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The RNA chaperone Hfq fulfills important roles in small regulatory RNA (sRNA) function in many bacteria. Loss of Hfq in the dissimilatory metal reducing bacterium Shewanella oneidensis strain MR-1 results in slow exponential phase growth and a reduced terminal cell density at stationary phase. We have found that the exponential phase growth defect of the hfq mutant in LB is the result of reduced heme levels. Both heme levels and exponential phase growth of the hfq mutant can be completely restored by supplementing LB medium with 5-aminolevulinic acid (5-ALA), the first committed intermediate synthesized during heme synthesis. Increasing expression of gtrA, which encodes the enzyme that catalyzes the first step in heme biosynthesis, also restores heme levels and exponential phase growth of the hfq mutant. Taken together, our data indicate that reduced heme levels are responsible for the exponential growth defect of the S. oneidensis hfq mutant in LB medium and suggest that the S. oneidensis hfq mutant is deficient in heme production at the 5-ALA synthesis step.
Citation: Brennan CM, Mazzucca NQ, Mezoian T, Hunt TM, Keane ML, Leonard JN, et al. (2014) Reduced Heme Levels Underlie the Exponential Growth Defect of the Shewanella oneidensis hfq Mutant. PLoS ONE 9(10): e109879. doi:10.1371/journal.pone.0109879
Editor: Roy Martin Roop II, East Carolina University School of Medicine, United States of America
Received: June 18, 2014; Accepted: September 3, 2014; Published: October 30, 2014
Copyright: © 2014 Brennan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.
Funding: Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number 2 P20 GM103430. IDeA website address: http://www.nigms.nih.gov/Training/IDeA/Pages/default.aspx. BJP's funding was awarded as a sub-project of the above grant through Rhode Island IDeA Network for Excellence in Biomedical Research (RI-INBRE, http://web.uri.edu/inbre/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The RNA chaperone Hfq is a highly conserved protein that mediates interactions between many regulatory sRNA molecules and their mRNA targets in bacteria (for reviews see [1]–[4]). Hfq protein monomers form a homohexameric ring capable of binding both regulatory small, noncoding RNAs (sRNAs) and their target mRNAs [5], [6]. These Hfq-RNA interactions stabilize sRNAs and help the sRNAs locate and interact with their mRNA targets. Because sRNA expression and subsequent changes in target gene expression help bacteria adapt to changing environmental conditions, Hfq plays key roles in regulating a wide variety of cellular functions.
Genetic loss-of-function studies have been widely employed to elucidate how Hfq functions to help regulate bacterial gene expression networks (for example see [7]–[10]). Consistent with a role in a multitude of cellular processes, loss of Hfq is typically pleiotropic, and hfq mutants exhibit a diverse array of bacterium-specific phenotypes. This suggests that, despite its high level of conservation, Hfq has evolved distinct roles in closely related bacteria. However, a common theme in the study of Hfq is that hfq mutants are typically less efficient at responding to the challenges of growth and stress conditions, which is consistent with the role of Hfq as a functional mediator for adaptive sRNAs. Though reduced sRNA function likely contributes to many hfq mutant phenotypes, the mechanisms by which loss of Hfq results in particular mutant phenotypes often remain obscure.
A null mutant of hfq in the dissimilatory metal-reducing bacterium Shewanella oneidensis was recently characterized [10]. Loss of hfq in S. oneidensis results in slow exponential phase growth, reduced stationary phase culture density, slow anaerobic growth, a reduced capacity for chromium reduction, and a greatly increased sensitivity to oxidative stress. S. oneidensis is of particular interest because anaerobically growing cells are capable of transferring electrons to a wide variety of extracellular terminal electron acceptors, including many soluble and insoluble metals [11], [12]. This capability has sparked interest in S. oneidensis as a potential bioremediating organism as well as an organism capable of producing electrical current in microbial fuel cells [13], [14]. Because bacteria in nature likely experience frequent metabolic changes, understanding how Hfq and sRNAs mediate adaptive cellular processes will provide useful insights into the genetic and physiological control mechanisms of S. oneidensis.
Heme molecules participate in a variety of important cellular processes. For example, heme is a key prosthetic group in cytochrome proteins used in electron transport chains. Heme also has other important physiological roles in iron storage and oxidative stress resistance. The importance of heme in bacterial growth is underscored by the fact that bacteria that are unable to synthesize heme, including many pathogens, must acquire it from their external environments [15]. Heme availability is particularly important for electron transport in S. oneidensis, which can potentially encode 42 different c-type cytochromes that are thought to confer the ability to use a diverse array of terminal electron acceptors [16], [17]. The predominant form of heme in S. oneidensis is heme C [18], which is covalently attached to two cysteine residues in cytochrome proteins by the activity of the heme lyase protein CcmF [19].
Heme biosynthesis is a well-conserved process (for reviews see [15], [20]). The first committed step in heme synthesis is the production of 5-aminolevulinic acid (5-ALA). In mammalian cells, yeast, and the α-proteobacteria, the HemA protein (5-ALA synthase) catalyzes the synthesis of 5-ALA in a single step from glycine and succinyl-CoA (the C4 pathway). In other organisms (the remaining eubacteria, plants, Archaea, and algae), 5-ALA is produced in a two step process (the C5 pathway). First, glutamyl tRNA reductase (GtrA/HemA) uses NADPH as a cofactor to reduce glutamate supplied by a charged glutamyl tRNA to a glutamate-1-semialdehyde (GSA) intermediate. GSA is then converted to 5-ALA by the enzyme GSA aminotransferase (HemL). Following 5-ALA synthesis, heme is produced using seven additional conserved enzyme-catalyzed reactions.
Regulation of heme biosynthesis in bacteria is largely focused on the activity of the first enzyme in the heme biosynthetic pathway (reviewed in [20]). Though bacteria modulate production of GtrA/HemA on the transcriptional level, this regulation is generally modest. In contrast, bacteria appear to exert a much larger influence on glutamyl tRNA reductase activity by posttranlationally regulating GtrA/HemA protein stability. For example, in Salmonella typhimurium, heme binds to the GtrA/HemA protein and promotes its proteolytic degradation via the Lon and ClpAP proteases [21]. Thus, high heme levels negatively regulate the first committed step in heme biosynthesis via anabolite repression.
In this study, we report the serendipitous discovery that reduced heme levels underlie the exponential growth phase defect of the S. oneidensis hfq mutant. Restoring heme levels in the hfq mutant by nutritional supplementation with 5-aminolevulinic acid or by exogenous production of the S. oneidensis glutamyl tRNA reductase (hereafter referred to as gtrA or GtrA to reflect the naming convention proposed by Panek and O'Brian [15]), the first enzyme in the heme biosynthesis pathway, restores exponential growth to wild type levels. Thus, the slow growth of the S. oneidensis hfq mutant in LB is due to reduced heme levels and not a combination of pleiotropic factors. In addition, the defect in heme synthesis in the S. oneidensis hfq mutant occurs at or before the 5-ALA synthesis step. Our findings represent an important advance in understanding the link between the S. oneidensis hfq mutant slow growth phenotype and a defect in a specific metabolic pathway.
Growth on blood agar substantially rescues the small colony defect of the S. oneidensis hfq mutant
Colonies formed by an S. oneidensis hfq mutant on LB plates are substantially smaller than colonies formed by cells containing a wild type copy of hfq (Figure 1A, [10]). Because the hfq mutant is highly sensitive to peroxide stress, we tested trypticase soy agar (TSA) medium containing 5% sheep blood as a potential qualitative measure to assay whether the hfq mutant produces higher levels of reactive oxygen species. Though the hfq mutant did not produce more heme oxidation (α hemolysis) than wild type cells, we were surprised to observe that growth on sheep blood agar substantially rescued the colony size defect of the hfq mutant (Figure 1B). The hfq mutant small colony defect on TSA without blood (Figure S1A) was comparable to the phenotype observed on LB (Figure 1A).
Colony size comparisons of MR-1/pBBR1-MCS2 (vector), and hfqΔ/pBBR1-MCS2 (vector) streaked to single colonies on (A) LB Km, (B) TSA containing 5% sheep blood [including higher magnification insets for colony size comparison: (B′) MR-1/pBBR1-MCS2 (vector) colonies and (B″) hfqΔ/pBBR1-MCS2 (vector) colonies], (C) LB Km supplemented with 50 µM FeCl3, (E) LB Km supplemented with 50 µM hemin, or (F) LB Km supplemented with 50 µM 5-aminolevulinic acid (5-ALA). Plates were photographed following 23–25 hours of growth at 30°C. (D) Quantification of total free iron in wild type MR-1 and hfqΔ mutant cells using the ferrozine reagent. Concentration of detectable free iron was computed as described in Materials and Methods. Data presented is the mean of three independent cultures. Error bars indicate standard deviations. The difference between iron levels in MR-1 and the hfqΔ mutant is not statistically significant (P = 0.22 in an unpaired two-tailed Student's t-test).
