text:
The Escherichia coli hlyE gene (also known as clyA or sheA) codes for a novel pore-forming toxin. Previous work has shown that the global transcription factors FNR and CRP positively regulate hlyE expression by binding at the same site. Here in vivo transcription studies reveal that FNR occupies the hlyE promoter more frequently than CRP, providing a mechanism for the moderate upregulation of hlyE expression in response to two distinct environmental signals (oxygen and glucose starvation). It has been reported that H-NS interacts with two large regions of the hlyE promoter (PhlyE), one upstream of the −35 element and one downstream of the −10 element. Here we identify two high-affinity H-NS sites, H-NS I, located at the 3′ end of the extended upstream footprint, and H-NS II, located at the 5′ end of the extended downstream footprint. It is suggested that these high-affinity sites initiate the progressive formation of higher order complexes, allowing a range of H-NS-mediated regulatory effects at PhlyE. Finally, the identification of a SlyA binding site that overlaps the H-NS I site in PhlyE suggests a mechanism to explain how SlyA overproduction enhances hlyE expression by antagonizing the negative effects of H-NS.
Recently, a novel pore-forming toxin, designated HlyE, ClyA, or SheA, was identified for Escherichia coli and Salmonella enterica serovars Typhi and Paratyphi A (2, 6, 7, 10, 16, 17, 21, 22, 35). The three-dimensional (3-D) structure of HlyE shows that it is a mostly α-helical, long (∼100 Å), rod-shaped molecule with a hydrophobic two-stranded antiparallel β-sheet at one end that is thought to allow an interaction between HlyE and target membranes (35). The HlyE protein forms pores in target membranes that appear in electron microscopy (EM) images as ring-shaped structures with internal diameters of 50 to 55 Å, when viewed from above, and as 100- to 105-Å spikes in a side view, suggesting that HlyE does not undergo large conformational changes during pore formation (35).
Two members of the CRP family of transcription factors control the expression of hlyE in E. coli K-12 by binding at the same site centered at −61.5 bp upstream of the hlyE transcriptional start (10, 36). Thus, CRP enhances hlyE expression in response to glucose starvation (36) and FNR enhances hlyE expression in response to oxygen starvation (10, 11, 23). Both CRP and FNR are ∼50-kDa homodimers that bind related inverted repeats with a TGANNNNNNTCA core motif (12). At the hlyE promoter (PhlyE), this site (TTTGATATTTATCATA) most closely resembles an FNR site (9 of 10 nucleotides match the FNR consensus, TTGATNNNNATCAA, compared to 8 of 10 matches to the CRP consensus, TGTGANNNNNNTCACA; discriminatory bases are underlined). However, it has been shown that in certain circumstances CRP can recognize FNR sites, although the affinity of CRP for an FNR site is 50-fold lower than that for an equivalent CRP site (27).
A further layer of regulation is provided by the nucleoid structuring protein H-NS (36). The H-NS protein influences the expression of many genes in E. coli K-12. It is a small ∼15-kDa protein that forms higher order complexes in a concentration-dependent manner (30, 34). The H-NS protein was shown to interact with a large region of PhlyE (from −137 to +172, relative to the transcription start site) to repress hlyE expression (36) after the observation that an hns mutant strain has a hemolytic phenotype (9).
A hemolytic phenotype was also conferred upon E. coli K-12 by the overproduction of either the E. coli or Salmonella enterica transcription factor SlyA (16, 21). The SlyA protein is a member of the MarR family of transcription factors that includes MarR and EmrR (E. coli), PecS (Erwinia chrysanthemi), HprR (Bacillus subtilis), and RovA (Yersinia enterocolitica) (20, 24). The 3-D structures of E. coli MarR and a SlyA-like protein from Enterococcus faecalis provide the structural archetypes for this family of proteins. The 3-D structures show that they are homodimers in which each subunit possesses a winged-helix DNA-binding domain (1, 37). A recent characterization of the Salmonella SlyA protein revealed that it is also a homodimer (∼32 kDa) that recognizes an inverted repeat sequence in target promoters (32). Site-directed mutagenesis of PhlyE led to the suggestion that a GC-rich sequence located between an unusual heptameric −10 element (TATGAAT) and a conventional −35 element might be the site of SlyA action (17). Here we show that the regulation of hlyE expression by H-NS is more complex than was previously thought and that the overproduction of SlyA enhances hlyE expression by antagonizing the negative effects of H-NS.
Bacterial strains, plasmids, and microbiological methods.Relevant characteristics of the bacterial strains and plasmids used are given in Table 1. Isogenic derivatives of M182 were constructed by P1vir-mediated transduction. Bacteria were grown in Lennox broth (L-broth; contains yeast extract, 5 g liter−1; tryptone, 10 g liter−1; and NaCl, 5 g liter−1) at 37°C supplemented with glucose (0.2% [wt/vol]), ampicillin (150 μg ml−1), tetracycline (35 μg ml−1), and chloramphenicol (20 μg ml−1), as appropriate. For β-galactosidase activity measurements (19), anaerobic cultures were grown in sealed bottles filled to the neck with medium, and aerobic cultures were grown in conical flasks (250 ml) containing medium (10 ml), with vigorous shaking (250 rpm) at 37°C. Hemolytic activities were estimated by measuring the areas of hemolysis surrounding individual colonies after 16 h of growth at 37°C on blood agar under aerobic conditions.
Gel shift assays.Initial H-NS gel shifts used a PCR-amplified 696-bp fragment of PhlyE containing all of the previously reported sequences protected by H-NS from DNase I digestion (36) plus additional upstream and downstream sequences. In subsequent experiments, this fragment was further resolved into four subfragments by restriction digestion with SspI, BsaMI, and DraI. Target DNAs (200 to 500 ng) were incubated with H-NS (0 to 6 μM) in 25 mM HEPES, pH 7.6, containing 0.1 mM EDTA, 10% (vol/vol) glycerol, 5 mM dithiothreitol, 50 mM KCl, and 0.01 mg of poly(dI-dC) ml−1 for 15 min at 25°C in a total volume of 10 or 20 μl. Complexes were separated in 6% (wt/vol) nondenaturing Tris-borate-EDTA-buffered polyacrylamide gels for the long PhlyE fragment or in 12.5% (wt/vol) gels for the dissected PhlyE fragments. DNA was visualized by staining with ethidium bromide. The interaction of H-NS and SlyA with PhlyE was also investigated by using PhlyE DNA amplified by PCR with pGS1064 (10) (Table 1) as the template and with pUC/M13 forward and reverse primers. The product was digested with BamHI and radiolabeled with Klenow enzyme and [32P]α-dGTP (26). For H-NS, ∼10 ng of DNA and 0.04 to 2.0 μM H-NS were coincubated for 5 min at 25°C in a solution containing 10 mM Tris-HCl (pH 7.6), 50 mM MgCl2, 2 mM spermidine, and 15 mM potassium glutamate (total incubation volume, 10 μl). For SlyA, ∼10 ng of DNA and 0.06 to 0.63 μM SlyA protein were coincubated for 2 min at 25°C in a solution containing 10 mM Tris-HCl (pH 9.0), 50 mM KCl, and 0.1% (vol/vol) Triton X-100 (total incubation volume, 10 μl). Reactions were then loaded onto gels for autoradiographic analysis.
DNase I footprinting.For H-NS, the reactions (total volume, 10 μl) contained radiolabeled PhlyE (∼10 ng), H-NS (1 μM), 10 mM Tris-HCl (pH 7.6), 50 mM MgCl2, 2 mM spermidine, and 15 mM potassium glutamate. For SlyA, the reactions (total volume, 10 μl) contained radiolabeled PhlyE (∼10 ng), SlyA (2.0 and 4.0 μM), 20 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 10 mM dithiothreitol, and 5% (vol/vol) glycerol. The mixtures were incubated for 2 to 5 min at 25°C, followed by digestion with DNase I (1 μl of a 1-U μl−1 solution for 5 to 60 s at 25°C). Reactions were stopped by the addition of 200 μl of 0.3 M sodium acetate (pH 5.2) containing 20 mM EDTA, followed by phenol-chloroform extraction. The DNA was ethanol precipitated and resuspended in 10 μl of loading buffer (80% [vol/vol] formamide, 0.1% [wt/vol] sodium dodecyl sulfate, 10% [vol/vol] glycerol, 8 mM EDTA, 0.1% [wt/vol] bromophenol blue, and 0.1% [wt/vol]) xylene cyanol) for electrophoretic fractionation on 6% (wt/vol) polyacrylamide-urea gels and autoradiographic analysis. Maxam and Gilbert G tracks were used to provide a calibration (18).
Other methods.The H-NS protein was provided by C. F. Higgins (MRC Clinical Sciences Centre, Hammersmith Hospital, London, United Kingdom), and the SlyA protein was isolated from E. coli strain JRG4385 (Table 1) as previously described (32). The manipulation of DNA, PCR, and plasmid constructions was achieved by conventional methods (26). Plasmid copy numbers were estimated by the method of Taylor and Brose (33).
Effects of the global transcription factors FNR, CRP, and H-NS on hlyE expression in liquid culture.Previous studies have shown that FNR, CRP, and H-NS contribute to hlyE expression when E. coli is grown on a solid medium (10, 36). Both FNR and CRP make positive contributions, whereas H-NS acts negatively under these conditions. Because bacteria grown on solid media are exposed to different microenvironments due to the generation of concentration gradients of nutrients and oxygen within colonies, the starting point for this work was to investigate the effects of all three global regulators on hlyE expression in liquid cultures by using a plasmid-based hlyE::lacZ gene fusion (pGS1629; Table 1). The reporter plasmid contained a fragment of DNA stretching from −299 bp to +82 bp relative to the hlyE transcript start site ligated with pRS415 (Table 1). Initially, transcriptional activity from PhlyE was estimated for anaerobic cultures of parental (M182), fnr, crp, and hns strains transformed with pGS1629 (Table 1), after growth in L-broth at 37°C for 16 h. The data revealed that both FNR and CRP made significant positive contributions to hlyE expression, whereas the hns mutation resulted in slightly reduced hlyE transcription under these conditions (Fig. 1a). It was predicted that the addition of glucose (0.2% [wt/vol]) to the medium would abolish the effects of CRP. Accordingly, hlyE expression was reduced in cultures of the parental strain containing glucose compared to those without glucose (Fig. 1b). Moreover, while hlyE expression was reduced in the fnr strain compared to that in the parent strain in the presence of glucose, it was, as expected, unaffected in the crp mutant (Fig. 1b). The transcription of hlyE in the hns strain under anaerobic conditions in the presence of glucose is presumably driven by FNR alone. Under these conditions, hlyE expression was greater for the hns strain than for the corresponding parental cultures, suggesting that H-NS has a negative effect on FNR-driven hlyE expression (Fig. 1b). Thus, we concluded that CRP, FNR, and H-NS all contribute towards the regulation of hlyE expression.
