text:
Bola Ahmed Tinubu (the revered APC chieftain) and Adewale Tinubu (the revered oil magnate of OANDO) are family relatives.
Adewale Tinubu is the nephew of chief Bola Tinubu, former governor of Lagos State and the National Leader of APC.
Adewale is Tinubu's front in most oil deals in Nigeria. This is an open secret.
NNPC's retail filling stations and more than 20 petroleum products marketing firms, are owing Federal Government at least N85 billion naira.
The debts arose from products PPMC gave these companies for distribution over the last 10 years.
NNPC retail filling stations and OANDO owes more than 50% of the entire debt.
Read Also: EXCLUSIVE: Presidency to investigate NNPC's boss for fraud?
NNPC debt stood at N22.56 billion while that of Oando stands at N25.05 billion. The remaining oil companies owe the rest.
The most indebted companies are owned by some of Nigeria's and Africa's richest billionaires.
Oando PLC, which owes the largest chunk of N25.05 billion, is owned by the Tinubu family.
The PPMC had allocated petroleum products to these companies for onward distribution nationwide.
They took the products, distributed them and refused to pay the Government for 10 years plus.
A law firm, B. I. Murtala & Co., petitioned EFCC, accusing top NNPC and PPMC officials of "abuse of office and economic sabotage.
Read Also: Baru returns NNPC to loosing trends.
"There exists a conspiracy between PPMC/NNPC and suspected oil marketers who deliberately withheld huge government revenues in respect of petroleum products received on credit without due payment or remittance into the Federation Account."
One PPMC source said the relationship between NNPC/PPMC and marketers allowed marketers to owe for a period without payment.
"Products are supplied and an average of two weeks grace given for payment.
"Depending on the time of reconciliation, there could be credit and debit here and there.
"There is always overlapping period before reconciliation.
"But, no marketer would owe for a long period of 10 years without NNPC taking action to recover monies from them," the source said.
Why the EFCC, NNPC and FG are yet to invite the Tinubu's and other oil moguls for questioning beats our imagination.


text:
Peach Nut Cluster Crunch
|Ripe peaches
|4 Large
|1 1⁄3 Tablespoon (1 Tablespoon Plus 1 Teaspoon)
|Cashew nut
|3⁄4 Cup (12 tbs), crushed
|Brown sugar
|18 Tablespoon (1/4 Cup Plus 2 Tablespoon)
|1 Teaspoon
|1 Dash
1.In medium saucepan melt 1 tbsp of butter.Add granola clusters
(crushed),vanilla extract and brown sugar. Cook on low heat until
sugar melts and coats granola.
2. Cut the top off of the peaches, and scoop out center
slightly to remove seed. Spoon granola mixture inside
peach and pack down. Sprinkle with cinnamon. Bake at 325
for 20 minutes until top is slightly brown.
3.Melt remaining tsp of butter and 2 tbsp of brown sugar and stir for 1
minute. Remove from heat. Pour brown sugar glaze over peaches. Serves 4
Recipe Summary
Rate It
Nutrition Rank
Nutrition Facts
Serving size
Calories 496 Calories from Fat 129
% Daily Value*
Total Fat 15 g23.4%
Saturated Fat 4.5 g22.5%
Trans Fat 0 g
Cholesterol 10.8 mg3.6%
Sodium 3.6 mg0.1%
Total Carbohydrates 93 g31%
Dietary Fiber 3.5 g13.9%
Sugars 83.8 g
Protein 6 g12.2%
Vitamin A 13.9% Vitamin C 19.5%
Calcium 2.2% Iron 11.5%
*Based on a 2000 Calorie diet

subset: commoncrawl


text:
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text:
Staphylococcus aureus pathogenesis is significantly influenced by the iron status of the host. However, the regulatory impact of host iron sources on S. aureus gene expression remains unknown. In this study, we combine multivariable difference gel electrophoresis and mass spectrometry with multivariate statistical analyses to systematically cluster cellular protein response across distinct iron-exposure conditions. Quadruplicate samples were simultaneously analyzed for alterations in protein abundance and/or post-translational modification state in response to environmental (iron chelation, hemin treatment) or genetic (Δfur) alterations in bacterial iron exposure. We identified 120 proteins representing several coordinated biochemical pathways that are affected by changes in iron-exposure status. Highlighted in these experiments is the identification of the heme-regulated transport system (HrtAB), a novel transport system which plays a critical role in staphylococcal heme metabolism. Further, we show that regulated overproduction of acidic end-products brought on by iron starvation decreases local pH resulting in the release of iron from the host iron-sequestering protein transferrin. These findings reveal novel strategies used by S. aureus to acquire scarce nutrients in the hostile host environment and begin to define the iron and heme-dependent regulons of S. aureus.
Virtually all bacterial pathogens require iron to successfully infect their human hosts. This presents a problem to invading bacteria because the majority of iron in humans is tightly bound by iron-binding proteins. To counteract this host defense, bacterial pathogens have developed elaborate mechanisms to acquire nutrient iron during infection. To gain insight into how the amount of available iron impacts the human pathogen Staphylococcus aureus, the authors identified proteins that increase or decrease abundance upon alterations in iron status. The authors found that under conditions of iron starvation, the Fur regulatory protein of S. aureus coordinates a redirection of the central metabolic pathways causing the bacteria to produce large amounts of acidic end-products. The accumulation of these acidic end-products facilitates the release of iron from host iron-binding proteins, in effect increasing the availability of this precious nutrient source. These findings provide a mechanistic explanation for how S. aureus alters its local microenvironment during infection to increase the availability of nutrient iron. Based on the well-established role for bacterial iron acquisition during pathogenesis, systems involved in iron acquisition represent excellent potential therapeutic targets against bacterial infection.
Citation: Friedman DB, Stauff DL, Pishchany G, Whitwell CW, Torres VJ, Skaar EP (2006) Staphylococcus aureus Redirects Central Metabolism to Increase Iron Availability. PLoS Pathog 2(8): e87. doi:10.1371/journal.ppat.0020087
Editor: Fred M. Ausubel, Harvard Medical School, United States of America
Received: March 31, 2006; Accepted: July 14, 2006; Published: August 25, 2006
Copyright: © 2006 Friedman 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.