Heme or 5-aminolevulinic acid substantially rescues the small colony phenotype of the S. oneidensis hfq mutant
Because most bacteria have the capacity to scavenge iron from hemoglobin to support vital cellular functions [27], we determined whether the addition of iron to the medium at concentrations approximating those found in trypticase soy agar plates containing 5% sheep blood could rescue the growth defect of the hfq mutant. Addition of 50 µM FeCl3 to LB plates, which already contain ∼17 µM iron [28], did not substantially alter the size of the hfq mutant colonies or the wild type colonies (Figure 1C), suggesting that iron is not the factor present in blood that rescues growth of the hfq mutant. An alternative possibility is that the hfq mutant might be defective in iron uptake, but not heme uptake, allowing the cells to scavenge iron by importing heme. However, exponentially growing hfq mutant and wild type cells contained similar levels of free iron per cell (Figure 1D). In addition, for both the MR-1 wild type and hfq mutant cultures we observed the same pattern of free iron levels. At lower cell densities, iron levels per cell are highest, but after culture density reaches an ABS600 of ∼4.0, the free iron content of the cells decreases (Figure S2C). Taken together, these data suggest that rescue of the hfq mutant small colony phenotype is due to a factor other than iron.
Since hfq mutant colonies are noticeably less pigmented than colonies of strains with a wild type copy of hfq (Figure 1A), and because the pink hue of Shewanella is largely the result of reduced heme in cytochrome proteins [29], [30], we hypothesized that growth rescue of the hfq mutant was due to the heme present in mammalian blood. Addition of 50 µM heme in the form of purified porcine hemin substantially rescued the small colony phenotype and partially restored the pink color of the hfq mutant (Figure 1E). This suggests that heme, but not iron alone, rescues the growth of the hfq mutant.
Synthesis of 5-aminolevulinic acid (5-ALA) is the first committed step in heme biosynthesis. 5-ALA synthesis is a critical focal point for regulation of heme biosynthesis, since 5-ALA is used exclusively for heme production [20]. Because exogenously-supplied heme partially rescues the hfq mutant small colony phenotype, we hypothesized that a defect in 5-ALA synthesis, and thus heme biosynthesis, results in slow growth of the hfq mutant. Addition of 50 µM 5-ALA to LB plates rescued the colony size defect of the hfq mutant to a similar extent as addition of 50 µM heme (Figure 1F). Because eight 5-ALA molecules are used to synthesize one heme molecule, we also determined whether higher levels of 5-ALA would increase the degree of colony size rescue. However addition of 400 µM 5-ALA rescued hfq mutant growth to a similar extent as 50 µM 5-ALA (Figure S1B).
Nutritional rescue by 5-ALA of the growth defect of the hfq mutant suggests that loss of hfq creates a heme-specific growth phenotype. This model is supported by our observation that increasing the nutritional content of LB by doubling the concentrations of the tryptone and yeast extract components failed to rescue the hfq mutant growth defect (Figure S1C). Because glutamyl tRNAGlu supplies the glutamate that is converted into 5-ALA, we also determined whether supplementing LB with a mixture of glutamate, arginine, and serine could rescue the hfq mutant growth defect. However, addition of these amino acids at the concentrations they are found in modified M1 medium (see Materials and Methods) did not rescue hfq mutant colony size beyond that seen on LB alone (Figure S1D). Taken together, our data suggest that the hfq mutant is defective in heme biosynthesis at the 5-ALA synthesis step.
The hfq mutant contains reduced levels of heme
The pigmentation difference between wild type S. oneidensis colonies and hfq mutant colonies on LB plates (Figure 1A) is more striking when cell pellets of the two strains are compared (Figure 2A). To determine whether the reduced pigmentation of hfq mutant cells is in fact due to reduced levels of heme, we performed the pyridine hemochrome assay [23] on exponentially growing wild type MR-1 and hfq mutant cells. In this assay, the reduced form of heme C, the dominant heme species in S. oneidensis [18], produces an absorbance peak at 550 nm. The height of the 550 nm absorbance peak as measured from the absorbance trough at 535 nm is directly proportional to the amount of heme in the sample [23]. Analyses of the heme spectra from wild type and hfq mutant cultures (Figure 2B) and subsequent statistical analyses of the quantities of heme present in the samples (Figure 2C) indicated that exponentially growing wild type cells contain significantly more heme than hfq mutant cells.
(A) Comparison of exponentially-growing MR-1 wild type and hfqΔ mutant cell pellet pigmentation. Both cell pellets are comprised of similar numbers of cells. (B) Superimposed heme assay subtraction spectra (reduced – oxidized) from single samples of exponentially-growing MR-1 wild type and hfqΔ mutant cultures. Data presented is typical of the difference observed for the two strains. (C) Quantification of heme concentrations from MR-1 wild type and hfqΔ mutant cultures. Concentration of detectable heme was computed as described in Materials and Methods. Data presented is the mean of three independent cultures. Error bars indicate standard deviations. *** indicate that the difference in heme levels between MR-1 and the hfqΔ mutant is statistically significant (P<0.001 in an unpaired two-tailed Student's t-test).
Increasing expression of gtrA rescues the small colony phenotype of the S. oneidensis hfq mutant
That addition of 5-ALA to the growth medium substantially rescued the small colony phenotype of the heme deficient hfq mutant suggests that a deficiency in heme production might be the result of a defect in 5-ALA synthesis. In S. oneidensis, 5-ALA is synthesized in a putative two-step process involving the genes gtrA and hemL (Figure 3A). GtrA protein converts glutamate donated by glutamyl tRNAGlu into glutamate-1-semialdehyde, which is then converted into 5-ALA by the HemL protein. Since 5-ALA is used exclusively for heme production, we hypothesized that the heme biosynthesis defect of the hfq mutant is due to reduced function of the gtrA gene, the hemL gene, or both.
(A) Putative two step pathway for the biosynthesis of 5-aminolevulinic acid in Shewanella oneidensis MR-1. (B and C) Colony size comparisons of MR-1/pBBAD-SP (vector), MR-1/pBBAD-gtrA (pgtrA), hfqΔ/pBBAD-SP (vector), and hfqΔ/pBBAD-gtrA (pgtrA), streaked to single colonies on (B) LB Km and (C) LB Km containing 0.005% arabinose. (D and E) Colony size comparisons of MR-1/pBBAD-SP (vector), MR-1/pBBAD-phemL (phemL), hfqΔ/pBBAD-SP (vector), and hfqΔ/pBBAD-phemL (phemL), on (D) LB Km and (E) LB Km containing 0.005% arabinose. Plates were photographed following 24 hours of growth at 30°C.
If lowered gtrA and/or hemL function underlies the heme biosynthesis defect of the hfq mutant, then increasing expression of gtrA, hemL, or both should rescue the mutant growth defect. To test this, we constructed arabinose-inducible plasmid expression vectors containing the gtrA gene, the hemL gene, or both the gtrA and hemL genes. We then constructed wild type and hfq mutant strains containing these plasmids and determined whether expression of gtrA, hemL, or both altered growth of these strains relative to wild type and hfq mutant strains containing the empty vector.
Exogenous expression of gtrA alone completely rescued the small colony phenotype of the hfq mutant (Figure 3B and 3C). In contrast, hfq mutant colonies expressing hemL alone were indistinguishable in size from hfq mutant colonies with vector alone (Figure 3D and 3E). Expressing both gtrA and hemL rescued hfq colony size to a similar extent as expressing gtrA alone (Figure S3). Taken together, our data suggest that reduced gtrA function underlies the colony size defect of the hfq mutant.
Increasing heme availability via nutritional supplementation or genetic manipulation rescues the exponential growth defect of the hfq mutant
The small colony phenotype of the hfq mutant could be due to one or more S. oneidensis hfq mutant phenotypes, including the defect in exponential phase growth and/or saturation at a lower terminal density [10]. To determine how nutritional supplementation and genetic manipulation of the heme biosynthetic pathway influence culture growth kinetics and increase the size of hfq mutant colonies, we performed detailed analyses of both the growth kinetics and heme content of wild type and hfq mutant cultures in which we manipulated heme availability via nutritional or genetic means.
Addition of 50 µM 5-ALA to LB liquid medium completely rescued the exponential phase growth defect of the hfq mutant (Figure 4A) and restored heme levels in the hfq mutant to wild type levels (Figure 4B). However, by 7–8 hours of culture growth, when heme levels in the unsupplemented hfq mutant were indistinguishable from those in wild type cells (Figure 4B and 4D), absorbance values for the hfq mutant plus 5-ALA stopped increasing and were no longer coincident with the MR-1 cultures (Figure 4A). The terminal density of stationary phase hfq mutant cultures was significantly increased by addition of 5-ALA, but these cultures never achieved the terminal density of wild type cultures (Figure 4A). This suggests that factors independent of heme levels are at least partially responsible for the reduced terminal density of the hfq mutant.
(A) Growth curves for MR-1/pBBAD-SP (MR-1) and hfqΔ/pBBAD-SP (hfqΔ) grown in either LB Km liquid medium or in LB Km liquid medium supplemented with 50 µM 5-ALA. (B) Heme content analysis of cells from MR-1/pBBAD-SP (MR-1) and hfqΔ/pBBAD-SP (hfqΔ) cultures grown for either 4 hours or 8 hours in either LB Km liquid medium or in LB Km liquid medium supplemented with 50 µM 5-ALA. (C) Growth curves for the wild type MR-1 strain and hfqΔ mutant strains containing either pBBAD-SP (vector/ev) or pgtrA grown in either LB Km or in LB Km containing 0.005% arabinose. (D) Heme content analysis of MR-1 and hfqΔ mutant cells containing either pBBAD-SP (vector/ev) or pgtrA grown in either LB Km or in LB Km containing 0.005% arabinose. Results presented for both growth curve and heme assays are the means from three independent cultures. Error bars in panels (A) and (C) indicate a 99% confidence interval (P = 0.01). Error bars in panels (B) and (D) indicate standard deviations. ** indicates that the difference between heme levels at 4 hours between MR1 and the hfqΔ mutant are statistically significant (P<0.0025 in an unpaired two-tailed Student's t-test).