Similar experiments were done with aerobic cultures. For these conditions, we predicted that the glucose-responsive effects of CRP would be retained, whereas the positive regulation by FNR would be abolished. The prediction for the CRP response was confirmed by the data (Fig. 1c and d). However, the data for the parental and fnr cultures suggested that even under aerobic growth conditions, some active FNR was present in the bacteria, and that even this small amount of active protein (relative to anaerobic conditions) was sufficient to interfere with CRP-mediated hlyE expression (Fig. 1c). Thus, these data suggest that under such conditions, FNR and CRP are in competition for binding at PhlyE, and that the simplest explanation of these observations is that FNR is more efficiently bound than CRP at PhlyE but is not as effective as CRP in activating hlyE transcription. Such an interpretation is consistent with the sequence of the FNR/CRP box in PhlyE, which lacks the discriminatory G-C base pairs that promote CRP specificity, and with the observation that, unlike CRP, FNR is a poor activator of class I promoters such as PhlyE (reviewed by Green et al. in reference 12). The effect of the hns mutation on hlyE expression in aerobic cultures was also interesting (Fig. 1c and d). In the absence of glucose, H-NS appeared to have a positive effect on hlyE expression (Fig. 1c). However, in the presence of glucose, H-NS appeared to have little effect on hlyE expression (Fig. 1d). Thus, under aerobic conditions, H-NS has a positive influence on CRP-mediated hlyE expression. This positive effect of H-NS was not observed under anaerobic conditions, presumably because FNR and not CRP occupies PhlyE under these conditions.
To further investigate the effects of the regulators on hlyE expression, we monitored the activity of the hlyE::lacZ fusion in pGS1629 in strains carrying multiple relevant mutations. Under anaerobic conditions in the absence of glucose, hlyE expression in an fnr crp hns triple mutant was low compared to that of the parental strain (Fig. 2a). Restoring hns did not affect hlyE expression. Similarly, restoring CRP did not enhance hlyE expression, despite the absence of glucose (Fig. 2a). However, the restoration of fnr produced a significant increase in hlyE expression under these conditions (Fig. 2a). This suggests that during anaerobic growth in liquid cultures in the absence of glucose, FNR is the major regulator of hlyE expression, and that CRP-mediated hlyE expression requires the presence of H-NS. The pattern of expression obtained in anaerobic cultures in the presence of glucose was similar to that obtained in the absence of glucose, except that hlyE expression in the fnr crp hns triple mutant and the crp hns double mutant was significantly higher than that for the corresponding cultures that lacked glucose (Fig. 2b). The hlyE expression patterns for equivalent aerobic cultures in the presence and absence of glucose were similar to those obtained under anaerobic conditions (Fig. 2c and d).
In summary, the data presented here suggest that FNR and CRP are positive regulators of hlyE expression in liquid culture in response to oxygen and glucose starvation, respectively. This is in agreement with observations made with cultures grown on a solid medium (36). However, rather than the approximately eightfold enhancement in hlyE expression observed in an hns mutant on solid medium (36), in liquid culture H-NS has a positive effect on CRP-driven hlyE expression, a negative effect on FNR-driven hlyE expression, and little intrinsic regulatory activity in the absence of FNR and CRP.
H-NS binds at two regions of the hlyE promoter with high affinity.Footprinting studies have shown that FNR and CRP activate hlyE expression from the same site centered −61.5 bp upstream of the SlyA-associated transcription start site (10, 36). A further footprinting analysis indicated that H-NS protects a large region of the hlyE promoter, extending from −137 to +172 (relative to the transcription start site) (36). To investigate how much of this extensive H-NS protection is required for the observed regulation of hlyE expression, we used two approaches as follows.
Firstly, a 696-bp region of the hlyE promoter region was amplified by PCR (Fig. 3a). Gel shift assays revealed that H-NS retarded the mobility of this fragment upon electrophoretic separation in Tris-borate-EDTA-buffered polyacrylamide gels (Fig. 3b). In the presence of 1 μM H-NS, retardation of some of the DNA target was observed as a smear in the gel. Upon the addition of further H-NS (2 μM), the free DNA was replaced entirely by a retarded smear. Only at 4 μM H-NS was a distinct retarded complex observed (Fig. 3b). The amplified fragment contains restriction sites for SspI, BsaMI, and DraI, such that a triple digest yields four fragments (Fig. 3a). Gel shift assays using a mixture of hlyE DNA fragments as the target DNA revealed that H-NS interacts with all four PhlyE fragments to some extent. However, the 147-bp DraI-BsaMI fragment was bound with the highest affinity, with 50% of this fragment retarded at H-NS concentrations of ∼3 to 4 μM (Fig. 3c and d). Thus, this region of PhlyE (−91 to +56) contains both the FNR/CRP box (−61.5) and the highest affinity H-NS site(s).
Secondly, for testing of whether the region of PhlyE containing the high-affinity H-NS site(s) was sufficient to account for the pattern of hlyE regulation observed in vivo, another hlyE::lacZ reporter plasmid was used (Table 1) (10). This second gene fusion (pGS1065) contains a minimal hlyE promoter beginning 18 bp downstream of the BsaMI site and ending 25 bp downstream of the DraI site and thus extends from −97 to +61 relative to the transcript start site in the low-copy-number (two to five copies per cell) vector pRW50 (Table 1). Cultures of strains M182 and M182 hns transformed with pGS1065 were grown under anaerobic conditions in the presence and absence of glucose at 37°C for ∼16 h. The transcriptional activity in vivo, as estimated by the measurement of β-galactosidase activity, indicated that this (pGS1065) low-copy-number reporter containing a minimal hlyE promoter responded in the same way as the longer medium-copy-number hlyE::lacZ fusion (pGS1629) to the presence or absence of H-NS (Table 2). Thus, in the presence of glucose, hlyE expression was enhanced in an hns mutant (365 Miller units) relative to the parent (271 Miller units) (compare with Fig. 1b). In the absence of glucose, hlyE expression was slightly lower in the hns mutant (399 Miller units) than in the parent (420 Miller units) (compare with Fig. 1a). Furthermore, there were no significant H-NS-related changes in reporter plasmid copy number (not shown), as judged by the method of Taylor and Brose (33). Thus, it was concluded that the BsaMI-DraI fragment of PhlyE contains all of the significant regulatory elements that control hlyE expression under the growth conditions used here and that the effects on hlyE expression observed with pGS1629 were not significantly affected by the copy number of the reporter plasmids.
Identification of the high-affinity H-NS sites in PhlyE.For determination of the number of H-NS sites within the minimal PhlyE sequence from pGS1065, further gel shift and footprinting assays were undertaken. The gel shifts showed that H-NS bound to PhlyE at concentrations close to those observed for other H-NS-regulated genes (∼1 μM) (3) and that two distinct retarded complexes were formed (Fig. 4a). The locations of the H-NS binding sites were determined by DNase I footprinting (Fig. 4b). Two protected regions were detected: H-NS I, consisting of an A-T-rich region (20 A-T/24 bp) stretching from −75 to −37 which overlaps the FNR/CRP box centered at −61.5; and H-NS II, between −22 and +11, overlapping the −10 element of PhlyE. The H-NS II site contains a hypersensitive base at position −17 that is separated by several unprotected bases (−16 to +1) from another protected A-T-rich region (8 A-T/10 bp) (Fig. 4b). Based upon the size and properties of H-NS and the extent of the DNase I footprint, it is likely that several H-NS molecules bind PhlyE.
Effects of H-NS on SlyA-promoted hlyE expression.Overproduction of the E. coli or S. enterica SlyA protein confers a hemolytic phenotype on E. coli K-12 by enhancing hlyE expression (16, 17, 21). To test the effects of H-NS on SlyA-driven hlyE expression, we measured β-galactosidase activities from cultures of isogenic parental and hns strains carrying the hlyE::lacZ plasmid pGS1065 and either a multicopy S. enterica SlyA expression plasmid (pGS1657; Table 1) or, as a control, the vector (pBluescript). As expected, irrespective of the addition of glucose, the presence of multicopy slyA increased hlyE expression in cultures of the parental strain, although the enhancement was greater for cultures lacking the glucose supplement (Table 2). In contrast, in the absence of hns, SlyA overproduction did not enhance hlyE expression (Table 2). This pattern of expression was supported by qualitative studies in which the same strains were grown on blood agar plates. These revealed that multicopy slyA enhanced hemolytic activity associated with the parental strain but not with the hns strain (not shown). These results confirm and extend the observations of Westermark et al. (36) and suggest that, either directly or indirectly, SlyA activates hlyE expression by modulating H-NS binding and ultimately by counteracting the negative effects of H-NS. This suggestion was supported by the observations that multicopy slyA did not enhance hlyE expression in a crp fnr double mutant (Table 2) and that SlyA did not promote open complex formation at PhlyE in vitro (not shown). Thus, it was concluded that SlyA, like H-NS, has no intrinsic activator role at PhlyE in the absence of FNR and CRP.
Interaction of SlyA with the E. coli hlyE promoter.The pattern of regulation reported above suggests that SlyA, like H-NS, interacts directly with the E. coli hlyE promoter. Therefore, gel shift assays were used to investigate the interaction of purified SlyA with PhlyE. These experiments revealed that the addition of 0.16 to 0.31 μM SlyA was sufficient to retard the mobility of PhlyE (Fig. 5a).