Funding: This research was enabled by Vanderbilt University Medical Center Development funds and U.S. Public Health Service grant AI69233 from the National Institute of Allergy and Infectious Diseases. VJT was supported by Ruth L. Kirschstein NRSA AI071487, and DLS was supported by T32 HL069765–05, from the National Institute of Allergy and Infectious Diseases.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: 2D, two-dimensional; DIGE, difference gel electrophoresis; DIP, 2,2′-dipyridyl; Fur, ferric uptake regulator; MS, mass spectrometry; PCA, principal component analysis; sRNA, small regulatory RNA; TCA, tricarboxylic acid; TSB, tryptic soy broth
Staphylococcus aureus requires iron to successfully colonize the host [1]. To ensure efficient uptake and metabolism of host iron sources, bacterial pathogens regulate a variety of genes in response to the levels of available iron. The canonical bacterial repressor responsible for this iron-dependent regulation is the ferric uptake regulator (Fur) [2]. S. aureus has a functional Fur which has been implicated in the iron-dependent repression of a subset of genes [3–5]. An S. aureus Δfur mutant has a significant defect in virulence in a mouse model of infection, underscoring the importance of iron metabolism to staphylococcal pathogenicity [6]. The consensus sequence to which the S. aureus Fur binds has been predicted using in silico techniques [7]; however, a global analysis of Fur and iron-affected proteins in this important human pathogen has not been reported. The demonstrated role for iron and Fur in staphylococcal pathogenesis emphasizes the importance of identifying the iron-dependent Fur regulon of S. aureus.
Heme is the preferred iron source of S. aureus, and heme acquisition contributes to staphylococcal infection [8]. We have proposed a model for heme-Fe acquisition that involves the hemolysin-dependent lysis of host erythrocytes followed by hemoglobin recognition, heme removal, and transport into the bacterial cytoplasm [9]. Once inside the bacterium, heme can either be degraded by staphylococcal heme monoxygenases [10,11] or segregated to the bacterial membrane, where it is likely incorporated intact into bacterial heme-binding proteins [8]. It is possible that the different potential fates for intracellular heme are dependent on the level of iron and/or heme exposure experienced by the bacterium in distinct host environments. If correct, this model suggests that bacterial pathogens monitor the level of intracellular heme and alter protein expression in response to changes in heme status.
Based on the demonstrated role for iron, Fur, and heme in staphylococcal pathogenesis, we sought to evaluate changes in global protein status in response to alterations in bacterial iron status using two-dimensional (2D) difference gel electrophoresis (DIGE). DIGE enables quantitative differential-display analysis with statistical confidence and is based on 2D gel separations whereby thousands of protein features can be resolved based on isoelectric point and by apparent molecular mass. It uses spectrally resolvable fluorescent dyes (Cy2/3/5) to prelabel samples that are then multiplexed onto the same gel, allowing for direct quantification of each resolved protein feature between the three dye channels without analytical (gel-to-gel) variation. Multiple samples from a complex experiment can be analyzed across several DIGE gels, whereby an internal standard comprised of every sample present in the experiment is included in each multiplexed gel [12–15]. Within each gel, quantitative measurements are made for each resolved protein feature relative to the cognate signal from the internal standard, which is then used to normalize the intragel ratios between gels in a coordinated experiment. Thus, DIGE enables multiple conditions with repetition to be quantitatively analyzed with statistical confidence. Proteins of interest are then identified using mass spectrometry (MS) and database interrogation. Multivariate algorithms, such as principle component analysis (PCA) and unsupervised hierarchical clustering, can now be applied to DIGE datasets to enable analysis of global expression patterns. Combining multivariable DIGE/MS with multivariate statistical analyses clusters cellular protein responses across distinct environmental conditions based on total expression profiles. These technologies can be combined to identify expression changes in coordinated biochemical pathways.
In this manuscript, we report the application of multivariable DIGE/MS to S. aureus cultures exposed to various biochemical and genetic manipulations in cellular iron status. We found that 21 distinct proteins undergo expression changes in response to exogenous hemin, representing the first reported global analysis of bacterial proteins that are affected by this host molecule. Further, through biochemical classification of proteins undergoing expression changes upon alterations in iron status, we observed an overrepresentation of proteins involved in the central metabolic pathways of S. aureus. Based on these observations, a series of experiments was performed revealing that the Fur protein of iron-starved S. aureus redirects central metabolic pathways to increase production of lactate as a fermentative end-product. This increase in lactate production contributes to a decrease in local pH, facilitating the release of iron from host transferrin. Results obtained from this study identify a novel strategy used by S. aureus to increase host iron availability and begin to define the iron- and heme-dependent regulons of S. aureus.
Changes in Iron Status Alter Staphylococcal Protein Expression Patterns
To identify proteins that are affected by alterations in host iron sources, we performed differential expression analyses on S. aureus cultures grown under various conditions of iron exposure. Cytoplasmic proteins were prepared from wild-type and Δfur mutant cells grown under either iron-replete conditions, after iron starvation elucidated by treatment with 2,2′-dipyridyl (DIP), or after exposure to hemin. Protein extracts from each of the four conditions were independently isolated in quadruplicate to control for nonbiological variation, and the resulting 16 extracts were simultaneously coresolved across eight DIGE gels that were coordinated by a Cy2-labeled 16-mix pooled-sample internal standard as described in Materials and Methods (Figure 1). PCA was used to group the 16 individual Cy3- or Cy5-labeled proteome maps based on the overall expression pattern from the more than 1,000 resolved protein forms under survey. PCA allowed for independent confirmation of distinct expression patterns from the four groups and demonstrated high reproducibility between the replicate samples. The four groups (control, iron-starved, Δfur, and hemin) all clustered into separate quadrants with only one proteome map from the hemin group clustering equidistant from the hemin group and the control group (Figure 1B). These assignments were reiterated in an unsupervised hierarchical clustering of the independent proteome maps presented as a heat map, where expression patterns of the individual proteins can also be compared (Figure 1D).
(A) False–colored representative gel from the eight-gel set containing three differentially labeled samples as described in Materials and Methods. Cy2-labeled internal standard (blue), Cy3-labeled control No. 1 (green), and Cy5-labeled iron-starved No. 1 (red) are overlaid for illustrative purposes.
(B) Unsupervised PCA properly groups the 16 individual DIGE expression maps differentiated by two principle components (PC1 and PC2) and demonstrates high reproducibility between the replicate samples within each group.
(C) Composites of DIGE expression patterns representing the five proteins that increase abundance in the presence of hemin (Class I, Table 1), the 29 Class II protein features negatively affected by Fur and iron (Table 2), and the 30 Class III protein features positively affected by Fur and iron (Table 3).
(D) Unsupervised hierarchical clustering of the 16 individual DIGE expression maps (groups, shown along top) and of individual proteins (shown on the left), with relative expression values for each protein displayed as a heat map using a relative scale ranging from −0.5 (green) to +0.5 (red). The gel number (1 through 8), samples (A, TSB; B, iron-starved; C, hemin; and D, Δfur), and Cy3/5 dye labeling for each sample are listed below.