Increasing gtrA expression using an inducible plasmid vector also completely rescued the hfq mutant exponential phase growth defect in LB liquid medium, as growth of the hfq mutant expressing gtrA was indistinguishable from growth of wild type cells expressing gtrA (Figure 4C). We consistently observed that expression of gtrA slightly slows the growth of both the hfq mutant and wild type cells, indicating that exogenous gtrA expression mildly compromises growth of S. oneidensis cultures. Expression of gtrA restored heme levels in exponentially growing hfq mutant cells to wild type levels (Figure 4D). Similar to nutritional rescue of hfq mutant growth, genetic rescue of growth waned between 7–8 hours (Figure 4C).
These data indicate that the exponential phase growth defect of the hfq mutant is strongly linked to heme levels 2–3 fold lower (see Figures 2C, 4B, and 4D) than those found in wild type cells. This heme deficiency persists only until cultures begin to enter stationary phase, at which point the heme levels in the mutant cells are equivalent to those found in wild type cells (Figure 4B and 4D). Increasing heme availability rescues exponential growth of the S. oneidensis hfq mutant in LB, indicating that slow growth is due to reduced heme levels. In addition, our genetic and nutritional data strongly suggest that the S. oneidensis hfq mutant's heme defect occurs at the 5-ALA synthesis step.
The hfq mutant contains modestly reduced levels of gtrA mRNA
To begin to elucidate the mechanism by which Hfq regulates gtrA function, we analyzed relative gtrA mRNA levels in exponentially growing wild type and hfq mutant cells. In 4 hour old cultures, we observed a modest, but significant reduction in gtrA mRNA levels in the hfq mutant relative to the wild type strain. When gtrA expression levels were normalized to 16S rRNA levels, the hfq mutant contained 0.63 fold the quantity (P<0.00002) of gtrA mRNA detected in wild type cultures (Data S1). When we independently normalized gtrA expression levels to recA mRNA levels, we observed that the hfq mutant contained 0.69 fold the quantity (P<0.0014) of gtrA mRNA detected in wild type cultures (Data S1). Thus, our data suggest that loss of hfq results in reduced gtrA transcription, an increased gtrA mRNA turnover rate, or both.
Here we report that the exponential growth defect of the S. oneidensis hfq mutant in LB medium is due to reduced levels of heme during exponential phase growth. The lowered heme levels are likely due to a defect in the 5-ALA synthesis step, as addition of 5-ALA to the medium or increased gtrA expression completely restores growth, while increasing the nutrient pool available to the cells does not restore growth. The fact that growth in nutritionally supplemented solid medium was not as robust as rescue in nutritionally supplemented liquid medium could be the result of local depletion of resources surrounding colonies on solid medium. However, increasing supplement concentrations did not further increase growth rescue on solid medium, suggesting that the difference in degree of growth rescue between solid and liquid medium is likely a reflection of differences in growth in the two media types.
That the hfq mutant exponential growth phenotype in LB is due to a deficiency in a single metabolic pathway is striking because loss of Hfq compromises many cellular processes. S. oneidensis hfq mutant cells growing in LB are partially auxotrophic for heme during exponential growth. Any other deficiencies that might influence exponential growth of the hfq mutant are not apparent when grown in LB liquid. A heme deficiency also contributes to the slow growth of the hfq mutant in medium other than LB, since heme supplementation of modified M1, a less rich defined medium, also substantially rescues the hfq colony size defect (Figure S4A and S4B). However in M1 medium, we observe a more modest rescue of hfq mutant growth with 5-ALA supplementation than we observe in LB (Figure S4C). This suggests that the hfq mutant has a reduced ability to convert 5-ALA into heme when it is grown on modified M1 medium. Indeed, supplementation of M1 medium with 400 µM ALA inhibits growth of the hfq mutant, possibly as the result of buildup of heme biosynthetic intermediates, some of which are toxic [31]. Thus, growth in less nutritionally rich medium appears to expose additional metabolic deficiencies of the hfq mutant.
Once hfq mutant cells reach the end of exponential phase, their heme levels are similar to those observed in similarly aged wild type cells. Thus, a heme deficiency cannot fully explain other hfq mutant phenotypes, such as the reduced stationary phase density or the late stationary phase survival defect of hfq mutant cultures. However, the terminal densities of hfq mutant cultures in which the heme defect has been rescued are significantly higher than the terminal densities of hfq mutant cultures alone. This indicates that the terminal density of a culture is in part, but not wholly, determined by the extent of its exponential phase growth. Further investigations into the mechanisms by which Hfq regulates heme levels should provide insight into why the hfq mutant has lower heme levels during exponential phase growth but not at the beginning of stationary phase.
An attractive model to explain slow exponential phase growth in the hfq mutant is that lower heme levels reduce the electron transport capacity of the cells, since heme is the redox center of cytochrome proteins. Reduced cytochrome function and diminished electron transport could also account for the slower chromium reduction kinetics and reduced anaerobic growth of the hfq mutant. Low heme levels could explain other S. oneidensis hfq mutant phenotypes. For example, because heme is a key part of the active site of the hydrogen peroxide degrading enzyme catalase, reduced heme availability could compromise catalase activity, making the hfq mutant more sensitive to oxidative stress. Heme is also a key part of the iron storage protein bacterioferritin, raising the possibility that changes in heme availability during exponential phase could impact iron homeostasis in the hfq mutant. However, regardless of bacterioferritin function, the levels of free iron in both wild type and hfq mutant cells are similar, suggesting that iron homeostasis may be minimally affected by loss of Hfq under the conditions tested.
Though Hfq appears to regulate heme production at the 5-ALA synthesis step, the mechanism by which loss of hfq results in lowered GtrA activity and thus reduced heme levels is not yet clear. Considering the modest reduction in gtrA mRNA levels in the hfq mutant during exponential growth, Hfq could function to stimulate transcription of gtrA and/or promote gtrA mRNA stability. However, though there is a statistically significant difference in gtrA mRNA levels between wild type and hfq mutant cells, it is not clear whether a ∼1.5 fold difference in gtrA expression is biologically significant in regard to heme levels. This raises the possibility that S. oneidensis regulates gtrA activity posttranscriptionally. For example, Hfq could promote the function of an unidentified sRNA that positively regulates the translation of gtrA mRNA by releasing attenuation due to a secondary structure that blocks the putative ribosome binding site. It is also possible that loss of Hfq could result in an increased heme or GtrA turnover rate, leading to lower steady state heme levels in exponentially growing hfq mutant cells. It is clear from our analyses that the regulatory mechanisms controlling heme levels in the hfq mutant differ substantially from those in wild type cells. We are currently investigating the mechanism(s) by which Hfq regulates heme levels in S. oneidensis.
Materials and Methods
Growth media, strains, and culture conditions
Unless otherwise specified, cultures were grown in LB medium (10g/L tryptone, 5g/L yeast extract, 10g/L NaCl) supplemented with kanamycin (Km). Other media used included modified M1 medium [10], [11], LB with double the normal quantities of tryptone and yeast extract (20g/L tryptone, 10g/L yeast extract, 10g/L NaCl), LB supplemented with 135.9 µM L-glutamic acid, 114.8 µM L-arginine, and 190.3 µM DL-serine, the concentrations of amino acids found in modified M1 medium [10], [11], trypticase soy agar (TSA), TSA supplemented with 5% sheep blood (Becton Dickinson), LB supplemented with 50 µM iron (in the form of FeSO4 or FeCl3), LB or M1 supplemented with 50 µM or 400 µM 5-aminolevulinic acid, and LB or M1 supplemented with 50 µM porcine hemin.
S. oneidensis strains used in this study are wild type strain MR-1 [11] and the MR-1 hfqΔ mutant [10] and their derivatives. S. oneidensis cultures and cultures containing both E. coli and S. oneidensis were grown at 30°C, while E. coli cultures were grown at 37°C. Antibiotics were used at the following concentrations: kanamycin (Km), 25 µg/mL; gentamicin (Gm), 5 µg/mL.
Bacteria grown on plates were streaked to single colonies in four phases from frozen permanent stocks or from colonies on streak plates that had been inoculated from frozen permanent stocks and grown overnight. For growth curves, overnight aerobic 5 mL LB Km cultures of S. oneidensis strains inoculated from frozen permanent stocks were diluted in LB Km to an ABS600 ≅0.1 and outgrown aerobically to mid exponential phase (ABS600 ≅0.4–1.0). These log phase cultures were then diluted to an ABS600 ≅0.1 in LB Km and grown aerobically in 125 mL Erlenmeyer flasks shaken at 250RPM. Culture turbidity (ABS600) was measured at regular intervals.