A consensus SlyA DNA target was recently suggested to exist (32), and an inspection of the PhlyE sequence revealed at least two possible matches. Therefore, the location of the SlyA binding site(s) within PhlyE was investigated by DNase I footprinting (Fig. 5b). A protected region, −70 to −38 relative to the previously determined SlyA-associated transcript start site (17, 36), was observed in the presence of 2 μM SlyA. This region of protection was extended further downstream to −31 when the concentration of SlyA was increased to 4 μM (Fig. 5b). Thus, SlyA protects a region of PhlyE similar to the H-NS I region (−75 to −37; Fig. 4b). The cross sections of the E. coli MarR and E. faecalis SlyA-like protein dimers are ∼70 Å (1, 37) and therefore would be expected to protect ∼20 bp of DNA. Thus, the protection observed here (up to 40 bp) suggests that two SlyA dimers are bound at PhlyE. An inspection of the DNA sequence of the protected region revealed two related sequences with partial dyad symmetry that resemble the proposed SlyA binding consensus; they are SlyA I (−61TTATCATATTAA−50, with 8 of 12 bases (bold) matching the SlyA binding site consensus TTAGCAAGCTAA) and SlyA II (−50ATAGAAATAAAG−39, with 6 of 12 bases matching the consensus). The putative presence of two SlyA dimers at PhlyE was consistent with the two distinct complexes with retarded mobilities in the gel shift assays (Fig. 5a). The site centered at −55.5 is the better match to the SlyA binding site consensus and may be occupied in preference to the site centered at −44.5, which accordingly is less similar to the consensus.
The starting point for the work described here was the observation that lesions in hns or the overproduction of S. enterica or E. coli SlyA confers a hemolytic phenotype on E. coli K-12 by enhancing the expression of the pore-forming toxin HlyE (9, 16, 17, 21). Here we have shown that H-NS and SlyA interact directly with the hlyE promoter. Both proteins occupy a common region of PhlyE that overlaps the binding site for the global transcription factors FNR and CRP, which are known to activate hlyE expression (10, 36). In vivo and in vitro evidence suggests that SlyA activates hlyE expression by antagonizing H-NS-mediated repression.
The in vivo transcriptional evidence presented here suggests that FNR occupies the hlyE promoter more frequently than CRP even under aerobic conditions. This is evident from enhanced hlyE::lacZ expression in aerobic cultures (compare parent, fnr, and crp strains in Fig. 1c and d). These observations may be explained by a mechanism in which FNR recognizes PhlyE efficiently but acts only as a relatively poor activator of hlyE transcription (10, 11), whereas, conversely, the recognition of PhlyE by CRP is poor, but once CRP is bound, CRP-mediated activation of hlyE expression is efficient. This reciprocity of binding site recognition and transcriptional efficiency provides a mechanism for the moderate upregulation of hlyE expression in response to two distinct environmental signals (oxygen and glucose starvation) rather than, for example, the much larger degree of upregulation when FNR and CRP act synergistically by binding at different sites within the ansB promoter (28). In principle, any pair of transcription factors that recognize similar DNA sequences but have different transcriptional efficiencies could adopt this strategy.
Under some of the conditions studied here, the effects of an hns lesion on hlyE::lacZ expression in liquid cultures were different from those reported by Westermark et al. for bacteria grown on a solid medium (36). This is perhaps not surprising considering the significant physiological differences between cultures grown in liquid and on solid media. In the previous report (36), H-NS acted as a strong repressor of hlyE expression, whereas here H-NS appears to have a positive effect on hlyE expression in the absence of glucose but an attenuated or negative effect on hlyE expression in the presence of glucose (Fig. 1). This suggests that, in general, H-NS inhibits FNR-driven hlyE expression (Fig. 1) but enhances CRP-driven expression in liquid cultures (compare Fig. 1b and c). Moreover, any enhancement of hlyE::lacZ expression in the hns strain observed here did not approach the eightfold increase observed on solid medium (36). Thus, it appears that H-NS modulates PhlyE activity both positively and negatively in response to the prevailing growth and/or environmental conditions, such as growth in liquid and on solid media. This may be a reflection of the formation of different H-NS-PhlyE complexes in response to environmental signals. This idea is supported by the identification of two regions within the larger previously described H-NS-PhlyE complex (36) that bind H-NS with high affinities. The first region (H-NS I) overlaps with, and extends downstream from, the FNR/CRP site (Fig. 6). The extent of protection associated with the H-NS I site (38 bp) suggests that more than one H-NS dimer is bound, and the formation of different subcomplexes could potentially act to modulate FNR/CRP-driven transcription activation either positively (occupation of the downstream portion of H-NS I; thick arrow in Fig. 6) or negatively (occupation of sequences overlapping the FNR/CRP-binding site; thin arrow in Fig. 6). While H-NS usually acts as a negative regulator of gene expression, hlyE is not the only example of a CRP-regulated gene whose expression is positively regulated by H-NS; for example, expression of both the malT and csiD genes in E. coli is stimulated by H-NS (8, 14). Thus, it would appear that a CRP family transcription factor plus H-NS is a versatile combination, providing the means to generate a range of regulatory effects.
The second region of H-NS protection (H-NS II) overlaps the basic promoter elements, and H-NS bound at this region would be expected to repress hlyE expression by promoter occlusion. The findings that SlyA does not enhance hlyE expression in hns cultures grown in liquid medium under anaerobic conditions and that SlyA appears unable to activate hlyE expression in the absence of FNR and CRP and the determination of the location of the region of PhlyE occupied by SlyA (Fig. 6) suggest that SlyA activates hlyE expression by antagonizing the negative action of H-NS. Previous studies have indicated that the intracellular levels of H-NS change with the growth phase. However, whereas one proteomics-based study suggested a fivefold increase in H-NS in stationary-phase cultures (31), a more recent immunological analysis suggested a twofold decrease (4). In addition, we have shown that intracellular H-NS levels increase as growth temperature decreases, as evidenced by an approximately twofold increase in H-NS immunoblot signal intensity from cultures grown at 20°C compared to those grown at 37°C (N. R. Wyborn and J. Green, unpublished data). Thus, it appears that intracellular H-NS levels are influenced by the environment. Therefore, we suggest that when environmental conditions dictate a low intracellular concentration of H-NS, the H-NS I region of PhlyE is occupied to modulate the activity of the upstream activator (FNR or CRP). As the intracellular levels of H-NS increase, H-NS II is occupied and hlyE expression is downregulated by promoter occlusion. The footprinting studies described by Westermark et al. (36) indicated that further increases in H-NS levels result in the formation of higher order complexes that occupy PhlyE from the −10 element to position +172 and from just upstream of the −35 element to position −137. Thus, it appears that these higher order complexes extend from the primary sites of interaction (H-NS I and H-NS II) identified here. Such higher order complexes, which effectively silence hlyE expression, appear to have directionality in that they extend upstream from H-NS I (to as far as position −137) to occlude the FNR/CRP site and downstream from H-NS II (to as far as +172) to occlude the basic promoter elements. This leaves the region between the two primary sites of interaction unoccupied. Thus, we suggest that the formation of different H-NS-PhlyE complexes offers the opportunity for H-NS, in combination with FNR and CRP, to control hlyE expression both positively and negatively. This type of behavior, in which specific patterns of protection are replaced by general protection as the concentration of H-NS increases, has been observed for footprints of gal promoter variants (25). It was also shown previously that this general protection was dependent on the polymerization of H-NS on the DNA and that this was more likely if there was an initial nucleation event on the DNA at specific sites at low H-NS concentrations (25). It appears that the H-NS I and H-NS II regions of PhlyE are such nucleation sites. By competing with H-NS for the region of PhlyE downstream of the FNR/CRP site, SlyA may prevent the formation of the negatively acting H-NS-PhlyE complexes by blocking a primary interaction between H-NS and PhlyE, allowing FNR and CRP to continue to activate hlyE expression. Presumably, the action of the E. coli SlyA protein will prove to be similar to that of the Salmonella protein studied here, given that their primary structures are 89% identical and that the overproduction of E. coli SlyA also enhances hlyE expression (16, 21).
In summary, we have shown that the H-NS-mediated regulation of hlyE expression in E. coli K-12 is more complex than was previously suggested because H-NS can contribute positively as well as negatively to hlyE expression. This range of regulatory activity is probably associated with the progressive formation of higher order H-NS-PhlyE complexes that extend from two primary sites of interaction to ultimately bring about silencing of the hlyE gene. The H-NS and SlyA footprints suggest that H-NS regulation is overcome by the overproduction of SlyA, which by binding at a site that overlaps one of the primary H-NS sites (H-NS I), prevents the formation of repressive H-NS-PhlyE complexes. Further detailed in vitro analyses will be required to fully analyze the complex relationships between H-NS, SlyA, FNR, and CRP and their consequences for hlyE expression.
We acknowledge A. J. G. Moir for DNA sequencing. We thank Jeff Cole and Steve Busby (University of Birmingham, Birmingham, United Kingdom) and Bernt Eric Uhlin (Umea University, Umea, Sweden) for providing bacterial strains. We thank Chris Higgins (Hammersmith Hospital, London, United Kingdom) for providing the H-NS protein.
This work was supported by the Biotechnology and Biological Sciences Research Council, Swindon, United Kingdom.
- Received 30 June 2003.
- Accepted 5 December 2003.
- Copyright © 2004 American Society for Microbiology