Proteins Affected by Hemin Independent of Iron and Fur
Proteins Negatively Affected by Fur and Iron
Proteins Positively Affected by Fur and Iron
DIGE analysis combined with subsequent PCA and hierarchical clustering allowed for the grouping of protein expression changes between these four conditions into the following five classes: (I) proteins that are affected by hemin independent of Fur or iron (Table 1, proteins that are more abundant as shown in Figure 1C), (II) proteins that are negatively affected by iron and Fur (Table 2 and Figure 1C), (III) proteins that are positively affected by iron and Fur (Table 3 and Figure 1C), (IV) proteins that are affected by iron independent of Fur (Table 4), and (V) proteins that are affected by Fur independent of iron (Table 5). We identified 29 resolved protein features (representing 20 distinct proteins including isoforms) under iron-dependent negative control by Fur, 30 distinct features (25 proteins including isoforms) under iron-dependent positive Fur-mediated control, and 21 distinct proteins that are exclusively affected by hemin.
Proteins Affected by Iron Independently of Fur
Proteins Affected by Fur Independently of Iron
Using the PCA and hierarchical clustering approach, we were able to further group the proteome expression maps into two primary clusters, one containing all eight samples prepared from bacteria grown in TSB or TSB containing hemin and a second containing all eight samples prepared from Δfur bacteria or bacteria starved for iron. This comprised the first principle component, which accounted for 62.3% of the variance in the system. These multivariate analyses of protein expression changes allow for a global representation of the similar patterns in protein expression that occur upon inactivation of fur versus those that occur upon iron starvation. Furthermore, this analysis reveals that exposure of S. aureus to hemin results in a vastly different, and less severe, change in cellular protein expression as compared to altering the iron status of the bacterium (Figure 1D).
S. aureus Proteins Regulated by Hemin Independently of Iron or Fur (Class I)
The reactive nature of heme presents a unique problem to bacterial pathogens, as they must maintain an intracellular heme homeostasis that prevents toxicity while internalizing enough heme for nutrient iron needs [8]. This raises the possibility that bacterial pathogens undergo a coordinated change in protein expression in response to exogenous heme. To identify proteins that respond to heme, we grew S. aureus in the presence of 10 μM hemin and identified proteins that change expression upon hemin exposure but not upon inactivation of fur or iron starvation. Twenty-one proteins were identified that responded exclusively to excess hemin with statistical confidence (0.04 > p > 0.0000037), 16 of which were down-regulated between 1.25-fold and 3.6-fold (Table 1). These proteins represent a variety of predicted biochemical functions without clear overrepresentation of any one physiological pathway. Only five proteins increased expression exclusively in response to hemin, comprising a hemin-activated regulon (Figure 1C). These proteins are involved in lactate metabolism (Ddh, 5.26-fold, p = 0.000067), gene regulation (SaeR, 1.34-fold, p = 0.02), and stress response (YaaD, 3.78-fold, p = 0.000037, ClpL, 1.58-fold, p = 0.033). The fifth protein, a putative conserved ABC transporter (SAV2359), exhibited a 45-fold increase (p = 0.0000037) upon exposure to hemin, and is predicted to encode for a conserved transporter with no demonstrated function. These experiments describe the first global analysis of the heme-regulon of a bacterial pathogen and identify a putative transport system that is highly up-regulated exclusively upon exposure to hemin.
The Hrt System Is Required for S. aureus Growth in Hemin
The protein demonstrating the most significant increase upon hemin exposure (45-fold increase) is an ATP-binding component (SAV2359) of a previously uncharacterized ABC-type transport system. The gene encoding for SAV2359 is located immediately adjacent to a predicted permease component (SAV2360) of the same transport system. Based on our proteomic observations, we have named these proteins the heme-regulated transporter ATPase (HrtA) and permease (HrtB). Importantly, genomic analyses demonstrate that this transport system is conserved across many pathogenic bacteria, including Bacillus anthracis, Listeria monocytogenes, and Enterococcus faecalis (unpublished data).
To explore the contribution of the Hrt system to heme transport, we investigated whether strains inactivated for hrtA or hrtB can grow when hemin is the sole available iron source. There was no detectable difference in the ability of wild-type, hrtA, and hrtB mutant strains to grow in medium where the sole iron source was FeSO4 (Figure 2). In contrast, compared to wild-type, the hrtA and hrtB mutant strains are severely impaired in their ability to grow when hemin is the sole iron source (Figure 2). To further confirm that the hemin-dependent growth defect exhibited by strains inactivated for hrtA and hrtB is dependent on the insertional mutations, and as an attempt to rule out the possibility that the observed growth defects were due to secondary mutations, we transduced the hrtA and hrtB mutations into a clean wild-type background as previously described [10]. Successful transductants exhibited identical phenotypes as the original hrtA or the hrtB mutants, suggesting that inactivation of the Hrt system is responsible for the observed inability to grow in the presence of high hemin concentrations (Figure 2). Taken together, these observations identify the HrtAB as a novel staphylococcal heme transport system that is critically important to proper heme metabolism.
S. aureus Newman (WT), SAV2359 mutant (hrtA), SAV2360 mutant (hrtB), transduced SAV2359 mutant (t-hrtA), and transduced SAV2360 mutant (t-hrtB) strains were grown in iron-free medium supplemented with iron (Fe) or with hemin (hemin). Bacterial growth was determined by measuring the optical density (O.D.600) of cultures. Results represent the mean ± SD from triplicate determinations. Asterisks denote statistically significant differences from wild-type as determined by a Student's t-test (p < 0.05).
S. aureus Proteins Negatively Regulated by Iron and Fur (Class II)
Proteins that increase abundance upon iron starvation or inactivation of fur (via release from repression) represent proteins negatively regulated by Fur in an iron-dependent manner, and hence comprise the canonical Fur regulon of the bacterium. We identified 29 distinct protein features comprising 20 unique cytoplasmic proteins that are increased from 1.3-fold to over 9-fold in the absence of iron or Fur (0.04 > p > 0.0000012, Table 2). These results demonstrate a strong correlation between expression changes upon iron chelation versus the absence of Fur. As expected, iron acquisition systems previously shown to be iron-regulated via Fur are up-regulated under these conditions including proteins involved in siderophore synthesis (SbnE isoforms exhibiting over 3-fold increases in minus iron or Δfur, 0.013 > p > 0.00013) [16] and transport (FhuA, over 8-fold increases, p < 0.0000016) [17].