Detection of free intracellular iron
Free, intracellular iron was detected via a modified version of a previously described protocol [22]. Overnight LB Km cultures of S. oneidensis strains were diluted to an ABS600 ≅0.1 and outgrown in LB Km. The equivalent of 5 mL of culture at an ABS600 of 1.0 was harvested for each strain after 4 hours of growth. Cells were pelleted in Eppendorf tubes and washed once by resuspending in sterile 0.85% (w/v) saline. The washed pellets were then resuspended in 1 mL of Bacterial Protein Extraction Reagent (B-PER, Thermo Scientific) containing 100 µg/mL lysozyme and incubated at room temperature for 15 minutes with regular mixing. 100 µL of 10 mM ferrozine reagent [3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4′, 4″-disulfonic acid sodium salt] (Sigma Aldrich) in 0.1 M ammonium acetate was added to each sample and mixed. 800 µL of this solution was then mixed with 150 µL of 1.4 M hydroxylamine hydrochloride in a fresh Eppendorf tube and incubated for 15 minutes at room temperature to reduce Fe(III) to Fe(II). The solution was then neutralized by adding 50 µL of 10 M ammonium acetate, pH 9.5. Samples were incubated for at least 4 hours at room temperature to allow full color development. Each sample was then pelleted for 2 minutes at maximum speed in a microcentrifuge. 600 µL of the supernatant was then transferred to a cuvette for ABS562 measurement in a spectrophotometer zeroed using a sample with assay reagents only. This assay is quantitative, as there is a linear relationship between iron concentration and ABS562 values (Figure S2A). Hemin alone did not produce a signal using this protocol (Figure S2B).
Detection of total intracellular heme
Bacterial heme was detected via a modified version of a previously described protocol [23]. Overnight LB Km cultures of S. oneidensis strains were diluted to an ABS600 ≅0.1 and outgrown in LB Km. The equivalent of 10 mL of culture at an ABS600 of 1.0 was harvested in duplicate for each strain at the indicated time points. Cells were pelleted in Eppendorf tubes and washed once by resuspending in sterile 0.85% (w/v) saline. The washed pellets were then resuspended in 840 µL of Bacterial Protein Extraction Reagent (B-PER, Thermo Scientific) containing 100 µg/mL lysozyme and incubated at room temperature for 15 minutes with regular mixing. 200 µL of pyridine and 100 µL of 1.0 M NaOH was then mixed with each sample. One paired sample was oxidized by addition of 10 µL of 1 M potassium ferricyanide. The second paired sample was reduced by addition of 2–5 mg of sodium hydrosulfite powder. After zeroing the spectrophotometer with a sample with assay reagents only, an absorbance spectrum for each sample at 400–700 nm was obtained. A subtraction spectrum for each sample pair was generated by subtracting the oxidized spectrum from the reduced spectrum. Heme concentration was calculated using the absorbance difference between the peak at 550 nm and the trough at 535 nm from the subtraction spectrum and the millimolar extinction coefficient for heme C of 23.97 [23].
Construction of plasmid vectors and arabinose induction
pBBAD-SP is an arabinose-inducible expression vector derived from the Kmr broad host range vector pBBR1-MCS2 [24]. A ∼1.7 kb fragment containing the araC gene, the PBAD promoter, the multiple cloning site, and the transcriptional terminators from pBAD18 [25] was PCR amplified using the primers TCCGAGATCTTTATGACAACTTGACGGCTACATC and AGCGCTCGAGAACAAAAGAGTTTGTAGAAACGCAAAAAGG. This PCR fragment was restricted with both BglII and XhoI and then ligated to BamHI and XhoI-restricted pBBR1-MCS2.
To construct plasmid vectors expressing gtrA (So_3834) alone, hemL (So_1300) alone, or both gtrA and hemL from S. oneidensis under control of an arabinose-inducible promoter, the appropriate open reading frame(s) was/were PCR amplified and cloned into pBBAD-SP. To construct pBBAD-gtrA, a gtrA PCR product generated using the 5′ primer GGCGAATTCCATAGGGCCCTAAGGAGGAAAAAAAATGAGCCTTGTAGCAATC and the 3′ primer GGCAAGCTTCCTTCATTTAACTCGCTAACC was digested with EcoRI and HindIII and ligated to EcoRI and HindIII-restricted pBBAD-SP. To construct pBBAD-hemL, a hemL PCR product generated using the 5′ primer GGCGAATTCCATAGGGCCCTAAGGAGGAAAAAAAATGACCCGTTCCGAAGC and the 3′ primer GGCAAGCTTGTAAATACTTAGTTTGCCGC was digested with EcoRI and HindIII and ligated to EcoRI and HindIII-restricted pBBAD-SP. To construct pBBAD-gtrA+hemL, a gtrA PCR product was generated using the 5′ primer GGCGAATTCCATAGGGCCCTAAGGAGGAAAAAAAATGAGCCTTGTAGCAATC and the 3′ primer GGCGAATTCCCTTCATTTAACTCGCTAACC, while a hemL PCR product was generated using the 5′ primer GGCGAATTCCATAGGGCCCTAAGGAGGAAAAAAAATGACCCGTTCCGAAGC and the 3′ primer GGCAAGCTTGTAAATACTTAGTTTGCCGC. The gtrA PCR product was digested with EcoRI, while the hemL PCR product was digested with both EcoRI and HindIII. These two fragments were ligated to pBBAD-SP restricted with EcoRI and HindIII to generate an arabinose-inducible gtrA-hemL synthetic operon.
S. oneidensis strains (wild type MR-1 and hfq mutant) containing the plasmids described above were constructed by first transforming the plasmids into the tra+ E. coli strain S17-1 λpir [26]. The plasmids were then mobilized into S. oneidensis via conjugal transfer. MR-1 transconjugants were isolated from modified M1 plates [10] containing Km, while hfq mutant transconjugants were isolated from LB Km Gm plates.
Induction of heme biosynthesis genes cloned downstream of the PBAD promoter was accomplished by addition of 0.005% (w/v) arabinose to the medium. This arabinose concentration was selected to optimize growth rescue and minimize negative effects of exogenous gtrA expression (see Figure 4C). The slight rescue of the hfq mutant growth phenotype by the pBBAD-gtrA plasmid (Figure 3B) and the significant rescue by the pBBAD-gtrA+hemL plasmid (Figure S3A) without arabinose present suggests that there is a low level of expression from the PBAD promoter in S. oneidensis on LB medium in the absence of induction.
Preparation of total RNA and QRT-PCR analysis
Aerobic, 5 mL LB Km cultures of S. oneidensis strains inoculated from frozen permanent stocks and grown overnight were diluted in LB Km to an ABS600 ≅0.1 and outgrown aerobically until harvest 4 hours later. Total RNA was prepared using the RNeasy Protect Bacterial RNA Kit (Qiagen) following manufacturer instructions.
cDNA reaction mixtures were prepared with the AffinityScript QPCR cDNA synthesis kit (Agilent Technologies) according to manufacturer's instructions. cDNA synthesis was primed using random oligonucleotide nonamers provided in the cDNA synthesis kit, and 100 ng of total RNA was used as the template for each 20 µL reverse transcription (RT) reaction. RT reactions were incubated at 25°C for 5′, 42°C for 15′, 55°C for 15′, 95°C for 5′, and then held at 4°C. Reactions were used immediately or stored at −20°C for later use.
Target-specific primers for QPCR reactions (Table S1) were selected using the PrimerQuest tool available on the Integrated DNA Technologies (IDT) web site. QPCR reactions were performed using the Brilliant II SYBR Green QPCR Master Mix reagent and the Mx3000P QPCR System (both from Agilent Technologies) as per manufacturer instructions. For each target, up to three different sets of QPCR primers were evaluated for quantitative amplification and amplification efficiency using four serial dilutions of cDNA samples spanning a 64 fold concentration range. For gtrA and recA primers, cDNA dilutions used were undiluted, 1/4, 1/16, and 1/64, while for 16S primers dilutions were 1/16, 1/64, 1/256, and 1/1024. Technical data for primer sets selected for QPCR analyses are contained in Data S1. All primers were used at a concentration of 600 nM.
Quantitative RT-PCR analyses were performed on three independent biological replicates. Three technical replicates were analyzed for each biological replicate. QPCR reactions for recA and gtrA were performed using 1/4 dilutions of cDNA, while 16S rRNA reactions were performed using 1/256 dilutions of the cDNA. Threshold cycle (Ct) values were determined as per the software defaults. gtrA data was individually normalized to either 16S rRNA levels or recA mRNA levels. All amplification data were efficiency corrected using the primer pair standard curve data found in Data S1 and Table S1, and relative target quantities were calculated as per the software specifications. Statistical analyses of QPCR data were performed using unpaired two-tailed Student's t-tests to compare the means of the technical replicate data for the three biological replicates. Technical data for the QPCR reactions is contained in Data S1. Despite the multiple physiological differences associated with loss of Hfq, both 16S rRNA and recA mRNA levels were similar between exponentially-growing wild type and hfq mutant cells for each of the biological replicates (Data S1).
Supporting Information
Data S1.
Data from QPCR experiments and primer set efficiency analyses.
Data S2.
Data for Figure 1D. Raw and processed data for total free iron assays.
Data S3.
Data for Figure 2B and 2C. Raw and processed data for heme assays.
Data S4.
Data for Figure 4. Raw and processed data for growth curves and heme assays.
Data S5.
Data for Figure S2. Raw and processed data from iron assay standard curve and from total free iron assays.
Figure S1.
Growth of wild type and hfq mutant strains on different media. Colony size comparisons of MR-1/pBBR1-MCS2 (vector) and hfqΔ/pBBR1-MCS2 (vector) streaked to single colonies on (A) trypticase soy agar (TSA), (B) LB Km supplemented with 400 µM 5-ALA, (C) LB Km containing double the normal concentrations of tryptone and yeast extract (2X LB), and (D) LB Km supplemented with DL-serine, L-glutamic acid, and L-arginine (LB + AAs - see Materials and Methods).
Figure S2.