text:
Himalayan Salt Cooking Tile Gift Pack
Now everyone can gift affordably with this two pack of Himalayan Salt cooking tiles. Perfect for single servings and easy use on the dinner table.
Keep all your guests happy as you serve those exciting starters, mains or even dessert on these Himalayan salt tiles.
Freeze them for a salt choc ganache dessert with toppings, Heat them like Himalayan salt cooking blocks for serving the steak on or keep them at room temp for your savoury bites..... The ideas are endless... You can even cook on them!
Size 20 x 10 x 2.5cm each
Weight estimate 900g per tile

subset: commoncrawl


text: Metal Angle 90degree 25x25mm For MF Ceiling 3000mm £3.54 (Inc. VAT) £2.95 (Ex. VAT) The MF Suspended System is ideal for commercial and domestic applications, where services are to be accommodated and when upgrading and protecting existing structures. Ceiling height may be varied to accommodate services or ducting, etc providing a high... Add to Cart


text:
Estimate Your Student Loan Payments This lesson will show you how to: Take stock of your current and future student loan debt, Get an idea of your monthly payment obligation You're probably in school for several reasons, but building a better future for yourself is likely high on the list You know that your career dreams are achieved through education No matter what line of work you'll go into, you'll benefit from the skills you'll gain through setting academic and professional goals, and then striving to reach them
In this video series we're going to cast our eyes toward that beautiful future We'll estimate your debt, envision your future income, and discuss how the decisions you make today impact tomorrow Let's start by getting a handle on your current student loan debt There are a number of places you can go to find your total student loan debt You can always speak with the financial aid office at your school to see how much you've taken out in student loans
They have access to information about your past and present student loans The Department of Education also will have your loan records You can visit studentaidgov to punch up your full inventory of federal loans Once you have your list of student loans, determine which loans are subsidized, meaning you are not responsible for the accruing interest while you're in school, which loans are unsubsidized meaning you ARE responsible for the accruing interest during school, and which loans are private
Be sure to note who your loan servicers are, what their contact information is, and what the interest rate is on all of your loans Then use a repayment calculator to determine your monthly payment on these loans Repayment calculators can be found in several places on the web Try the Repayment Estimator at StudentLoansgov
It'll estimate your future payments and give you a repayment plan comparison Every loan borrower's situation is unique, so let's use a hypothetical scenario Let's say Joe just pulled up his loan data He has $15,000 in debt, but he's only half way through his program Using a repayment calculator, he figured his current debt would equal a roughly $150 a month payment on a 10-year standard repayment plan
His next move is to estimate how much he'll need to borrow to finish his program, and how his interest will accrue while he's completing it When you're doing this exercise, remember to factor in tuition and cost of living increases Continuing our hypothetical, Joe doubled his debt over the second half of his degree program With interest accruing, his full student loan debt on graduation day was $32,000 Now Joe's payment is closer to $325 per month on a standard 10-year repayment plan
When loan repayment begins, Joe, as federal student loan borrower, has the option to explore various federal loan repayment plans A standard repayment plan means his monthly payment is fixed for 120 payments Yet, there are several other options and we'll explore those later You can always speak to your federal loan servicer if you have questions about federal repayment plans Okay, our hypothetical is over
Let's get back to reality It's time for you to go through the same exercise as Joe Find your current debt load, estimate the amount of loans you'll need to finish, and then calculate your estimated monthly repayment amount How do you feel about this estimate? Does it seem like it will be manageable? How much money you plan to earn after school may factor in to how you answer those questions We'll talk about calculating your future income in the next video
Join us, won't you?


text:
Over the past year, I have added over 20lb to my already 40lb overweight body. I am 5'1 at 186lb. When I first started college, I was 145lb and, even though I was still overweight, I was pretty happy with my body. Fast forward to my senior year and I have added over 40lb to my weight. For a long time, I was in denial. When I hit 160lb I was still mostly okay with how I looked and felt. At 165 I told myself okay I am going to get disciplined and lose the weight before it gets out of hand. Unfortunately, I have always been a lazy person so I never got disciplined and avoided the scale so I wouldn't feel guilty. That was the biggest mistake I could have made.
Although I do not feel like I look good at almost 200lb, I am mostly worried about how my health is being affected. Lately, I've had issues like growing chin hairs and general exhaustion. I can't even walk up a flight of stairs without breathing heavily for minutes afterward. I feel an increase in urgency because the older I get the less my body is able to recover from abuse and the greater my chances of serious chronic illness. Because of these things I have decided to get myself back on track. Writing this blog will help me become a more healthy weight by holding me accountable. I will also be able to vent my struggles and celebrate my successes.
I hope that by this time next year I will be a functional adult with a healthy body.


text:
Cromwell gets 40 applications for senior center director
CROMWELL >> Town officials have begun the process of filling the position of director of human services/senior center director.
The post has been filled on an interim basis by James Gere since mid-April, when the previous director, Theresa Strong, was dismissed from town service. The town received 40 applications from potential candidates for the position, according to Director of Human Resources Thomas A. Roohr.
"We're still going through the process and reviewing the applications," Roohr said.
Roohr has assembled a committee to begin reviewing the applications in an effort to pare the number down to a manageable few who will then be interviewed in person.
In addition to Roohr, the other members of the review committee include Gere and First Selectman Mertie L. Terry. The final two members of the committee are Selectman Enzo Faienza and Director of Finance Marianne Sylvester.
Strong had worked for the town in one form or another for 25 years. This spring, she became embroiled in a controversy involving a town-owned computer which she frequently used.
Town officials believed she was misusing the computer to conduct personal business.
A police investigation was begun. But it faltered when Strong could not produce the computer and subsequently insisted it had been stolen.
The Board of Selectmen sat in judgment on Strong. A majority of the members found her explanation unsatisfactory. The board eventually voted 5-2 to let Strong go.
Gere, who had chaired a citizens' advisory committee, took over the role on an acting basis. Gere, a former superintendent of schools, served in that capacity for almost a month before the Board of Selectmen voted to approve his interim appointment.
Roohr said Monday he is anxious to fill the position with a new full-time replacement "as soon as possible."


text:
Code: #WB3200CNC
Not yet reviewed. Be the first to write a review
In Stock and available for immediate delivery.
Not what you were looking for?
We have new machinery and equipment arriving weekly; feel free to contact us today to discuss our latest stock!
|Wadkin Bursgreen
|Panel Saw
|Rectified cast iron fixed table dimension
|570 x 1000
|Sliding table dimension
|380 x 3200 mm
|Main saw bore
|30 mm
|Max. cutting height with blade at 90º
|105 mm
|Max. cutting height with blade at 45º
|Main blade speed
|3000/4000/5000 rpm
|Scoring saw blade bore
|Ø 22 mm
|Scoring blade speed
|8000 rpm
|Cutting width adjustment
|CNC Control
|Blade tilting adjustment
|CNC control (0 º~45 º)
|Main saw height adjustment motorize
|CNC Control
|Overhead saw guard
We've put together a selection of products that we think you might also be interested in.
You might find the answer to your question in the FAQ, but if not - we are only too happy to help. You can call us on 0844 844 9949 or use the form below.