Five of 21 proteins that were classified as having Class II expression patterns with mostly moderate increases (approximately 1.5-fold) are enzymes of the glycolytic pathway including fructose 1-P kinase (FruB), fructose bisphosphate aldolase (FbaA), triosephosphate isomerase (Tpi), glyceraldehyde 3-phosphate dehydrogenase (Gap), and transketolase (Tkt) (Table 2). This observation is consistent with a systemic up-regulation of glycolysis upon iron starvation, which would lead to a commensurate increase in pyruvate for subsequent use in the tricarboxylic acid (TCA) cycle or as a substrate for fermentative metabolism (Figure 3).
A subset of the predicted central metabolic reactions of S. aureus is shown. Proteins shown in red are up-regulated in the absence of iron or upon inactivation of fur. Proteins shown in blue are down-regulated in the absence of iron or upon inactivation of fur. Proteins shown in green are down-regulated in the absence of Fur independent of iron status. The red arrows predict the direction of reactions upon iron starvation, while the blue arrows predict reactions that are inhibited upon iron starvation.
S. aureus Proteins Positively Affected by Iron and Fur (Class III)
Fur is traditionally considered a repressor of iron-regulated gene transcription. However, recent work has highlighted a role for Fur in the direct and/or indirect activation of a small subset of genes in Helicobacter pylori [18], Vibrio cholerae [19], Neisseria meningitidis [20], Escherichia coli [21,22], and Bacillus subtilus [23]. We identified 30 distinct protein features representing 25 unique proteins that were positively affected by Fur in an iron-dependent manner as measured by decreased detection in the absence of Fur or upon iron starvation (Table 3).
Numerous regulatory factors were positively affected by Fur in an iron-dependent manner including RsbU. RbsU, down 2.12-fold (p = 0.0014) and 1.48-fold (p = 0.016) in Δfur and iron-deplete conditions, respectively, controls the expression of a variety of virulence genes and regulatory systems. Based on this pleotropy, small changes in RbsU expression may have profound affects on cellular metabolism. In particular, RbsU activates acetate catabolism; therefore, the Fur-dependent activation of RbsU is consistent with a down-regulation of the TCA cycle upon iron starvation or fur inactivation [24].
The value of iron to the bacterium is underscored by the Fur-mediated iron dependent increase of four separate proteins that are predicted to contain iron-sulfur clusters. Three of these proteins are the TCA cycle enzymes succinate dehydrogenase (SdhA, decreased 6.42-fold in Δfur, p = 0.0015 and 3.8-fold in iron-depleted, p = 0.00053), aconitate hydratase (CitB, decreased over 4-fold, p < 0.0002 in both conditions), and fumarate hydratase (CitG, decreased over 2-fold in both conditions, p < 0.00025). A fourth TCA cycle enzyme, phosphoenolypyruvate carboxykinase (PckA), which coverts oxaloacetate to phosphoenypyruvate during gluconeogenesis, was decreased approximately 1.7-fold upon inactivation of fur (p = 0.0023) or iron depletion (p = 0.037). Two additional proteins associated with central metabolism and demonstrating group III expression patterns are d-fructose-6-phosphate amidotransferase (GlmS) exhibiting 2.29-fold (p = 0.00063) and 1.8-fold (p = 0.0038) decreases in Δfur and iron-depleted conditions, respectively, and glyceraldehyde 3-P dehydrogenase (GapB), exhibiting 14.5-fold (p = 0.00052) and 6.1-fold (p = 0.000088) decreases in Δfur and iron-deplete conditions, respectively. GlmS converts fructose 6-P to glucosamine-6-P, and hence depletes substrate for phosphofructokinase in effect antagonizing glycolysis. GapB is a second glyceraldehyde 3-P dehydrogenase of S. aureus and based on its function in Bacillus subtilus, is predicted to possess an enzymatic GAPDH activity involved in gluconeogenesis [25]. These results support the Fur-mediated up-regulation of glycolysis upon iron starvation and suggest a commensurate systemic and regulated inhibition of the TCA cycle. Together, these findings support a model whereby in iron-starved S. aureus, excess pyruvate produced as a result of an up-regulation of the glycolytic pathway is shuttled into fermentative pathways as opposed to the TCA cycle.
In keeping with the above model, Class III proteins associated with fermentative metabolism were represented by alcohol dehydrogenase (Adh1), butyryl-CoA dehydrogenase (between 1.5- to 7-fold decreases for the two conditions, 0.05 > p > 0.00002), and three distinct isoforms of formate acetyltransferase (PflB) (between 3.6 to 7.3-fold decreases for the two conditions across isoforms). These three enzymes are involved in the conversion of pyruvate to distinct end-products of fermentative metabolism: formate, ethanol, or butyrate, respectively. These decreases upon iron chelation suggest that iron-starved S. aureus metabolize pyruvate through fermentation to metabolic end-products other than formate, ethanol, or butyrate. These results suggest that excess pyruvate produced as a result of increased glycolysis is converted to other predicted products of staphylococcal fermentative metabolism, such as 2,3-butanediol and/or lactate (Figure 3).
S. aureus Proteins Regulated by Iron Independently of Fur (Class IV)
In addition to Fur, S. aureus possesses the metal-dependent regulators Zur [26], PerR [27], and MntR [28]. The presence of multiple metal-specific regulatory factors raises the possibility that additional as-yet-unidentified factors other than Fur respond to changes in cellular iron content. We identified 22 unique proteins that were positively or negatively affected by iron starvation, but whose expression was not affected by inactivation of fur (Table 4). Four of these proteins are associated with cellular respiration including formate dehydrogenase (Fdh) which was up-regulated 22.32-fold in the absence of iron (p = 0.015), Hpr Kinase (HprK), NAD synthase (NadE), and a single isoform of formate acetyltransferase (PflB). Thus, we have identified a significant pool of proteins that are affected by iron independently of Fur, raising the possibility that an additional transcriptional regulator exists in S. aureus to monitor intracellular iron status. It should be pointed out that DIP binds divalent cations other than iron which might be responsible for some of the Class IV expression changes that were observed.
S. aureus Proteins Regulated by Fur Independently of Iron (Class V)
We also identified 24 unique proteins that changed expression upon inactivation of fur without any significant changes in iron availability status (Class V, Table 5). One Class V protein, GapR, is an activator of Gap expression and is increased in the absence of Fur (1.24-fold, p = 0.05). This moderate increase in expression may contribute to the increase in Gap expression observed upon inactivation of fur (as much as 2.27-fold, p = 0.0048; Table 2). Inactivation of fur leads to down-regulation of PckA (2.85-fold, p = 0.00039) and isocitrate dehydrogenase (CitC; 3.85-fold, p = 0.0018), consistent with our previous observation of Fur-mediated activation of the TCA cycle. Furthermore, acetoin reductase (ButA) is decreased by inactivation of fur (1.55-fold, p = 0.024) implying a commensurate decrease in the production of 2,3-butanediol upon iron starvation. Taken together with results described above, this suggests that a major metabolic end-product of carbohydrate metabolism produced by iron-starved S. aureus is lactate (Figure 3).