Total iron detection: standard curve and cell culture trends. (A) Standard curve generated from ferrozine assays performed using known concentrations of FeCl3. The blue trend line from a linear regression analysis with the y-intercept set at zero indicates that the assay is quantitative within the indicated concentration range (ABS562 = 0.0182 * [Fe] in the sample). For this data set the coefficient of determination (R2) = 0.99705. (B) Results of iron assays using samples containing either 5 µM FeSO4 or 5 µM hemin. Data is the mean of three independent samples. Error bars indicate standard deviation. **** indicates that the difference between iron detected is statistically significant (P<0.0001 in an unpaired two-tailed Student's t-test). (C) Plot of ferrozine assay results (ABS562 values) from iron assays versus culture densities (ABS600 values) at time of harvest. Data is pooled from multiple independent experiments using both MR-1 wild type cells (blue data points) and hfq mutant cells (red data points).
Figure S3.
Exogenous expression of both gtrA and hemL rescues the colony size defect of the hfq mutant. Colony size comparisons of MR-1/pBBAD-SP (vector), MR-1/pBBAD-pgtrA+hemL (pgtrA+hemL), hfqΔ/pBBAD-SP (vector), and hfqΔ/pBBAD-pgtrA+hemL (pgtrA+hemL) streaked to single colonies on (A) LB Km and (B) LB Km containing 0.005% arabinose. Plates were photographed following 24 hours of growth at 30°C.
Figure S4.
Growth of wild type and hfq mutant strains on solid M1 medium supplemented with heme or 5-ALA. Colony size comparisons of MR-1/pBBR1-MCS2 (vector) and hfqΔ/pBBR1-MCS2 (vector) streaked to single colonies on (A) M1 Km medium, (B) M1 Km medium supplemented with 50 µM hemin, (C) M1 Km medium supplemented with 50 µM 5-aminolevulinic acid (5-ALA), and (D) M1 Km medium supplemented with 400 µM 5-ALA. Plates were photographed following 72 hours of growth at 30°C.
Table S1.
Oligonucleotide primers used for QPCR analyses.
We thank Fr. Nicanor Austriaco, O.P. and Jennifer Gervais for thoughtful discussions and critical reading of the manuscript. We also thank Marla Tipping for technical assistance with the QRT-PCR analyses.
Author Contributions
Conceived and designed the experiments: CMB NQM TMH BJP. Performed the experiments: CMB NQM TM TMH MLK JNL SES ENB BJP. Analyzed the data: CMB NQM TMH BJP. Contributed reagents/materials/analysis tools: SP. Wrote the paper: CMB NQM TM TMH BJP.
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Skip to comments.Woman's Scent Can Lure Or Repel
Posted on 01/15/2006 4:51:09 PM PST by blam
Woman's scent can lure or repel
Men said the women's odour was most intense during menstruation
A woman's body odour can help her attract men when she is at her most fertile and repel them when she is not, scientists have said. According to a report in the journal Ethology, when a woman is at the most fertile part of the menstrual cycle her armpit odour is at its mildest.
But when she is having a period, and not ready for pregnancy, the smell changes to an acute, repellent odour.
The researchers studied 12 women who wore armpit pads for 24 hours a day.
A number of primate species display changes during their fertile period, but the majority of scientists believed that this was not the case with humans.
Subconscious cue
The 12 women were restricted from eating certain foods and from using deodorant or hormonal contraceptive.
They provided odour samples on the armpit pads, which were then presented to 42 men, who sniffed them and assessed the attractiveness of the scent.
"Axillary odour from women in the follicular phase was rated as the most attractive and least intense," the study's leader Dr Jan Havlicek, from Charles University in Prague, Czech Republic, said.
"The results suggest that body odour can be used by men as a cue to the fertile period in current or prospective sexual partners," he added.
A previous study by the same team suggested that women subconsciously prefer the aroma of dominant men when they are at the most fertile stage of the menstrual cycle.
Then the researchers asked 48 men to assess how dominant they felt.
The men then wore cotton pads in their armpits for 24 hours, which were subsequently presented to 65 women.
Those who were ovulating rated the "dominant" men as sexiest, but there was no similar pattern among women at other stages of their menstrual cycle.
Women smell like heaven (when their friend aint visiting). Men smell like apes.
And all this time, I thought it was just a dog thing...
Crap ! You beat me to posting it's picture ! :-P
I've found the scent which best attracts women is the odor of the "One Karat Diamond..."
Men smell like apes.And the French (men and women), smell like garbage.
"who sniffed them and assessed the attractiveness of the scent."
I'm trying to think how much you'd have to pay me to do that........
What the...?I must be missing something here.
Dominent men were designated by refusal to sniff armpit pads.
It's a dish.......
And she says those THREE MAGIC WORDS.
What about these women? Equally brave, IMHO!
***I've found the scent which best attracts women is the odor of the "One Karat Diamond..."***
Too many Playboy cartoons jump into my mind!
A beautiful girl wears a big diamond and says to her friend
"This is the famous HOPE diamond!:" then pointing to an ugly short man, "And this is the curse that goes with it!"
A short bald fat man sets at a bar talking to a sweet young thing He says, " I'm not really this tall. I'm setting on my wallet!"
Okay I'll go out on the limb here and say that
I like the scent of my husband. I like his sweat scent too, it turns me on. Both of us use Mitchems so we don't have a problem really.
Hey, why does it always have to he a short, fat, BALD man?
no deordorant? yep,that would repel me
Too much information....[sawing rapidly through the limb]
don't take it personally, kiddo. A lot of us women like bald men.
Now I wonder how exactly a man can smell "dominant"? And how smelling a woman's armpit sweat can ever be attractive?
Get off me, Snardius.
Because men tear their hair out wondering why they wound up short and fat
Those Pine Tree car scent things work well. Just weave the string into your pit hair.
From "Diamonds are Forever";
PO- "I'm Plenty."
Bond- "But of course you are"
PO- "Plenty O' Toole"
Bond- "Named after your father perhaps"
[sawing rapidly through the limb]
Actually, a receding hairline is caused by thigh-wear which is worsened by repeated contact with the headboard.
are you kidding? I have no armpit hair. I take care of it all.
What do you think I am?
Lieutenant Colonel Frank Slade - Unavailable for comment.
"Actually, a receding hairline is caused by thigh-wear which is worsened by repeated contact with the headboard."
You can say that will all confidence. Don't gloat, my dear.
***Bond- "Named after your father perhaps"***
I hope it wasn't B.O. Plenty from the Dick Tracy comic strip!
Would that mean that 'Peter O'Toole' had two daddies? :~0
Or...Diamonds, because you thought oral sex was worth $5000...
Would that mean that 'Peter o'Toole' had two daddies?
I remember when I was a little boy, every time my Mother would hug me, she always smelled so good. I have often wondered is it is something genetic, or maybe she just smelled good.
'Bite them on the spine'?
Not my quote...I heard it in a bar...from a short, fat, bald guy...
Mysteries of Odor in Human Sexuality.
BTW did you know that a hunter bee can smell a flower from distances of over a mile? Once a flower patch is found they can lead the worker bees, who collect the pollen, by using their extremely sensitive sense of smell. But that's another story.
Dogs prefer to sniff an area quite a bit different than an armpit.
Nam Vet
I could make a fortune selling a laundry soap with the smell of menstrual cycle sweat to wash your teen age daughters clothes in. And a second fortune selling some that smelled like dominate men to combat it.
And the odor that most turns a woman's sex drive cold is wedding cake.
Nam Vet
'I take care of it all'
Let me dream on that one ;-)
Just a sec,as I ask
Tony the Tuna what the odor smells like.
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October 18 2012 08:18PM
When it comes to negotiation, there are several styles that can and will work. It all depends on what your side wants and the other side desires. Then, you have to use the things available to you--time, money, bullets, assclowns--to force the issue. At some point, the other side gives in, sells the plant or moves everything to China.
One of the best negotiators I've ever met was my old boss. In the interests of anonymity, we'll call him OTTO. Otto felt that the only good negotiation was one that left both sides feeling a little uncomfortable with the final agreement. The more both sides stretch, the more likely the agreement was truly beneficial.
Most of us live in a world that doesn't allow these long transactions. The amount of lawyering involved in the NHL/PA negotiations could sustain third world countries for a century, but folks like you and me can't stay the course over a long period.
Last time the NHL and the NHLPA got together it cost the players one season without much in the win column. Now, the NHL succeeded by so much that everyone made money but that's different than winning a negotiation. I'm no expert in the field, but this--right where we are now in the process--is the point where I believe the 'PA lost it all last time.
The players side were convinced of one thing: the NHL didn't want to lose an entire season, they would not go that far. The owners called their bluff, and after that all that was left to do was buy presents and wait for Gary to cancel the season. I think Don Fehr has decided to turn the owners ploy from last time into the players ploy this time: the players as a group are acting like they're somewhat indifferent as to the outcome of the negotiation.
Fehr's offer today was described as 'disappointing' by Gary Bettman. Sure! They haven't learned the lesson of last time! From the Fehr pov, that's probably the best word of the day: disappointment.
The NHLPA gave in to a salary cap last time and this time the issue is the percentage to cap. If the NHLPA didn't say no last time and they don't say no this time--what then? At some point you have to show the courage of your convictions and the players appear to feel this is the time.
I can't say I blame them, but that doesn't really matter. The point is this: just like in childhood, when you are challenged by a bully, the choices are stand up for what you believe in or give in and lose a piece of yourself.
I believe that's what players are fighting for this time.