text:
Take one look at the bottomless candy bowl in our showroom if you need some proof! We even have the privilege of designing t-shirts for local sweet spots, like Candy Craze and Rick's Bakery! Below, we've included a list of some of our favorite go-to spots when we're looking to satisfy our sugary cravings. Be sure to try them out if you're ever in Fayetteville!
With two locations at opposite ends of town, this cupcake shop is easy to access and hard to resist. Boasting flavors such as chocolate milkshake, blackberry vanilla bean and pancake breakfast, there's sure to be a choice for any craving. If you want a real sugar high, grab a frosting shot for later!
This rustic looking coffee shop serves fabulous espressos and savory meals. However, they also have a special section devoted to sweet crepes. If you'd like to start your day with a Nutella crepe and coffee, you should stop at Arsaga's ASAP!
A fan favorite. If you want to surprise a special someone (or if you're just looking to treat yourself,) Rick's is the place to turn! They have all types of tasty treats that can be personalized for any occasion. Rick's serves donuts, pastries, decorated cakes and cookies, muffins, bagels, dessert cakes, cupcakes, petit fours and more. Step inside and you're sure to see an employee wearing a custom t-shirt designed especially for Rick's Bakery!
Hopefully you won't overdose on sugar with these suggestions, but then again would that really be so bad?


text:
Threat Events
A threat event report is a report of traffic that has been dropped, or that has generated an alert, after matching one of Cisco Talos' intrusion policies. In most cases, there's no need to tune IPS rules. If necessary, you have the option to override how an event is handled by changing the matching rule action in CDO.
Note the following behaviors of the Threats page:
- Threat events that are displayed are not live. Devices are polled hourly for additional Threat events.
- Threat events that are not included in the Live or Historical view are not part of Cisco Security Analytics and Logging.
- To see Threat events that you've hidden from view, click the filter icon and check the view hidden option.
- From the navigation pane, select Monitoring > Threats. You can filter what events are shown and search by source IP address.
- Click on a threat event to expand the details panel on the right.
- For more information on the rule, click the Rule Document URL in the Rule Details section.
- To hide this event, check the toggle switch for Hide Events. The event handling continues as is, but you won't see it here, unless you click View Hidden or un-hide this event.
- To edit rule overrides, click Tune Rule. When you change a rule action in CDO, the override applies to all the pre-defined policies. This is different than in the FDM where each rule can be different from policy to policy.
- In the Override All devices pull-down, select an action and click Save.
- Drop-This choice creates an event when this rule matches traffic and then drops the connection. Use this action to tighten security of certain rules. For example, specifying Drop would make security stricter when the Talos rule is matched even if the "Connectivity over Security" policy is specified for the access control rule.
- Alert-This choice creates an event when this rule matches traffic, but it does not drop the connection. A use case for "Alert" is when traffic is blocked, but the customer wants to allow, it and look at the alerts before disabling the rule.
- Disabled-This choice prevents traffic from being matched to the rule. No events are generated. The use case for "Disabled" is to stop false positives in reports, or remove rules that do not apply to your environment, like disabling Apache httpd rules if you don't use httpd.
- Default-This choice returns a rule to the default action it was assigned by Talos, for the intrusion policy it is listed in. For example, when you return an intrusion rule to "Default" that may mean its action returns to "Alert" in the "Connectivity over Security" policy and "Block" in the "Balanced Security and Connectivity" policy.
- To edit rule overrides by device, check the Advanced Options slider. This section shows you the configured rule action for each device, which you can change by checking the affected device, selecting an override action, and clicking Save.
- Affected Devices does not indicate the source devices. Instead, it shows the FTD devices reporting the event.
- Review and deploy now the changes you made, or wait and deploy multiple changes at once.


text:
Officials said that Modi could reach Colombo on March 12 or 13 as part of a four-nation tour and pay a visit to Jaffna on March 14, adding that the prime minister is also planning to visit Trincomalee where India is constructing a power plant.
Anuradhapura, in north-central Sri Lanka, where Emperor Ashoka's son Mahendra delivered two public speeches on Buddhism in 3rd century BC, is the first place where people beyond the shores of India embraced Buddhism.
India in August 2012 took Kapilavastu Relics preserved in Delhi's National Museum to Sri Lanka.
With the introduction of Buddhism, architecture in Anuradhapura gained more prominence. (Times of India)


text:
A Poor Young Boy and The Dog Short Moral Story
Empathy For Others
A Motivational story to share with kids and even adults that story can change the habits of the world can change the biggest issues in the world right now, all we need a good mindset and encouraging people all around us all the bad things happening in the world right now is due to lack of moral education and empathy for others, we can bring the change in society by only good education but good education is a big issue itself will write another blog on that so you enjoy the story of a poor young boy and the dog share in comments what you learned from this story,.
A few days ago I landed at the Bombay Airport (India) and took a cab to my scheduled destination in South Bombay.
I was enjoying the busy traffic with people rushing in every possible directions then we got stranded at a very busy intersection.
As we waited for the signal to turn green, my eyes met up with a poor young boy, about 12 years old. He removed a piece of bread from his pocket and took a bite.
As he was about to take his next bite, a stray dog wagged his tail looking at him. Without hesitation, he sat down and put the bread on the road for the dog to eat.
You May Like Also: Best top 8 Stories with moral & With Valuable Knowledge
The dog sniffed the bread and walked away. The boy waited until he was sure the dog was gone then he picked up the bread and ate it!
My heart cried and wanted to walk up to the boy but before I could open the door the signal turned Green and our car drove away. I kept thinking about the boy and later during my evening meal I realized that I was thinking of approaching the boy but never did, I could have stopped the car and walked up to him which again I never did.
All I did was "thinking" and this poor little boy who had only one piece of bread without any hesitation sharing it with the dog, though he himself seemed to be very hungry.
I learnt one of the biggest lessons in my life which that boy taught me without a conversation. He taught me to share with love and happiness. I am so blessed to have learnt this beautiful lesson from my 'little unknown master'. It is my moral duty to share this incident with all my friends across the globe and be blessed with happiness.
Thank you friends for making this world a beautiful place to dwell.


text:
a short distance away, annoyed us very much. We were engaged nearly all of the time during the day, and fired as fast as the sharpshooters would permit as to load our pieces. We expended 200 rounds of ammunition; had 1 man killed and 1 wounded, 1 horse killed and 1 wounded. At night the battery was relieved and we marched with our corps to the left of the army, and went into position the next day on the extreme left flank of the line. We remained in position the next day on the extreme left flank of the line. We remained in position the 23rd and until dark the 24th, when were relieved and went into camp. From the 25th to the 29th of June we remained in camp. On the 29th marched to Reams' Station. 30th, remained in park near the station.
July 1, we returned from Reams' Station and went into the camp occupied by us before we marched to the station. From the 2nd to the 7th remained in camp. On the 7th moved a short distance to the left, and occupied a fort on the left flank of the line, but were relieved in the afternoon and returned to camp. On the 9th marched to City Point; arrived there on the morning of the 10th, at 7 o'clock. From the 10th to the 13th remained in camp near City Point. On the 13th embarked on transports at City Point for Baltimore. Arrived in Baltimore on the afternoon of the 15th; camped for the night in the streets near the landing. Next morning early moved through the city and took the cars for Washington; arrived there at dark, and marched to Camp Barry and camped for the night. Next day, the 17th, moved from Camp Barry at 4 p.m. to Seventh-street Wharf. There we embarked in transports for City Point. On account of the roughness of the weather did not arrive at City Point until the afternoon of the 20th. We immediately disembarked and went into camp near City Point, where we remained until the 26th. On the 26th, at 4 p.m., [marched] to near Petersburg and went into camp in rear of the Ninth Corps, and remained there until the night of the 29th, when we moved into position in front of Petersburg.
I am, sir, very respectfully, your obedient servant,
Captain, Commanding Battery.
Colonel TOMPKINS,
Chief of Artillery, Sixth Corps.
No. 167. Report of Captain Greenleaf T. Stevens, Fifth Maine Battery, of operations June 12-July 10.
On the march across the Chickahominy nothing worthy of note occurred. June 16, ordered into position with the corps, covering the crossing of the army at the James. On the 18th went into position before Petersburg, and at once remodeled an old rifle-pit of the enemy, making it appropriate for artillery, and opened, expending eight shot and twenty-four spherical case. By direction of Colonel C. H. Tompkins, one section of the battery was withdrawn at night and moved some 900 yards to the front and placed in position on the right of the road running parallel with the Appomattox. Nearly the same operations were repeated here as at Cold Harbor. The guns were unlimbered and run
*For portion of report [here omitted] covering operations from May 4 to June 9, 1864, see Vol. XXXVI, Part I, p.760.