Iron-Starved S. aureus Produce Excess Lactate Leading to a Decrease in Local pH
Iron-starved S. aureus are known to restrict oxidative capacity by oxidizing glucose with the accumulation of much lactate and minor amounts of pyruvate, acetate, and acetoin [29]. The coordinated expression changes of the staphylococcal central metabolic pathways identified using DIGE/MS are summarized in Figure 3 and provide a mechanistic explanation for this observation. Our results suggest that iron-starved S. aureus undergo a Fur-mediated redirection of central metabolic pathways leading to the production of lactate as a primary end-product of fermentative metabolism.
To test this hypothesis, we measured the amount of lactate produced by S. aureus after growth in either iron-replete conditions, upon iron starvation elicited by DIP, or upon fur inactivation. S. aureus grown under iron-starved conditions produced approximately 3-fold more lactate than S. aureus grown in the presence of iron. Similarly, inactivation of fur increased lactate production by approximately 2-fold in iron-replete medium (Figure 4A). To test whether this increase in lactate production contributes to a commensurate decrease in pH, we measured the pH of medium from cultures of iron-replete, iron-deplete, Δfur, and Δfur containing a full-length copy of fur provided in trans (Δfur + fur). These experiments demonstrated a drop in the pH of the iron-starved culture from 7.2 to 5.2 upon either iron starvation or fur inactivation (Figure 4B). Providing fur in trans complemented the pH decrease of the Δfur strain linking the observed decrease in pH to an absence of fur. When subjected to identical growth conditions, iron-replete cultures increased pH to close to 8.0 (Figure 4B). From these data, we conclude that S. aureus elaborates a Fur-mediated redirection of central metabolism under iron starvation to increase lactate production and decrease pH.
(A and B) pH and lactate concentrations of fresh sterile medium (TSB 1), sterile medium after 15 hour incubation at 37 °C (TSB 2), fresh sterile medium containing 1mM DIP (2,2′ dipyridyl), spent medium from wild-type staphylococci grown under iron-replete conditions (iron replete), spent medium from wild-type staphylococci grown under iron-starved conditions (iron deplete), spent medium from the Δfur mutant grown in TSB (Δfur), or spent medium from the Δfur mutant with full-length fur provided in trans (Δfur+fur). Asterisks denote statistical significance as determined by the Student's t-test. A single asterisk represents a comparison to iron-replete cultures, while dual asterisks represent a comparison to iron-deplete or Δfur cultures.
(C) Iron release from transferrin mediated by various media samples. A decrease in optical density signifies a release of iron from transferrin.
(D) Iron release from transferrin mediated by various lactate concentrations shown as molar values. The corresponding pH of the media containing the listed lactate concentrations is shown.
Transferrin-iron represents a viable iron source to invading bacterial pathogens. In order to utilize transferrin-iron as a nutrient iron source, the iron must be dissociated from transferrin and transported into the bacterium. Free Fe3+ is more readily utilized as a nutrient source than transferrin bound iron, and iron is known to dissociate from transferrin upon changes in pH [30]. We hypothesized that the Fur-dependent redirection of central metabolism by iron-starved S. aureus facilitates the release of iron from transferrin through a decrease in local pH. To test this hypothesis, we measured iron release from transferrin mediated by spent medium from either iron-replete, iron-deplete, Δfur, or Δfur + fur staphylococcal cultures. We found that medium from iron-starved or Δfur staphylococcal cultures significantly increased the rate of iron release from transferrin and this phenotype was partially complemented by providing fur in trans (Figure 4C). A similar pattern of iron release was observed upon incubation of transferrin in the presence of lactate (Figure 4D). Taken together, these results demonstrate that the Fur-mediated production of lactate by iron-starved S. aureus facilitates the release of iron from host iron-sequestering proteins.
In this study, we have analyzed changes in the cytoplasmic protein profile of S. aureus upon genetic (Δfur) and biochemical (iron chelation, hemin treatment) alterations in iron exposure. Using large-format, high-resolution DIGE with mixed-sample internal standards, we simultaneously surveyed the S. aureus proteome in response to these manipulations versus control in quadruplicate to provide for statistical confidence. Overall, 156 protein features of interest, specifying approximately 120 individual proteins (including changes in post-translational modification) were identified by mass spectrometry and placed into functional groups defining Fur-dependent and independent iron regulation as well as hemin-affected proteins.
The hemin-affected proteins were particularly of note because this class of proteins has not previously been characterized in bacterial pathogens, despite the identification of heme-regulated proteins in other bacterial pathogens such as Corynebacterium diptherhia [31] and Bordetella sp. [32,33]. The majority of the 21 proteins in this class were decreased upon hemin exposure. However, a few notable exceptions were identified, including a dramatic 45-fold increase of a single protein component (SAV2359) of a putative transporter system which we have named the heme-regulated transporter (HrtAB). This dramatic increase in abundance in the presence of hemin suggests a role for the HrtAB system in heme transport. As a preliminary test of this hypothesis, we individually inactivated hrtA and hrtB and monitored the ability of these strains to grow in the presence of hemin as a sole iron source. These experiments demonstrated a severe growth restriction on hemin upon inactivation of hrtAB and identify the HrtAB system as a critical component of staphylococcal heme metabolism. The Hrt system joins the heme transport system (HtsABC) and iron-regulated surface determinant system, as a third membrane-associated heme transporter [4,8]. The presence of three separate membrane-associated transport systems with roles in heme transit underscores the value of heme metabolism to S. aureus.
The response regulator SaeR was also increased upon exposure to hemin, whereby the modest 1.34-fold increase (p = 0.02) may well have profound affects on gene transcription of target proteins. SaeR together with SaeS activates the transcription of several exoproteins including α-hemolysin and β-hemolysin [34], two proteins with potent erythrocyte lysis activity. It is tempting to speculate that the recognition of host heme up-regulates SaeR expression, in turn activating α- and β-hemolysins, which would lead to an increase in local erythrocyte hemolysis and free hemoglobin concentrations. This might represent a positive regulatory circuit used by S. aureus to increase local heme concentrations, and hence nutrient iron availability.