At some point, you have to make a choice. Whether it is minimum wage for $6M a year, the choice is the same: get busy living, or get busy dying. NHL hockey players may not be happy, but they are free. Free from taking a smaller piece of the pie on someone's say-so, or because they've been bullied into it.
I don't believe I have the right to tell a man to accept a deal he feels is demeaning or unjust. And that's the way it is. Sometimes we as fans lose perspective on these things, and I believe this is a time to remind ourselves it is a game, these men have skill that earns them a fine living, and that allowing the process to play itself out is probably the best plan.
I miss the NHL, and I very much miss the Oilers. This, I feel, is a bigger issue. And since it doesn't involve me--I can't pay to see a movie that hasn't been made, and I can't watch a league that can't gather its employees--then I choose to spend my money in other areas and wait for a day when this is settled.
I'm not angry at NHL players tonight. I would request that if they are serious they follow through. I'm not going to choose one side or another, but would like to express my respect for these men and how they play the game.
Godspeed, see you someday.
Unless the system is fixed I would not sign the agreement.
Forget what their chosen career is and what their income is.
If my employer came to me durind record profit years and renegotiated my commission rate so that I would make less and the company would make more for the same thing I have always done for them; it would be very hard to take.
Then 6 yrs later after posting record profits they come to me again telling me I am making too much and wanting to reduce my commision again. I don't care what the $ amount is I would be pissed and would let them know exactly what I thought of their idea. If they insisted I would be looking for work with another employer, preferably one that competes against my previous employer.
The only way I would sign is if the problems were fixed to the point that I was comfortable that I would not be asked to take another reduction when the next CBA expires.
Which 'other employer' would the players go to?
I don't care whet is 'fair'. Fair is some poor kid in Malawi getting the $10 he needs for school fees so he doesn't have to live in extreme poverty his whole life and can get a good job paying $75 a month.
What I care about is leverage in a business deal and the players have put themselves in a position where the owners have more leverage. Fighting for something you can't win because you don't hold the cards is dumb and a waste of my( hockey fans) time. If I felt the players had leverage here, I would be pissed off at the owners for not realizing it.
It's not about fair, it's about being stupid.
These players are already wealthy from the league and team owners.
They should thank the owners and sign the CBA.
If any group should be thanked for players excessive salaries and owners greed it is the poor suckers, the fans, who pay exorbinate prices for tickets, beer and NHL wearables.
As to Webber's contract, it was the Philadelphia Flyers organization that made an indecent offer to a RFA and along with other rich teams continues to screw up the salary system.
October 20 2012, 12:51AM
This a false comparison, like comparing government financing to household financing. Workers, even very skilled workers, can almost always be replaced and so they have very limited leverage, although the latter obviously have more.
Truly elite talent is different. The NHL players are the 700 best players in the world and the gap between them and the next 700 best is massive. They are, in essence, irreplaceable, which gives them immense leverage that does not exist in Ma and Pa companies or huge ones either, so stop making that comparison as it tells us NOTHING about the NHL labour dispute.
Yes, you could probably replace the bottom fifth or even quarter without total disaster, but there are no other Crosbys or Ovechkins, or Toews, or Quicks, or Webers, or Charas or Sedins out there somewhere. Grinders yes, stars no, and that makes all the difference. Even the rubes in Phoenix could tell the difference.
Sorry David. You bust your ass and take home 100 K, which is good dough, although if you really work about 100 hours a week you REALLY, REALLY need to get a life. You know what Socrates said. The unexamined life is not worth living. Time foe a little self examination bro.
Sorry but the players have something almost all of us lack. An incredibly elite talent that people will big huge bucks to watch, because they have phenomenal and unique skill, courage and determination. Do you hate movie stars? All pro athletes? Rock stars? Bankster crooks? Lottery winners? People who inherit? Vulture capitalists? I detect a whiff of jealousy here mate. Maybe if your mum had put you in power skating....Nah.


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Lightweight 80GSM custom tote bag is sized to take on everyday essentials and holds up to heavy use because the seams are designed for added strength.
Water resistant and roomy, these flat custom tote bags have reinforced stitching to carry more.
Made of 600d Poly Canvas provides this custom tote bag with a solid structure to transport all your gear and company logo.
This custom tote bag is 100 % cotton and a great price with lots of colors to choose from.
Made of 100% natural cotton canvas, this 8 oz. imprinted tote bag is perfect for everyday use.
These custom tote bags are made of recycled materials and can be easily folded up in a zip.
These non-woven promotional totes are perfect for outdoor markets especially during the rainy season.
This logo tote bag is large enough to hold a whole day's worth of shopping while using an alternative to plastic shopping bags.
This non-woven custom totes are constructed of non-woven polypropylene and feature a die cut style handle.
A custom bag is lightweight and inexpensive which makes it great for schools, stores, trade shows and events!
Jumbo reusable logo tote bags made of 100% non-woven polypropylene with two side gusset pockets and a Velcro closure
This Polyester Shopper Promotional Tote Bag is a great moving billboard every time your clients go out shopping.
With its stylish colors and cool features, this custom tote bag will get your logo out fast at your next trade show or event.
Made of Non-woven Polypropylene with extra long handles, this custom tote bag is perfect for daily shopping.
Go eco-friendly on your next grocery trip with this promotional tote bag that used to be a plastic bottle.
Non-woven logo tote with die cut carry handle or shoulder straps, so you can carry this bag in the way that's easiest for you.
If you are looking for tote bags that will definately get your brand noticed than these large square custom tote bags are exactly what you want to put your message on!
This non-woven printed shopper tote bag designed to replace your disposable paper and plastic shopping bags.
We will help you create trade show & event bags that will make your company stand out from the crowd. Potential customers will flock to your booth, lured in by the quality custom tote bags printed with your logo. Getting attendees to head to your booth is the first critical step in generating interest and building new business.
Choose the perfect logo bag from dozens of choices and customize the size, color, and finish. You'll find plenty of ways to utilize these trade show bags. Use a few to hold door or raffle prizes, or add tissue paper and employee Christmas gifts during the holiday season. We even carry laminate wine bags to help with gifting the perfect bottle of wine to your favorite clients.
You'll want to keep a stack of our wholesale custom tote bags with you on every business trip. They'll help you publicize your brand whether you're off to a small conference with a few key company partners, or headed to a convention bursting with thousands of potential new customers. Stuff trade show bags with more promotional materials, and you'll have a gift bag everyone will want to receive.
Don't let conference and convention guests slip through your fingers. With customized bags to hold their maps and miscellany, you'll get your brand right WHERE you want it: in the palms of their hands!


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Riviera Armchair
- Overview
Cast of strong, lightweight aluminum, our Riviera Armchair has the subtle curves and substantial presence of classic resort furniture. Click to read an article on recommended care.
- Cast entirely of rustproof, weather-resistant aluminum.
- Features a hand sanded finish antique bronze finish.
- Thick, box-style seat cushion and knife-edged back cushion; imported. (Sold separately.)
- Cushion covers remove for machine washing.
- Armchair: 26.5" wide x 24" deep x 36" high
- Dimensions & Care
- Overall: 26.5" wide x 24" deep x 36" high
- Seat: 26.5" wide (front), 24" wide (back) x 23.5" deep Seat height above floor: 13"
- Backrest: 26.5" wide x 22" high
- Arm length from backrest: 21"
- Arm height from floor: 22"
- Seat Cushion: 22.5" wide x 23.5" deep x 4.5" thick
- Back Cushion: 22" wide x 24" deep x 4.5" thick
- Maximum Weight Capacity: 300 pounds
To maintain outdoor metal furniture, we recommend the following care tips:
- Wash frame with a mild solution of soap and water; rinse with clean water and dry thoroughly.
- Wipe spills immediately with a clean dry cloth.
- Cover furniture with outdoor covers (sold separately) when left outside during incliment weather or when not in use.
- Store indoors during long periods of inclement weather.
- Before storing, drain all water that may have accumulated around frame. Make sure furniture is completely dry before storage.
- Store cushions separately from furniture to prevent possible transfer of moisture.
- Shipping & Return Info
This is an upholstered furniture item.
View our Shipping Information
Return Policy
At Pottery Barn, we take great pride in the quality and craftsmanship of our products. Attention to design, materials, safety and construction is our priority. We carefully inspect your order prior to shipment. Upon receipt, please inspect your purchase and notify us of any damage; we will arrange for a prompt replacement. If, within 7 days for Quick Ship upholstery and 30 days for all other products, you are dissatisfied for any reason, you may return your purchase for a refund of the merchandise value.
We cannot accept returns on monogrammed, personalized, made to order items, or on items damaged through normal wear and tear. We also cannot accept returns of final-sale items which are identified by a price which ends in .99.
For returns of items purchased from your Pottery Barn registry, we will gladly provide a refund or exchange for the merchandise within 90 days of your event or within 90 days of purchase, whichever date is later.
You may return merchandise through the mail for a refund or replacement. For detailed procedures for returning items through the mail, please contact Customer Service at 1.888.779.5176, 7 days a week, 5:00am - 9:00pm (PT).
Armchair Frame
Armchair Cushion
Please Select Fabric: Sunbrella® - Solid
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Ryan Lambert
April 10 2009 06:02PM
Round 1 of The Battle of Alberta doubleheader goes tonight in what was originally going to be an absolute blowout final two games between the Oil and the Flames. That was before GDB No. 1, when hope ran high. Fast forward to GDB 81 and this game suddenly means less than a game of chess between Paris and Nicky Hilton. It will also be just as futile and pathetic.