text:
Regular price
Imagine creating a different and delicious snack with just a potato! The Potato Spiral Cutter allows you to make spiral potatoes for your wonderful BBQ experience or twisted potato chips.
You know it's time for a change in the routine, and cooking creative snacks should be the beginning of that!
• Material: Plastic + Stainless steel
• Excellent for making original potato spirals
• Easy operation
• Easy to wash
• Shipping takes from 10 to 25 days


text:
Create an Account - Increase your productivity, customize your experience, and engage in information you care about.
The Morgan's Point Resort City Story
This film was used by the Great Western Investment Corporation in the mid 1960's to sell lots in Morgan's Point Resort.
A brief history of Morgan's Point Resort
Early Morgan's Point Resort Advertisement
Armored Sentinel (Temple, Tex.), Vol. 23, No. 6, Ed. 1 Friday, April 30, 1965
Morgan's Homestead..."A Site To See" (PDF)
This article about the original James Morgan homestead was written by Rudy Purificato and published on Friday, March 26, 1982 in a supplement to the Morgan's Point Mariner newspaper.
A brief history of Morgan's Point Resort (PDF)
Armored Sentinel (Temple, Texas), Volume 23, Number 6, Edition 1 Friday, April 30, 1965


text:
Mini Montenegro
Excursion to the most southern fiord in Europe is the opportunity to explore all the uniqueness and natural beauty of the bay and to find out about its history, culture, architecture…
This trip is the opportunity to visit the oldest settlement in Montenegro – Risan, where is the location of Roman Mosaics
(of Roman God Hypnos) that originate from the 2nd century AD, the old town of Kotor (part of World Heritage and under the protection of UNESCO),
the old town of Budva, with numerous churches, museums, galleries as well as shopping malls and different bars and coffee shops that create a unique and specific atmosphere. Undoubtedly, you will enjoy in shade of old olive trees and picturesque view on Sv.Stefan, from the fifteenth century, nowadays known as Town-Hotel thus representing truly recognizable landmark of Montenegrin tourism.
Excursion description
- The trip starts at 8 am from the main bus station in Herceg Novi. On the way to Perast, you are about to see one of the 25 most beautiful bays in the world – The Boka Kotorska Bay where we will have a photo break at the narrowest part of it, for you to memorize.
- Around 8:30 am we reach Risan – the oldest settlement in Montenegro, where we have 20 minutes to visit Roman Mosaics.
- You have almost two hours at your disposal to visit Kotor, UNESCO protected part of the country.
- From Kotor, there is a 40-minute ride to Sv.Stefan and during the ride you will enjoy the shade of olive trees and view on the world-wide known peninsula.
- Budva is our next stop. There, besides visiting the historic and religious sites, you can relax and enjoy the atmosphere in shopping malls and restaurants and bars in the heart of the Old Town.
- Return is planned around 5 pm.
Price: 25,00 €


text:
You are not permitted to download, save or email this image. Visit image gallery to purchase the image.
Schools are being accused of going too far in what they teach children about sex. Children as young as 12 are being taught about oral sex and, in other cases, 14-year-old girls are being taught how to put condoms on plastic penises.
The often graphic nature of today's sexual education lessons is considered perfectly acceptable, and necessary, by some parents, but many others are shocked and say it has gone to far.
One concerned father, who did not want to be identified contacted Newstalk ZB last week saying he took his 12-year-old son out of a sexual education class after he came home upset about what had happened during one of the lessons at his all-boy school.
Other parents who phoned the radio station agreed sex education had gone too far.
One said her 14-year-old daughter came home on Tuesday saying "she had been applying yucky and sticky condoms to a plastic black penis".
Secondary Principals' Association president Patrick Walsh said sex education was taught as early as intermediate school, and was different from what many parents would have learnt.
The focus was no longer on reproductive health but on sexually transmitted diseases, sexual practices and keeping safe.
"It's becoming a bit more graphic and a bit more hands-on, I guess."
Mr Walsh said there was considerable latitude in what schools taught, and the things the father of the 12-year-old boy described were not uncommon.
"I don't know if I am being prudish but I have some difficulty with it as well."
He said deciding how far to go was difficult, but the message from those teaching the classes was "they have to be graphic and be upfront to get the messages across".
Family Planning's health promotion director, Frances Bird, said New Zealand had one of the highest teenage pregnancy rates and children as young as 12 were sexually active.
She said international research showed a good, quality, comprehensive programme could make a significant difference.
"It delays the first time people have sex, it reduces the number of partners they will have, it reduces the frequency of intercourse ... and it also increases condom and contraception use, so programmes are particularly effective if they begin before young people have sex."
The Ministry of Education's acting curriculum, teaching and learning group manager, Margaret McLeod, said while schools could decide on the kind of sexual education they taught, they were expected to consult their communities first.
• Today's sex education is less about human reproduction and more about sexually transmitted diseases, sexual practices and keeping safe.
• Schools can decide when and how they give sex education and what they teach, but they are expected to consult their communities before developing the programme.
• Parents have to sign a consent form and can withdraw children from a programme if they are unhappy with it.
- Elizabeth Binning


text:
Clam-O-Rama! Wild Edibles from the Marsh
The Trustees of Reservations invites families to an edible adventure for the whole family! Ipswich has long been renowned for it's delicious soft-shell clams. Join us as we wander into Fox Creek to find out firsthand where our food comes from! We'll dig for a variety of clams – and maybe find some vegetables to go with them – and then hike to the Pine Grove to cook and eat what we've gathered. You may want to bring a bag supper to supplement our harvest – clamming is harder than it looks!


