbsp chives or mint
- 1 tbsp lemon juice and zest
- salt and pepper to season


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You have the desire but perhaps something is stopping you.
You understand the benefits of getting active but are constantly putting it off. You've heard the individuals, organisations and celebrities extolling the virtues of an active lifestyle. You understand but feel overwhelmed.
I'd like to share my experience of getting fit. I'm biased here as I have always been active in some shape or form. I just love exercise and try to fit in what I can when I can and perhaps you can do it too.
First of all, start small, then make incremental increases. Starting small can mean doing chores at home; gardening, climbing the stairs between ironing, dancing to music, parking your car a little further away from the your local supermarket or carrying reasonably heavy shopping back to the car.
Increase the frequency; have different days of varying intensity such as light, moderate and tiring. There is a concept in health and fitness called "Perceived Exertion Level". This basically means trust your judgement as to how intense or physically demanding the activity is you are taking part in. If you feel it's making you tired then the chances are you are working your body – and that's a good thing.
It's a fairly reliable indicator of exertion in the absence of any wearable tech. Use that as a guide and – use your common sense. Keep a diary of your activity. This can be a pen and paper journal or electronic notepad. Most mobiles come with software that allow your to make notes or journal. Once you've completed the activity make a note or perhaps put it into somesort of calendar for reference.
One you have tracked your activity, this will give you the facts on frequency. Make a note of how tired you feel afterwards. You can also start cataloguing your sleep and your moods if you are going through a stressful period. It all helps with the 'Mind – Body' connection.
Keep going. Keep making incremental increases or undertake more challenges once you've built up enough confidence. You'll find that soon you'll build a virtuous cycle. Do not beat yourself up if things get in the way and you have not been as active as you'd like to. It's normal to have emergencies, lazy days or otherwise known as rest days. This is an invaluable opportunity to recover, rest, recharge and regroup. Taking adequate rest is an important part of the health and wellbeing equation.
Do not underestimate the power of nutrition and sleep. If you're a family and have a baby, or young children then you may think this isn't going to work for me. Just think about the activity of loading and unloading a buggie from your car, take turns as parents to push it round a circuit of your local park or as you wind round Ikea.
You may be able to ask your partner for some time off from running your household to go out for a walk or cycle ride. Indeed this will benefit your family in the long run as you are acting as a team to support your household and it's important that as a couple you both takes turns to be rested. You'll both be more resilient in the long run and you'll be doing your bit for the environment.
So, just get started and ACT for your own health and wellbeing and for your kids.
If you want to read more about the subject of health and wellbeing I recommend anything by Patrick Holford and Dr. Michael Mosley.


text:
Lobbying continues to be a good business to be in.
According to the Center for Responsive Politics, organizations spent $3.42 billion on lobbying in 2018. This was the biggest amount in a decade. Here are more details.
It was the U.S. Chamber of Commerce which ponied up the most: $95 million. Because the trade association is well-funded it is to be feared by liberals.
Currently, conservative lines of thought have the upper hand in decision-making by so many institutions such as the U.S. government.
But that could be changing with the Democrats having a majority in the U.S. House. And there's no telling how Election 2020 will play out. If the stock market goes to hell (right now the DOW is down 342+) the powerful could sour on conservatism.
In fact, the whole gilded age could implode. The loudest voices in the culture could be ordinary people.
Brilliantly, AOC has positioned and packaged herself as EveryWoman. Her influence will continue to increase, as long as she doesn't begin to chase after money. Without money, a politico (and everyone else) has nothing to lose. Disruptive policy proposals can rip.
Get amazing results through out of box communications strategies/content. Complimentary consultation email@example.com. I help, not sell.


text:
If water damage strikes your home, defective parts or faulty installation or repairs could be the cause.
If water damage to your home is caused by defective parts, or faulty installations or repairs, we will hold any negligent parties responsible. Successful recovery from a negligent party may result in a refund of a portion or all of your deductible.
For this reason, be sure to save parts, product manuals, purchase receipts and any applicable repair/installation invoices for your claim representative. Ask repair technicians to provide you with any defective pipes or other parts that were replaced, and provide them to your Farmers claims representative for use in claiming reimbursement from a negligent party.
Defective items could include (but aren't limited to):
Note: This scenario is only an example and not a promise to pay. All claims are handled based on the facts of the loss, the applicable policy language and applicable state law. All factors are taken into consideration prior to payments being issued.
Farmers' Emergency Mitigation Referral Program (E M R P) works with vendors certified in the cleaning and disaster restoration business. We work with several networks and will refer a vendor to you on claims that qualify.
Farmers' Emergency Mitigation Referral Program vendors provide:
Farmers' Emergency Mitigation Referral Program vendors are not affiliated with the Farmers Insurance Group of Companies.
The convenient way to report losses while at home or on the go.
Do you have a claims related question?
Chat is Available 8am - 9pm CST


text:
Price: £25.00
Quantity: 1 available
Book Condition: Good
8vo. Volume 2 only (of 2). Ex library with usual library markings. Eps and frontis foxed with sporadic foxing throughout. Good otherwise in original dark grey cloth gilt. A little worn withexternal splits to cloth at hinges (although book remains sound) and gilt library class mark to spine.
Title: Memoirs of Viscountess Sundon, Mistress to the Robes to Queen Caroline VOLUME 2
Categories: Top Floor,
Publisher: London, Henry Colburn: 1847
Binding: Hard Cover
Book Condition: Good
Size: 8vo - over 7¾" - 9¾" tall
Item: 1.00 Item
Seller ID: 16180
Keywords: Memoirs Viscountess Sundon Queen Caroline George II Mrs Thomson Biography Letters,