But even though the games mean nothing to the season overall, emotionally this game still means a lot to hardcore Oilers and Flames fans. Both camps are bitterly disappointed in their squads of late, yet Flames fans will continue to see playoff games featuring their favorite team. The OilersNation, again, will not.
TLP and Wanye have been accused of being the same person in the past. A quick search at the "imaginary internet people directory" shows they are very different. One hails from the Boston area, the other from the frozen tundra of E-town. One loves Hannah Montana, the other Billy Ray Cyrus. Both believe that their respective teams have completely dropped the ball and are willing to discuss tonight:
TLP: I have complied a list of five Reasons why the Flames have been the bigger disappointment, and by "compiled," I mean, "hastily scribbled down on the back of my suicide note while I read a pamphlet on noose preparation."
- Miikka Kiprusoff has a 2.85 GAA and .892 sv % since the deadline and has lost in nine of his 16 appearances.
- Dion Phaneuf, a Norris Trophy nominee just last year, has been on the ice for 116 goals against this year and is currently a -11.
- Despite a much better group around him, Jarome Iginla's goal total has dropped from 50 in 82 games in 2007-08 to 35 in 80 this year.
- Calgary hasn't scored a power play goal since March 23 and has given up five or more goals 16 times this year, including two eight-goal nights. The two teams that scored eight were Toronto and Tampa. Yes, you read that correctly.
- Calgary's likely playoff opponent, barring a literal miracle taking place and the Lord Our Savior Jesus Christ intervening and preventing the Canucks from earning two points this weekend, is Chicago. The Flames went 0-3-1 against the Blackhawks this year and allowed 19 goals in four games. They also only scored seven.
So, y'know, the Flames are pretty much boned.
Wanye: Be that as it may one must remember that you Flames fans get little sympathy in the OilersNation because: a) we hate you and b) you are at least going to be able to see a first round exit. Up here? Not so much.
So here are five reasons why the Oilers have been the bigger dissappointment, by Wanye von Gretz IV.
- The Edmonton Oilers have not won more games than they've lost in any season since the lockout.
- No Oiler has scored 30 goals since Ryan Smyth was traded for a) a forward with 29 total points b) a forward prospect who has spent the entire year in the AHL, with 1 goal to show for it and c) a draft pick spent on a defenseman who may not even get an entry level contract.
- The entire organization is screwed. The Oilers farm team is so bad that of all the players to spend the entire season there, only one has a positive plus-minus — a 32-year old journeyman who spent last season in Austria.
- Mathieu Garon, expected to be the starting goaltender, floundered so badly that he lost his job and was traded midseason. The 24-year old prospect who replaced him as backup not only has worse numbers but is trusted so little that he hasn't started an NHL game since November.
- Outside of Ales Hemsky, no Oilers' forward has even 20 goals. Only one team in the NHL has both a worse penalty kill and a worse powerplay than the Oilers and only two teams have scored fewer goals at home than the Oilers.We just can't stop at five, either...
- The Oilers best goal-scorer is in a nine-way tie for 73rd in the NHL.
- Andrew Cogliano is on pace to be the NHL's worst faceoff man in the last decade. Sam Gagner is only marginally better.
- No Oilers player managed to reach a point per game pace this season.
- The Oilers best defensive prospect at the start of the season, Taylor Chorney, is -29 in the AHL. The Oilers are so bad that he's currently on their NHL roster.
- Having said all of that they are still better than the Flames, who can expect back to back ass kickings from the Oilers en route to a first-round exit.
TLP: Yeah, you're right. You guys really are the worst.
Enjoy the game, everyone.
April 10 2009, 06:12PM
This post made me sad on the inside.
sounds like a battle of epic suckage..
April 10 2009, 06:18PM
Hey TLP remember when Derek Morris was your captain?
thats how I feel right now.
5 games of this home stand was enough for me... gave tonights away... me and MacT have tapped out.
Wow. So I went over to the Oilers site to catch the pre-game interviews. There's Cogliano, a bunch of Springfield Falcons and Charlie Huddy. This game looks like it's gonna be an old fashioned as* kicking and not in the good way. I'm not even sure if MacT is going to be on the bench.
Sad times.
the nation is so empty tonight....
The nation is probably so drunk tonight.
Sam's got the jump tonight! I feel bad for him, not making the second season.
April 10 2009, 07:36PM
this is what I think of the Flames.
No matter how bad the Oilers have stunk at different times. We have never been this bad.
GOILERS 09/10 Stanley Cup Champions.... oh god I sound like a leaf fan, the season can't end soon enough.
have faith Nationeers, it's always darkest before the dawn.
April 10 2009, 07:41PM
this is what I think of the Flames.
No matter how bad the Oilers have stunk at different times. We have never been this bad.
hxxp://www.youtube.com/watch?v=f9eF6DVI0tk (change the x's to T's)
GOILERS 09/10 Stanley Cup Champions…. oh god I sound like a leaf fan, the season can't end soon enough.
I mowed my lawn today and so did two of my neighbors. Got the hotub drained and scrubbed and filled and got the patio furniture out. I was rollin in a tshirt and was hot a few times and I saw people in shorts and wife beaters while I was out gettin propane. Felt like fricken summer today! It also felt like Saturday. Werd.
JDD playing well. Sure makes you think who the goalie next year is going to be
And noe I understand why Hemsky said checker. Look at the way "HORC" is playing. Either shoot or pass
Hocr is our 7m dollar man. It must be nice to be friends with the owner. Anyone want to be my friend
albundy wrote:
JDD playing well. Sure makes you think who the goalie next year is going to be
Yes Desjarlais is playing great, WHat ifs hey
i like how JD covers the net. I was trying to figure out why Roli looked so good and why weren't down at least one nothin when I noticed it was actually JD in net! lol
WOW WHAT A SAVE BY Desjarlais , What if....
Kotalik is here to play
Kotalik tryin to earn a contract.
Sandra wrote:
Yes Desjarlais is playing great, WHat ifs hey
His name is Jeff Drouin- Deslauriers.... Not Desjarlais.
Fingers crossed he plays solid back to back games. Would love to see the guy finish strong. expecially considering how long it's been since he has started.
Crowd seems really into it
Harlie wrote:
i like how JD covers the net. I was trying to figure out why Roli looked so good and why weren't down at least one nothin when I noticed it was actually JD in net! lol
Yes hopefully Rolison will go to detroit, we need him out od the dressing room. In my opinion he is not a team guy. Why? He was mentally , physically, tired also hurt but he would not tell MacT. "What ever Coach MacTavish says", Yah but your burnt old man let Desjarlais play , or what he might play well and you lose out on a contract next year, Retire at the Lacalta Lodge.
and they wouldn't play JD. Futtin idiots.
@ albundy: That's what is awesome with the Battle of Alberta. Brings people together.... no matter how disappointed they are in their team.
And plus if we can stick it to the flames and their division title hopes it's always a bonus. Try and finish the season on a high
Crowd should chant " morley scott"
@ Sandra:
I totally agree with you. Roli is tired and I'm tired of him.
BIG MAC!!!!! See what happens when we have strong tending!? The team can ROLL!
Have they shown MacT even once
albundy wrote:
Have they shown MacT even once
wow they just did. And he turned his head from the camera.
Sully has some of the sweetest stick handling I've ever seen. Captain Kangaroo takes him out after the play.
@ Harlie: now that they are leading.... i don't think they showed him in the first though. not that i noticed anyways.
solid second period. who is this team? second periods usually terrify me.
Harlie wrote:
and they wouldn't play JD. Futtin idiots.
becaus Roli kept Quiet, ruined MacT's career here, Real team guy.
Morley Scott is back?
Pemmer is playing like Horse horcoff, maybe his last game with the Oil?
Vaughn wrote:
@ albundy: That's what is awesome with the Battle of Alberta. Brings people together…. no matter how disappointed they are in their team. And plus if we can stick it to the flames and their division title hopes it's always a bonus. Try and finish the season on a high
Better to play the Hawks then the Jackets, Flames know what they are doing.
@ Sandra:
HA! Bad asset management any way you slice it. Roli did have a few great games but he had a lot of stinkers and more than a few times he had a terrible softy per game which killed our team's momentum. Here JD is covering the net, not floundering around with his stick missing and he is giving our team confidence which translates into OFFENSE.
Currently at the game. I really really hope the oil can keep it up and screw calgary over
Hopefully Oil will not bring back Roli. We honestly don't need him. Time is up. Morely not back yet but hoping he is soon. Fred takes over for Rod and they bring back Morely. I wonder how Rod felt when Katz fired Morely
Maybe Garon coming back
@ 17:
what's the atmosphere like?
if you haven't seen it BIG Mac does a dead on impression of Bubble from Trailer Park Boys. Just go to youtube and it's there.
albundy wrote:
Hopefully Oil will not bring back Roli. We honestly don't need him. Time is up. Morely not back yet but hoping he is soon. Fred takes over for Rod and they bring back Morely. I wonder how Rod felt when Katz fired Morely
I miss Morley's voice, at times it sounds like Rod has had enough. Do you think that Morley would be back when "Fred" takes over next year?
i hope that doosh mini kipper is at the home games in Calgary during the playoffs sp he can hex the flames.
Harlie wrote:
i hope that doosh mini kipper is at the home games in Calgary during the playoffs sp he can hex the flames.
LOL, yah that kid was a joke
@ Harlie: Not bad. Best part for chants so far were the fans going back and forth with LETS GO OILERS/FLMES in the gallery
This sure is going to be a doozy.