text:
Tagged: Wordpress
Tag – You're It!
So today I was nominated for the Beautiful Blogger Award by the lovely Jennifaye. Yay! Although this reminded me that I had also been tagged by nosugarjustspice WEEKS ago and I totally forgot! So, I'm starting with the earlier tag and, if I have time, will roll right on through to the more recent one!
First up, the one I am going to refer to as 'Blog Tag', has the following rules:
1. You must post the rules
2. Answer the questions the tagger set for you in their post
3. Create eleven new questions to ask the people you've tagged
4. Tag eleven people with a link to your post
5. Let them know you tagged them
And the questions posted by nosugarjustspice and my answers are:
1. What's the most exciting thing you've done in the last year?
Ooohhhh, quit my job, which I did just a few weeks ago! Exciting times are ahead!
2. Favourite 80's movie?
Girls Just Want to Have Fun!
3. Do you still have toys from when you were a child?
Yes, and sadly some are still in my bedroom. I just can't let them go and keep telling myself that they'll mean so much to my kids when I pass them on (note – I have no kids and don't expect to have any for quite a while still!)… except Sealy Lorne, who is my number 1!
4. You're being sent to Biosphere 2 for a year and can only take one person, who would it be?
Hmmm… only for a year? Would I have any means of communication with other people? Argh, I can't decide!
5. If you could be a member of the opposite sex for a day, would you?
No way! Haha!
6. Is there anything in the area surrounding you right now that could be used to fight zombies in the apocalypse?
Yep, a MASSIVE aerosol can of Fudge Skyscraper hairspray.
7. How many alarms does it take to wake you up of a morning? (It takes me 5)
Two alarms, which start at 7.15 and 7.20 and keep getting snoozed until about 7.50-8am. I HATE mornings!
8. Hollywood is remaking your favourite movie ever and wants you to play your favourite character from that movie, which movie is it and who are you playing?
Amelie and Amelie!
9. What are 5 terms/names/etc you wish you had never heard and could be removed history?
The first thing to come to mind is too horrid to share, but I had to ask blogger Lyndon Keane to translate and we both regret googling as soon as we had done so… no further comment on that.
Second, third, fourth and fifth would be babe (as in people refer to each other as this), moist, Hugh (as in the name, because I can't pronounce it) and… Mash (just the mention of it makes me feel sick)!
10. What's the stupidest thing you've done when drunk?
Oh, there's way too many to narrow it down… In recent months projectile vomiting all over my luggage and the hotel room floor would probably be the worst of it.
11. If you could wake up tomorrow and walk into your dream job what would it be?
Professional world traveller and gazillionaire 🙂
And eleven new questions:
- What's one city in the world that you've always wanted to visit and why?
- What is your biggest fear?
- Have you ever seen a ghost?
- What is your guilty pleasure?
- If you could have any animal in the world as a pet, what animal would it be?
- Are you a dog or a cat person?
- If you could have one superpower, what would it be?
- What's your favourite song of all time?
- When you were young, what did you plan to be when you grew up?
- Chips and salad or roast potatoes and vegetables?
- If you won a million dollars, what would you do/buy first?
And the eleven blogs that I tag are…
- www.frugalistablog.com (for her great Kristen Stewart impersonation)
- www.findingravity.com
- www.somethingswedish.wordpress.com
- www.housewifedownunder.wordpress.com
- www.paltrymeanderings.com
- www.beetleandswan.wordpress.com
- www.pithypants.com
- www.mikesilvia.wordpress.com
- www.disseminatedthought.wordpress.com
- www.torinelson.wordpress.com
- www.simonandfinn.com (the baby mouse deer made me cry :()
I hope you enjoy these blogs as much as I do!
Okay, having just done this one and with seven more blogs to nominate, I'm going to take a few days to complete the Beautiful Blogger Award! Phew!
Will Write for Food
Last week I quit my job.
Most people who know me saw it coming, but for the many people I know through work, it was completely unexpected. So far, everyone has reacted with 'Omg, what's happened?' and I suspect, knowing my tendency for dramatics, they are assuming that I cracked without warning, slamming the manual lift door while screaming 'I QUIT!' and storming off down La Trobe Street.
Alas, it was not so exciting and unlike the time someone put spag bol all over the bin in the midst of a bug infestation or the time I realised someone was stealing the toilet paper (I still have my suspicions, but no proof), there was absolutely no hysterics.
Resigning was a massive relief and although I had a TINY panic attack in between being offered my new job and quitting my current one, it wasn't long before I was getting excited about taking a huge step off my current career path and throwing caution to the wind.
There's something liberating about having absolutely no idea what the heck you will be doing a year from now, and, to quote my ever-unreliable Fortune Telling Fairy Cards, I am moving forward fearlessly!
Gone are the days of work-related panic attacks and swatting bugs as I sit at my desk – it's time for freedom, creativity and full, uninterrupted nights of sleep! Woo hoo!
As mentioned above, I do have a new job lined up, which I am very excited about, but leaving my current role will also allow me to look into new opportunities, to focus on my blog and what I really want to do – to DANCE!
Okay, that was totally a joke, but I have spent a fair chunk of the past few days thinking about the future. Now is the time to work out exactly what I want to do and how to do it… But in typical Tennizzle-style, I have become overwhelmed by the decision.
Ideally I would win lotto and spend the next few years jetting around the world, renovating my house and volunteering my time to the greater good… however accepting that this is not going to happen and that the chances of anyone paying me to hang out with my dogs for a living are just as low, I am going to need a more realistic plan.
I have decided to focus on copywriting, but without it being part of my everyday work.
So far, I have taken the massive steps of purchasing my blog domain (check it out, I've dropped the 'wordpress' from my site, la di da!) AND getting my own personal domain for the future… I haven't quite worked out how to set up a website or get it hosted, but I'm on my way! I can feel success in the air!
I'm starting with a bit of blatant self-promotion to people I know professionally and am hoping to start putting a portfolio together in the coming months. As a result, my blog will be growing and although my regular, neurotic posts will continue, I will also be using this page as a means of collating writing until I have a proper website.
In the meantime, if you see someone with a sandwich board reading 'Will Write for Food' standing outside Flinders St station next month… please stop by and say hi!
Are You Insane? Let Me Guess How You Found My Blog… And a Little Bit of Blog Love Too!
Okay, so this week I am being totally boring because I'm having bloggers block and my attempts at writing my next post are not getting me anywhere! So I have decided to take the opportunity to respond to a blogging award nomination and also share some random nonsense, to justify this as a REAL post for the week!
So, when I can't finish any of the gazillion drafts sitting in my blog, there is only one way out – to write a list! This week I have mainly been inspired by the weird and slightly disturbing search terms that have been coming up on my blog, but also by the many other blogs I have been reading and what I have learnt since I started blogging just over two months ago. The following are the top ten of these:
1. People, or more specifically, people who write and read blogs, are awesome and they brighten my life!
2. They are also sometimes insane… as is evident in the following search terms which have led people to my blog.
– Tenis player not wear pant – okay, I guess I was asking for this one with all that talk of Marat Safin and his flood pants, but seriously, who were you hoping to find?
– What is hi fives porn – If this is something that exists (and by the number of referrals, I'm guessing it does), I can guarantee this is NOT something you will be finding here. Move along!
– Eating my ex on the couch – I'm fairly certain I do not ever want to meet you…
– High five? Potato – If this was 'high five? Mashed potato' I would assume you were my arch nemesis. Alas, I have no idea what you were hoping to achieve. High five? Potato!
– Ecards about stupid people – I think I have Googled this exact search term, let's be friends!
– What is the fear of high fives called – Oh. My. God! I am not alone!
– Potatoes with cotton buds – I'm confused. Why does the potato have cotton buds?
– I can count to potato – Good for you!
– Women crapping – I am not even joking!
– Some people just need a high-five. In the face. With a chair – Wow, you have a way with words. I suspect you may be the reason for my fear of high fives…
– You call it madness but I call it love – Yes, and I call you insane. Have we dated?
– Green balls – I think you should get off Google and get to the doctor… Now!
– What is this shitload of mashed potatoes day – I think I had a nightmare along those lines just last week… a whole day of being chased around by a giant plate of mashed potato with legs… it's all coming back to me…
3. I am not alone in my pug love – Yep, my blog has received visits from people using 31 different Pug search terms so far! Some of my favourites are 'pug with backpack' 'thank god it's Friday pug' and 'pug true love'. I feel my heart warming just reading them! This is from one particular morning several weeks ago:
4. WordPress is my friend – It's true. I've actually found I spend more time with WordPress these days than with most of the people in my life. It makes me happy!
5. Twitter is not my friend – Also true. I Tweet… I get sweet nothing back… but I shall persist….
6. I wish I was cool enough to have a Facebook page for my blog – Alas, I am not, but I shall continue to sit back and silently envy those of you who do… Sitting there all smug in your popularity…
7. I want to start taking photos for my blog – I'm making this one of my missions in life. Having broken my iPhone camera and with minimal chance of lugging my SLR around with me each day, the likelihood of this happening is slim to none – so I'm aiming to post one photo I have taken by the end of 2012. Achievable? Achievable!
8. www.someecards.com is a sarcastic bloggers best friend – Seriously, the best resource ever! And they're FREE!
9. There's a fairly good chance I will fail in my attempt to blog every week of 2012… although I remain 100% committed to trying my hardest!
10. Blog awards are great for the ego – and a great excuse for spending an entire day reading other blogs… And on that note:
The rest of my entry today is to accept and pass on a little blog love with four (yep, FOUR) awards rolled into one… I don't even know how this happens and am slightly overwhelmed, but I'm going to go with it and see what happens!
So, first and foremost, many many thanks to No Sugar, Just Spice for nominating me… in perusing your blog I see we share some embarrassing old music tastes… well, taste, I should say… I'm not at the point of publicly admitting to such a thing, but just for you *ahem*:
"Have you ever stood outside a picket fence… you'll see through… but you can't get to the inside… oohhhhhhh!"
I still remember all the words… how embarrassing… let's never mention this again!
So, the awards are:
- Mrs. Sparkly's Ten Commandments Award
- The Sunshine Award
- The Candle Lighter Award
- The Liebster Award
To roll all the questions into one, I'm just going to answer EVERYTHING in one go and then nominate those blogs I would like to pass the love onto.
So, here goes:
1. Describe yourself in seven words.
Small, Energetic, Loyal, Passionate, Impatient, Blunt and High-Pitched (although that's kind of eight!)
2. What keeps you up at night?
Blog writing!
3. Whom would you like to be?
Noone, really. I'm pretty happy just being me.
4. What are you wearing now?
Tracksuit pants, a hoodie and my ugg boots… exciting, I know!
5. What scares you?
Mice and Horses.
6. What are the best and worst things about blogging?
Best: Interacting with other bloggers and getting feedback on my posts.
Worst: Writing a post when I've given myself a deadline. I always leave it until the last minute!
7. What was the last website you looked at?
I was Googling the lyrics to the song quoted above… I can't believe I actually second-guessed myself!
8. If you could change one thing about yourself what would it be?
I guess being a bit more tolerant to stupid people…?
9. Slankets, yes or no?
I think you mean Snuggie… and I would go with a no.. except on little kids and dogs, have you seen the dog snuggie? So cute! I wonder if there is a photo of a pug in a snuggie somewhere out there…
Oh. My. God…
10. Tell us something about the person who nominated you.
She sorts her fries by size. True story.
11. Favorite Color:
12. Favorite Animal:
Dogs, Elephants and Seals.
13. Favorite Number:
14. Favorite Non-Alcoholic Drink:
Sparkling water.
15. Facebook or Twitter:
16. My Passion:
17. Getting or Giving Presents:
18. Favorite Pattern:
Pattern?? Weird question… any of Cath Kidston's floral prints?
19. Favorite Day of the Week:
20. Favorite Flower:
And ten blogs to nominate:
Blog Love, Galeanthropy and an ABC Award!
Okay, so there's been some crazy shizz going on with WordPress lately.
When I logged in on Sunday morning I was shocked to realise that somehow, in my sleep my blog had doubled its daily view record (whatever it's called). After some slight confusion and a bit of investigation, I realised that not only one or two but FIVE of the blogs I actively follow have been Freshly Pressed in the last week! Yay for y'all, I've been clicking like wherever possible and hope you've all enjoyed the fame and fortune that I imagine it brings!
But – this was not where my joy ended! The lovely Lyndon of The Dissemination of Thought had nominated me for The Awesome Blog Content (ABC) Award! YAY! I am thrilled to pieces, even though it's taken me ages to find all those words!
So, as the rules of the award go, I am to:
- Thank the person who gave me the award
- Choose one word that describes me for every letter of the alphabet (for some I just chose words I like)
- Pass it on and nominate blogs which I think deserve fame and notoriety
So, first up, many, many thanks to Mr Keane and The Dissemination of Thought for nominating me! One of my favourite bloggers so far, Lyndon is flipping hilarious and his posts never fail to make me laugh. He's also kind of a big deal, so I'd recommend you become his friend and WordPress stalk him before he hits it big and forgets us little people!
And for the alphabet of ME:
*I don't actually think I'm a cat, but how good is that word?
**I'm also not a pug, but I love them!
***I can't actually yodel, but I had no other Y. I'll give it a shot if you pay me!
****I also wouldn't describe myself as plump or full-figured, but it sure beat zany
And for the blogs I would like to nominate:
www.pithypants.com – I just discovered this one today, very funny and clearly an Arrested Development fan! I can't wait to read more from her!
www.imnotfamousandneitherareyou.com – I nominated Emily last time, but she's still hilarious, and still reminds me of me.
www.dampsquid.wordpress.com – Another one I nominated last time, every time I visit this blog I end up giggling at something on there!
www.sarahpalma.wordpress.com – This bloggers name is Sarah Palma, which reminds of both Twin Peaks and Chicken Parma, what more can you ask for?
Sorry for only nominating four, it's bedtime and I'm starting to type crazy things as I doze off in front of my computer screen!
Thanks for reading my blog and for all the wonderful new followers I have gained over the past few days! Yay!
Ending the Week on a High!
So I'm not counting this as a REAL post and will post some usual ranting mid-next week, however I did have to share the highlights of my Friday.
I woke up yesterday and undertook my usual weekday morning ritual: Hit snooze – fall asleep – hit snooze – fall asleep – hit snooze – roll over – have a silent whinge to myself about how evil any hour prior to 9am is – fall asleep – hit snooze – check Facebook – check gmail – check work emails – roll out of bed.
On Facebook, I had been sent this little treasure:
Considering the last similarly themed linked I got from this particular FB friend, which was something along the lines of 'I've found you a husband: http://www.dailymail.co.uk/news/article-2071127/Bankers-1-615-word-email-woman-didnt-back.html', I wasn't expecting much. But this had me giggling out loud to myself.
In all honesty, it wouldn't surprise me if my dogs were running a similar operation of sabotage in my life. In the current absence of a boy in my life, they have free reign of the house, get unlimited attention and half of everything I cook (I suffer serious eyes-bigger-than-stomach issues). When I am dating someone, they become lounge-room sleepers, are made to sit on their own couch, get fewer walks and have to feign interest in a new person for at least a few hours every time they see them. It's a hard life and they're not exactly subtle about their disapproval when someone new comes into it.
Having worked myself into a slightly paranoid state and half expecting them to be watching the video over my shoulder while taking notes and giving each other knowing looks, I turned to see what the little darlings were actually up to. Ginger Dog was curled up at the end of the bed, like the perfect puppy that he is… while Midget Dog was sprawled out next to me on my bed, with her head ON my pillow, fast asleep. Note to self – this sleeping arrangement is not conducive to ever meeting a mentally sound husband.
I went to move her off… looked at her taking her little breaths, remembered the difficult night she'd had on Thursday, which involved an itchy leg and an unplanned haircut after I realized her overgrown fringe was impacting her ability to judge distances (only after she twice underestimated the jump from floor to couch and slammed head-first into the side of it) and decided to leave her where she was… I'll train her off the pillow on the weekend.
So back to the ritual – next step was my emails and this is where the day really kicked off. I admit that I've become somewhat addicted to WordPress lately and after smashing my own visits-per-day record on Thursday, I was thrilled to see I had a new comment on my most recent post. Then it got even better… a lovely blogger called blondgirl008 had nominated my little, semi-psychotic blog for the Versatile Blog Award! Considering I have been blogging for less than a month, this has not only made my day, but my week, month AND 2012 so far! Woo hoo!
It has taken me a bit of researching to work out exactly what this is, but to be honest, I am so thrilled that anyone is even reading my blog that I would take any award you're willing to give me… including the crazy dog lady of the year award. But alas, the Versatile Blogger Award is a way to show your support for blogs of all varieties. So, here goes my response to my much-appreciated nomination:
First up many thanks for the nomination, Blondgirl008. Everyone should check out your hilarious blog at:
Then, for seven things about me:
- I am very new to blogging. Yep, although I set up my account back in September 2010 (from memory), it wasn't until December 2011 that I actually started blogging. Needless to say, I am addicted and commited to posting every week for the next 12 months.
- I have two dogs (mentioned above) who I am fairly obsessed with. Ginger dog is 11 and is a little ball of Pomeranian-Cross love. He enjoys food, cuddles, staring longingly into peoples eyes and once punctured my eyeball in a moment of cuddle grabbing desperation, which saw me end up in the Eye and Ear Hospital and him feel quite guilty for a few hours. Midget Dog is equally lovable and if she was a human, I'm fairly certain she would actually be me. She is only 3 and is strong-willed, bossy and squeals when she doesn't get what she wants. However, at the end of the day she is a happy little girl who has a pretty sweet life and just wants to eat lots and curl up on the couch.
- I live in Melbourne, Australia and I love it! Although I travel a lot, and have lived overseas in the past, I don't imagine I will live anywhere else for a prolonged period of time. I also hope to stay in the same local area I currently live in and am very grateful to have grown up in such a diverse, beautiful and fun city.
- I am a dating disaster. It's true. I won't go into too much detail here, I have a separate (anonymous) blog all about the many crazy dating experiences I have had in my life. Having said that, I am a firm believer in learning from your experiences and that the worst dates always make for the funniest stories!
- I am totally addicted to soda water and sparkling water and I really need to invest in a Soda Stream this year. This addiction has developed since I quit soft drink last year. I just can't get enough of the bubbly goodness.
- I am attempting to be an owner-renovator. So far, it's been challenging and my house is currently covered in about 3cms of plaster dust. However, I love my little house and get excited by the smallest things. Note – do not ever get me talking about blinds, light switches or any other house-related item. I guarantee I won't stop talking, you'll be confused and you will regret ever starting the conversation.
- I have an inability to keep things brief. Most of the posts on my blog (including these seven points) were intended to be short and sweet. Eek! I attribute this to having a lot to say and expect that as the weeks roll on, I might stop crapping on so much! Taking a moment to pause, I realize this post is already at 1137 Words… Sorry!
Finally, fifteen blogs that I would in-turn nominate (most of which I am already following and a few I have sourced for this purpose!):
NeverContrary – Funny, open hearted ponderings on life
36ixty5 – Every time these images pop up on my 'Read Blogs' page they make me happy
Fibromy-Awesome – A lovely blog
I'm Not Famous and Neither Are You – This girl reminds me of myself, haha! I think her name is Sarah (I joke, I joke!)
The Dissemination of Thought – Hilarious
The Insanity Aquarium – Also very funny
Viciously Sweet – Originally I wanted to illustrate my blog. I failed and admire anyone with such artistic flair!
Cramp My Style – Recently on Freshly Pressed
Brooke and Mckenzie – I just discovered this one yesterday
Blank Stares and Blank Pages – So, so funny
Damp Squid – I don't really know how to explain this one
Hyperactive Inefficiency – I've only just started reading this one
The Waiting – A blog about pregnancy with a really cute 'About' page
The Middest Sister – Another funny one with pictures
Consider the Sauce – Omg, a blog dedicated to food in the Western suburbs! What more could a West-side Melbournian need?
Thanks again for the nomination, it was a great chance to discover even more awesome blogs!