Another noteworthy class of proteins identified in our study was decreased upon inactivation of fur in an iron-dependent manner, suggesting a Fur-mediated increase in abundance of these proteins. In gram-negative bacteria, the mechanism for Fur-mediated positive regulation of proteins has been elucidated by elegant studies beginning with the work of Masse et al. [35]. These and other investigations have identified the small regulatory RNA (sRNA) RhyB as being responsible for Fur-dependent protein activation in E. coli [35], Pseuodomonas aueruginosa [36], V. cholera [37], and Shigella flexerni [38]. The targets of RhyB include some of the same genes identified in our study as being positively regulated by Fur, including the TCA cycle enzymes aconitase (acnA), fumarase (fumA), and succinate dehydrogenase (sdhCDAB) [21]. This observation suggests that a similar mechanism of iron-dependent gene regulation is occurring in S. aureus, however we were unable to identify any potential homologues to RyhB in any Gram positive bacterial genome using traditional BLAST analyses (unpublished data). S. aureus has been reported to express at least 12 sRNAs with predicted roles in translational regulation through message stability [39]. It is likely that an as-yet-undiscovered sRNA-mediated regulatory system exists in S. aureus responsible for iron homeostasis through targeted mRNA degradation.
Our data indicate that iron starvation leads to the reversible inactivation (or down-regulation) of TCA cycle enzymes including aconitase, the down-regulation of which has been implicated as a survival response to oxidative stress induced during the host-pathogen interaction [40]. In S. aureus, downregulation of the TCA cycle through aconitase inactivation prevents maximal expression of the virulence factors lipase, staphylococcal enterotoxin C, and α- and β-hemolytic toxins and therefore alters the interaction between S. aureus and the host [40]. Additionally, inactivation of the TCA cycle or growth in iron-deplete conditions leads to a decrease in production of formylated delta-toxin, a potent neutrophil attractant [41]. Combined, these two factors have led to the suggestion that down-regulation of the TCA cycle may protect against host immune-mediated recognition of infecting S. aureus [41].
We propose a model whereby upon iron starvation, such as would be encountered inside the host, S. aureus up-regulates glycolysis through the release of Fur-mediated repression of glycolytic enzymes. Based on the simultaneous Fur-mediated down-regulation of TCA cycle enzymes, pyruvate does not enter the TCA cycle but instead is acted on by fermentative pathways. We have demonstrated here that four separate branches of fermentative metabolism are down-regulated under iron starvation, which we predict funnels excess pyruvate into acidic fermentative end-products including lactate (Figure 3).
The production of the acidic end-product lactate contributes to a decrease in the local pH of the microenvironment surrounding infecting staphylococci, a hypothesis supported by the observation that the pH of the spent medium from iron-starved or Δfur staphylococci is significantly more acidic than that of spent medium from iron-replete cultures (Figure 4B). This overproduction of lactate and subsequent decrease in pH dissociates iron from host iron-sequestering molecules (Figure 4C and 4D). Further, the decrease in the local pH combined with a commensurate decrease in Eh (oxidation reduction potential) would be expected to change the oxidation state of host iron atoms converting the insoluble ferric iron to the more bioavailable ferrous iron. An increase in local ferrous iron concentrations would significantly relieve the iron stress placed on the bacterium and provide a growth advantage to invading staphylococci. The Fur-mediated redirection of central metabolic pathways to increase iron availability is supported by published results showing that the uptake of iron (presented as 59FeSO4) by S. aureus is twice as great at pH 4.7 as it is at a pH 7.4 [42].
It is possible that additional acidic end-products of fermentative metabolism contribute to the acidification of the culture medium upon growth of iron-starved S. aureus. For instance, the production of formate as a fermentative end-product would contribute to a decrease in the pH of the microenvironment surrounding iron-starved S. aureus. Acetyl-CoA is converted to formate by formate acetyltransferase (PflB), an enzyme that was identified in our proteomic analyses by three separate isoforms exhibiting decreased abundance upon iron starvation or fur inactivation (Table 3). These results suggest that formate does not significantly contribute to the acidification of spent medium from iron-starved staphylococcal cultures. However, we did identify a single and separate isoform of PflB that increased expression upon iron starvation (2.03-fold; Table 4). Formate dehydrogenase, which subsequently converts formate to NADH, H+, and CO2, also exhibited an increase in abundance upon iron starvation (22.32-fold), consistent with the possibility that appreciable amounts of formate are formed by iron-starved staphylococci. We were unable to detect a significant increase in formate accumulation in the medium of iron-starved staphylococcal cultures (unpublished data), suggesting that if formate is being accumulated as a result of iron starvation, it is a transient increase due to catabolism by Fdh.
Although the experiments described here were performed in vitro, the severe iron restriction encountered by S. aureus once inside the host strongly supports an in vivo relevance for these findings. These fundamental changes in metabolic function potentially provide a survival advantage to S. aureus by preventing maximal activation of the immune system while the bacteria struggle to alter its microenvironment to access host iron.
Materials and Methods
Bacterial strains and growth conditions.
S. aureus clinical isolate Newman was used in all experiments. Prior to cytoplasmic extraction, bacteria were grown in TSB for 15 h at 37 °C with shaking at 180 rpm. Iron starvation was achieved by addition of 1 mM DIP to the growth cultures prior to inoculation. Hemin treatment was achieved by addition of 10 μM hemin to the growth cultures prior to inoculation. All cultures were incubated in the dark to maintain the integrity of the hemin. To avoid differential gene expression due to growth phase, the cultures were harvested at comparable optical densities during early stationary phase. Newman Δfur was created through transduction of the previously created Δfur allele from RN4220 [43] to strain Newman with the transducing phage Φ-85 as previously described [10].
S. aureus strain Newman Δfur was complemented by providing a full-length copy of fur (SAV1498) under the control of its native promoter in the context of a promoterless pOS1-derived vector. fur was PCR amplified from S. aureus Newman genomic DNA using a 5′ primer containing an EcoRI site and a 3′ primer containing a BamHI site. The PCR product was cloned into pCR2.1 (Invitrogen, Carlsbad, California, United States) and excised by digestion with EcoRI and BamHI (New England Biolabs, Beverly, Massachusetts, United States). pOS1 was digested with EcoRI and BamHI and fur was inserted, yielding pOS1fur, where fur is under the control of its native promoter. pOS1fur and pOS1 were electroporated into the restriction deficient primary recipient RN4220, after which they were electroporated into appropriate electrocompetent secondary recipient strains (Newman and Newman Δfur for pOS1, Newman Δfur for pOS1fur). S. aureus strains harboring plasmids were selected on and grown in either tryptic soy agar or tryptic soy broth containing 10 μg/ml chloramphenicol.
hrtA and hrtB mutants were obtained from the Phoenix (N) library, clones PhiNE 03177 (SAV2359) and PhiNE 01762 (SAV2360) [44]. The Phoenix mutant isolates are derivatives of the clinical S. aureus isolate Newman that has been mutagenized using the transposon bursa aurealis transposon. The exact site of transposon insertions have been determined by DNA sequencing and inactivated genes annotated using the S. aureus Mu50 genome [44]. The bursa aurealis insertions in hrtA and hrtB were transduced into wild-type S. aureus Newman with the transducing phage 85 as previously described [10].