23 save Desjarlais , Roli would have let one in already. Chant Desjarlais
@ 17:
Sandra wrote:
23 save Desjarlais , Roli would have let one in already. Chant Desjarlais
There's no "Desjarlais" on our team.
@ Sandra: How bout we don't chant Desjarlais and chant DESLAURIERS because THAT is his name. Not Desjarlais.
Lets chant JDD


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Showing 1-24 of 437 items found in Places to Stay
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If you've never been one for convention, ACME HOTEL is the right choice for you in downtown Chicago. Located 2 ½ blocks from the Magnificent Mile in the River North neighborhood, ACME is downtown Chicago's all new boutique hotel alternative. If you appreciate an eye for design and dig a high-tech vibe, you'll feel right at home. We'd like to call ourselves cool, or hip, because we are, but that just feels like bragging. But mostly, ACME is what we consider approachable - in more ways than one. The fact is, we want your experience at our downtown Chicago hotel to blow your mind, not your budget.
Over 130 rooms which range from jacuzzi suites for couples to comfortable family-style rooms.
Urban cool accommodations, located in the heart of River North, Chicago's most exciting neighborhood for dining & nightlife. Blocks from the Magnificent Mile and many other Chicago top spots.
110 rooms. 2 suites. 25 non-smoking rooms. Room service. Luxury hotel accommodations. Hotel located near the Hyatt and downtown hotels.
America's Best Value Inn & Suites - Waukegan
Indoor Heated Pool, Free Wireless Internet, Free Continental Breakfast, all rooms with refrigerator, iron/ironing board, hair dryer, and coffee makers.
Americas Best Value Inn & Suites - Waukegan/Gurnee
This hotel is known for its friendly, spacious and comfortable environment and is located a few miles from Six Flags, Gurnee Mills, Great Lakes Naval Training Center, downtown Waukegan, area attractions and the best sport facilities.
Americas Best Value Inn-Barrington/Chicago West
Welcome to Americas Best Value Inn Barrington/Chicago West located only thirty-two miles northwest of Chicago, Illinois. Take advantage of our hotel's easy access to area attractions such as the Museum of Contemporary Art -Chicago, Arlington Heights Historical Museum, Lords Park Zoo, and A Fantasy Amusement Co. Our hotel is also near Harper College, Roosevelt University, and The Illinois Institute of Art - Schaumburg. Enjoy outdoor activities at Goebbert's Farm and Garden Center or the Arboretum of South Barrington. Enjoy a delicious meal at our on-site restaurant, The Blue Heron Cafe and Lounge.
AmericInn Hotel & Suites - Schaumburg
The AmericInn Hotel & Suites Schaumburg Hotel provides overnight lodging accommodations in the heart of Schaumburg Illinois. Our entire staff will welcome you and your partner with a hot, home-style breakfast, free hotel-wide wifi, and warm, Midwestern hospitality. Shop at nearby Woodfield Mall, order room service from Wildfire Restaurant, or just relax in our cozy lobby. Your room is ready. We are located near several major Interstates including 90 (NW Tollway), 290 (Eisenhower Expressway) and 294 (TriState Tollway).
Arlington Heights Downtown Business Association
Stroll through our 8-block Downtown Business Association and find a great array of shops, many of which have provided quality services to our community for decades. The Downtown Business Association is home to a pharmacy and grocery store, to jewelry stores and art galleries, to clothing stories and home décor boutiques. Transportation services are also available if you wish to take in other Illinois sites.
CITY:Arlington Heights
This three-story hotel offers 142 guestrooms accessible via interior corridors. All rooms at the Baymont Chicago offer work desks with ergonomic chairs and phones with voice mail. Wireless Internet access and local calls are complimentary. Located eight miles from O'Hare International Airport. The hotel provides 24-hour front desk, free continental breakfast and a gym/fitness center. Near I-294.
Platinum winning property for exceptional quality accommodations and unsurpassed guest service. This hotel is ideally located just minutes from many nearby points of interest, including Six Flags Great America, Naval Station Great Lakes, College of Lake County and the Libertyville Sports Complex. The hotel offers standard rooms, king whirlpool suites, and specialty theme rooms. Take advantage of the hotel's heated outdoor pool, hot tub, sauna, fitness center and an on-site cocktail lounge. Free Continental Breakfast, game room and high speed Internet included with room.
Stay at this Gurnee, Illinois hotel located less than one mile from Six Flags Great America. Ideally situated off Interstate 294 in Northern Illinois, the Baymont Innl & Suites offers the best in accommodations, customer service and location. This Gurnee hotel features 65 well-appointed guest rooms, each offering cable satellite television and free wireless high-speed Internet access for guests' convenience. This 100 percent non smoking hotel offers a variety of amenities including a complimentary continental breakfast, an indoor heated swimming pool, fitness center, guest laundry and more.
Benedictine B & B offers a comfortable, spacious, private suite of rooms within the grounds of a real urban monastery. Monks have been welcoming travelers for centuries and you will be given the same warm welcome by the monks of the Monastery of the Holy Cross.
52 guest rooms, extended stay available. Amenities: business center, fitness center, outdoor heated pool. Complimentary full hot breakfast. Free wi-fi. Winner of Trip Advisor's Certificate of Excellence.
CITY:St. Charles
A smaller, intimate hotel catering to the business traveler with personal service and attention to detail. We feature two restaurants, banquet facilities, and a fitness center.
248-room full-service hotel conveniently located between O'Hare and downtown. Outdoor pool, fitness center, 24-hour restaurant, lounge, room service, fax and copy service. 14,000 sq.ft. of meeting/banquet space
The Best Western Des Plaines Inn is a first-class hotel located only 4½ miles away from O'Hare Airport and very convenient to Donald E. Stephens Convention Center, home of the Rosemont Expo, and All State Arena, home of the Rosemont Horizon. We offer you deluxe accommodations with complimentary breakfast, an indoor heated pool, fitness center, meeting room and free parking. There are several major shopping centers located from 3 to 8 miles from the Best Western Des Plaines Inn.
CITY:Des Plaines
The Best Western Grant Park Hotel is ideally located on Michigan Ave., directly across from Grant Park. Our property has 172 newly renovated guest rooms, an outdoor pool, and a fitness center.
Best Western Hawthorne Terrace
This Boystown vintage hotel is located close to historic Wrigley Field (home of Chicago Cubs baseball), as well as gay-friendly restaurants and nightclubs. Enjoy breakfast on the terrace during warm-weather months.
Best Western Inn of St. Charles
Convenient to area restaurants, downtown St. Charles shopping. Each guest room includes a mini-fridge & microwave. New executive suite wing.
CITY:St. Charles
Best Western Palwaukee Inn-Wheeling
Stay with us at our 3-Diamond rated facility a member of the largest hotel chain in the world. We are conveniently located, offer competitive affordable rates-WIFI included and complimentary continental breakfast. We have all the amenities to meet the needs of any traveler.
Best Western Plus Antioch Hotel & Suites
The Best Western Plus in Antioch, Illinois offers an unsurpassed combination of suburban comforts, neighborhood charms and magnificent views. Both vacationers and business travelers will appreciate the all the extra touches including a complimentary full hot breakfast buffet each morning, complimentary high-speed internet access, the new state-of-the-art 24-hour fitness room, and access to the new business center. Relax in the full-sized heated indoor swimming pool, oversized whirlpool and spacious sun deck. Stylishly decorated guest rooms features an upgraded luxurious bedding, flat panel HDTVs, microwaves & refrigerators. Enjoy a cocktail at Fox's Lounge overlooking Lake Antioch with indoor and outdoor seating including a 2,000 sq. ft. heated patio. And count on superior service from our 24-hour front desk associates to ensure every detail is perfect.
Best Western Plus Oakbrook Inn
DuPage County, Chicago's Western Suburbs - 45 fully-appointed rooms in a boutique hotel setting. Every guest room features goose down comforter, cd/dvd player, refrigerator, microwave, Evening homemade choc.chip cookies, and complimentary breakfast buffet.
Centrally located in downtown Evanston just blocks from the lake. Upscale amenities abound at this Best Western including free Wi-Fi, a modern fitness center, continental breakfast, courtesy shuttle and outdoor pool all for a great value. The 159 guest rooms feature Sealy Posturepedic pillow-top mattresses, luxury linens and coffee makers.


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The family of Leah Justesen will be celebrating her 90th birthday Aug. 16 at the Emery County nursing home.
Leah was the sixth child born Aug. 20, 1913 in Orangeville to Ole William and Caroline Tatton Sitterud, she is the last living member of her family. Leah married Russel Justesen on Oct. 29, 1936. Together they had six children, Annalee (Dick) Jones, Russel, who died as an infant, Karl (Gloria) Justesen, Alice (Tim) Blackham, Ralph (Becky) Justesen, and Kathy (Tracy) Addley. Leah also has 13 grandchildren, six great-grandchildren, and celebrates five generations of girls with her two great-great-granddaughters.
Family and friends are encouraged to stop by and wish Leah a happy birthday.


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|Posted by Steve on October 24, 19100 at 09:23:12:
I have a back-up somewhere in my main sewer line, and im planning on installing an outside cleanout. I was wondering if you had any ideas on footage where the cast iron would meet the clay tiles, i figure this would be my entry point to install a pvc cleanout, breaking the first clay tile out. I figure to use hydraulic cement on the pvc to clay tile and a rubber connector from the pvc to cast iron. The house was built sometime in the middle 60's. Does this sound like a sound plan.
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