text:
Guys, this hot chocolate is legit! It's dairy free, but there's a magic (and completely healthy) ingredient that makes it so creamy you'd never know!
Archives for October 2018
Is Yacon Syrup a Good Sugar Substitute?
We're heading into the holiday season! It's that wonderful time of the year that's filled with delicious comfort foods, plenty of excuses to eat said comfort foods, and cold weather that totally merits wearing sweat pants. Which brings us to yacon syrup.
Spicy Chicken Goulash
Spicy chicken goulash is a hearty meal that can be made in your instant pot or crock pot in one easy step, just dump the ingredients and go! With onions, tomatoes, chicken, and broccoli slaw—it's as healthy as a salad!
Cashew Cream – A Heavy Cream Substitute
You may be asking if this is legit—can cashew cream really fill the big, delicious shoes of 100% heavy whipping cream? Surely it's too good to be true…right?
Green Banana Flour for Gluten Free Cooking and Celiac Health
Are you on a grain free diet? Are you unable to use almond flour due to a nut allergy? Are you looking for gluten free flours that make fluffy baked goods? If so, green banana flour may be a great option for you!
[Read more…] about Green Banana Flour for Gluten Free Cooking and Celiac Health
Grain Free Pancakes
Looking for an easy recipe for grain free pancakes? Green banana flour is a fantastic ingredient to use instead of grain for fluffy, delicious pancakes!
Simon & Garfunkel Chicken Stew
This chicken stew recipe is a favorite in my house. It's sooo tasty and easy to make. I cook this in big batches and always have a couple containers of it on hand in the freezer. If you don't have an Instant Pot, you can easily cook this in a crock pot!