Preparation of cytoplasmic fractions.
Cytoplasmic extracts were prepared upon completion of 15 h of bacterial growth. Cells were pelleted by centrifugation at 6,000 g for 15 min. Pellets were resuspended in sucrose buffer (100 mM Tris-HCl [pH 7.0], 500 mM sucrose, 100 mM MgCl2) and incubated at 37 °C for 45 min in the presence of 1 mg of lysostaphin. Following cell wall digestion, protoplasts were isolated by centrifugation at 13,700 g and washed once and resuspended in 20 ml of buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 100 μM phenylmethylsulfonyl fluoride [PMSF]). To lyse the protoplasts, samples were subjected to two rounds of French Press mediated lysis at 20,000 psi. Insoluble material was removed by ultracentrifugation at 100,000 g for 45 min. The collected supernatant, representing the cytoplasmic fraction, was used in subsequent analyses. Successful fractionation was confirmed by immunoblot using fraction specific antisera which recognize the cytoplasm (α-IsdG), membrane (α-IsdE), and cell wall (α-IsdB) of S. aureus [10].
Quadruplicate samples from the four conditions were independently prepared as described above. For each sample, 0.25 mg of protein was separately precipitated with methanol and chloroform [45] and resuspended in 30 μl of lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris, 5 mM magnesium acetate). The NHS-ester dyes Cy2/3/5 were used for the minimal labeling protocol using an internal standard [12,13,46]. Briefly, one-third of each sample (10 μl, 83 μg) was removed and combined into a single tube to comprise the pooled-sample internal standard (1,330 μg total). The remaining two-thirds of each individual sample (20 μl, 167 μg) was individually labeled with 200 pmol of either Cy3 or Cy5, while the pooled-sample was labeled en masse with 1,600 pmol of Cy2. The samples were quenched with 10 mM lysine (2 μl for each 200 pmol) for 10 min on ice, followed by the addition of equal volume 2× rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 4 mg/ml DTT). Pairs of Cy3/Cy5-labeled samples were mixed with an equal aliquot of the Cy2-labeled internal standard according to the schema in Figure 1. Tripartite samples were brought up to 450 μl with 1× rehydration buffer (same as 2× buffer but with 2 mg/ml DTT and 0.5% IPG buffer 4–7) and passively rehydrated into 24-cm 4–7 immobilized pH gradient (IPG) strips for 24 h (total of 500 μg per gel).
All 2D DIGE-associated instrumentation was manufactured by GE Healthcare/Amersham Biosciences (Piscataway, New Jersey). First-dimensional separations were performed on a manifold-equipped IPGphor first-dimension isoelectric focusing unit, and second-dimensional 12% SDS-PAGE was carried out using hand-cast gels that had one plate presilanized (to ensure subsequent accurate robotic protein excision) using an Ettan DALT 12 unit, both according to the manufacturer's protocols. Cy2/3/5-specific 16-bit data files were acquired at 100 μm resolution separately by dye-specific excitation and emission wavelengths using a Typhoon 9400 Variable Mode Imager, and the gels were stained for total protein content with SyproRuby (Molecular Probes/Invitrogen) per the manufacturer's instructions.
The DeCyder v6.5 suite of software tools (Amersham Biosciences/GE Healthcare) was used for DIGE analysis. The normalized volume ratio of each individual protein spot-feature from a Cy3- or Cy5-labeled sample was directly quantified relative to the Cy2-signal from the pooled-sample internal standard corresponding to the same spot-feature. This is performed for all resolved features in a single gel where no gel-to-gel variation exists between the three co-resolved signals. The individual signals from the Cy2-standard were then used to normalize and compare Cy3:Cy2 and Cy5:Cy2 abundance ratios across the eight-gel set, enabling statistical confidence to be associated with each change in abundance or charge-altering post-translational modification using Student's t-test and ANOVA analyses (Table S1). Unsupervised PCA and hierarchical clustering was performed using the DeCyder Extended Data Analysis (EDA) module.
Proteins of interest were robotically excised, digested into peptides in-gel with modified porcine trypsin protease (Trypsin Gold; Promega, Madison, Wisconsin, United States) and peptides applied to a stainless steel target using an integrated Spot Handling Workstation per the manufacturer's recommendations. Matrix assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS) and data-dependant TOF/TOF tandem MS/MS was performed on a Voyager 4700 (Applied Biosystems, Framingham, Massachusetts, United States). The resulting peptide mass maps and the associated fragmentation spectra were collectively used to interrogate S. aureus Mu50 sequences to generate statistically significant candidate identifications using GPS Explorer software (Applied Biosystems, Foster City, California, United States) running the MASCOT search algorithm (http://www.matrixscience.com). Searches were performed allowing for complete carbamidomethylation of cysteine, partial oxidation of methionine residues, and one missed cleavage. Molecular Weight Search (MOWSE) scores, number of matched ions, number of matching ions with independent MS/MS matches, percent protein sequence coverage, and correlation of gel region with predicted MW and pI were collectively considered for each protein identification (all data are presented in Table S1).
Growth curve assays.
S. aureus cultures were grown overnight under low-iron conditions by inoculating strains in RPMI supplemented with 1% casamino acids plus 200 μM DIP. Overnight cultures were then subcultured in NRPMI (Chelex- treated RPMI) containing 500 μM DIP, and inoculated into NRPMI+ (NRPMI containing 25 μM ZnCl2, 25 μM MnCl2, 1 mM MgCl2, 100 μM CaCl2) supplemented with 500 μM DIP, and 20 μM iron sulfate or 10 μM hemin as indicated. Cultures were grown at 37 °C with aeration in a round-bottom 96-well plate and bacterial growth was monitored by increase of absorbance (O.D.600) over time.
Measuring pH, transferrin-Fe release, and lactate.
Bacteria were grown in 10 ml of tryptic soy broth (TSB) in a 50-ml flask for 15 h at 37 °C with 180 rpm shaking. All strains containing derivatives of pOS1 were grown in the presence of 10 μg/ml chloramphenicol to ensure successful maintenance of the plasmid and to normalize growth conditions. Iron was chelated from the media by adding DIP to a final concentration of 1 mM. After 15 h, the cultures were centrifuged and the supernatants were collected. All pH values were measured using a S20 SevenEasy pH meter (Mettler Toledo).
Measuring the release of iron from transferrin was performed as previously described [47]. Iron-bound transferrin exhibits an absorption peak at 470 nm. As iron dissociates from transferrin the intensity of the peak at 470 nm absorption decreases. Absorption at 470 nm was measured every 30 s for 15 min upon introduction of the samples. Transferrin stock solutions of 400 μM were prepared by suspending human transferrin (Sigma, St. Louis, Missouri, United States) in distilled water. Transferrin stock solutions were added at a final concentration of 40 μM to all samples. All absorption readings were measured using a Cary 100 UV-Vis spectrophotometer (Varian).
To measure lactate concentrations, bacteria were grown in 5 ml (TSB) for 15 h at 37 °C with 180 rpm shaking. Lactate levels were measured using a Lactate Assay Kit according to manufacturer's recommendations (Department of Biochemistry at The State University of New York at Buffalo). This kit measures the lactic acid–dependent production of formazan which exhibits an absorption maximum of 492 nm and is directly proportional to the concentration of lactate.
Supporting Information
Table S1. Master Table of All DIGE Profiling and MS Database Search Results
We would like to thank members of the Skaar lab for critical reading of the manuscript and the Vanderbilt Academic Venture Capitol fund for Proteomics. We would also like to thank Dr. Dominique Missiakas for providing the Phoenix library derivatives.
Author Contributions
DBF, DLS, GP, VJT, and EPS conceived and designed the experiments. DBF, DLS, GP, CWW, VJT, and EPS performed the experiments. DBF, DLS, CWW, VJT, and EPS analyzed the data. DBF, DLS, and EPS wrote the paper.
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- 19. Mey AR, Wyckoff EE, Kanukurthy V, Fisher CR, Payne SM (2005) Iron and fur regulation in Vibrio cholerae and the role of fur in virulence. Infect Immun 73: 8167–8178.
- 20. Delany I, Rappuoli R, Scarlato V (2004) Fur functions as an activator and as a repressor of putative virulence genes in Neisseria meningitidis. Mol Microbiol 52: 1081–1090.
- 21. Masse E, Vanderpool CK, Gottesman S (2005) Effect of RyhB small RNA on global iron use in Escherichia coli. J Bacteriol 187: 6962–6971.
- 22. Dubrac S, Touati D (2000) Fur positive regulation of iron superoxide dismutase in Escherichia coli: Functional analysis of the sodB promoter. J Bacteriol 182: 3802–3808.
- 23. Baichoo N, Wang T, Ye R, Helmann JD (2002) Global analysis of the Bacillus subtilis Fur regulon and the iron starvation stimulon. Mol Microbiol 45: 1613–1629.
- 24. Somerville GA, Said-Salim B, Wickman JM, Raffel SJ, Kreiswirth BN, et al. (2003) Correlation of acetate catabolism and growth yield in Staphylococcus aureus: implications for host-pathogen interactions. Infect Immun 71: 4724–4732.
- 25. Fillinger S, Boschi-Muller S, Azza S, Dervyn E, Branlant G, et al. (2000) Two glyceraldehyde-3-phosphate dehydrogenases with opposite physiological roles in a nonphotosynthetic bacterium. J Biol Chem 275: 14031–14037.
- 26. Lindsay JA, Foster SJ (2001) zur: A Zn(2+)-responsive regulatory element of Staphylococcus aureus. Microbiology 147: 1259–1266.
- 27. Horsburgh MJ, Clements MO, Crossley H, Ingham E, Foster SJ (2001) PerR controls oxidative stress resistance and iron storage proteins and is required for virulence in Staphylococcus aureus. Infect Immun 69: 3744–3754.
- 28. Horsburgh MJ, Wharton SJ, Cox AG, Ingham E, Peacock S, et al. (2002) MntR modulates expression of the PerR regulon and superoxide resistance in Staphylococcus aureus through control of manganese uptake. Mol Microbiol 44: 1269–1286.
- 29. Theodore TS, Schade AL (1965) Carbohydrate metabolism of iron-rich and iron-poor Staphylococcus aureus. J Gen Microbiol 40: 385–395.
- 30. Okada S, Rossmann MD, Brown EB (1978) The effect of acid pH and citrate on the release and exchange of iron on rat transferrin. Biochim Biophys Acta 543: 72–81.
- 31. Bibb LA, King ND, Kunkle CA, Schmitt MP (2005) Analysis of a heme-dependent signal transduction system in Corynebacterium diphtheriae: Deletion of the chrAS genes results in heme sensitivity and diminished heme-dependent activation of the hmuO promoter. Infect Immun 73: 7406–7412.
- 32. Kirby AE, Metzger DJ, Murphy ER, Connell TD (2001) Heme utilization in Bordetella avium is regulated by RhuI, a heme-responsive extracytoplasmic function sigma factor. Infect Immun 69: 6951–6961.
- 33. Vanderpool CK, Armstrong SK (2003) Heme-responsive transcriptional activation of Bordetella bhu genes. J Bacteriol 185: 909–917.
- 34. Giraudo AT, Cheung AL, Nagel R (1997) The sae locus of Staphylococcus aureus controls exoprotein synthesis at the transcriptional level. Arch Microbiol 168: 53–58.
- 35. Masse E, Gottesman S (2002) A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli. Proc Natl Acad Sci U S A 99: 4620–4625.
- 36. Wilderman PJ, Sowa NA, FitzGerald DJ, FitzGerald PC, Gottesman S, et al. (2004) Identification of tandem duplicate regulatory small RNAs in Pseudomonas aeruginosa involved in iron homeostasis. Proc Natl Acad Sci U S A 101: 9792–9797.
- 37. Davis BM, Quinones M, Pratt J, Ding Y, Waldor MK (2005) Characterization of the small untranslated RNA RyhB and its regulon in Vibrio cholerae. J Bacteriol 187: 4005–4014.
- 38. Oglesby AG, Murphy ER, Iyer VR, Payne SM (2005) Fur regulates acid resistance in Shigella flexneri via RyhB and ydeP. Mol Microbiol 58: 1354–1367.
- 39. Pichon C, Felden B (2005) Small RNA genes expressed from Staphylococcus aureus genomic and pathogenicity islands with specific expression among pathogenic strains. Proc Natl Acad Sci U S A 102: 14249–14254.
- 40. Somerville GA, Chaussee MS, Morgan CI, Fitzgerald JR, Dorward DW, et al. (2002) Staphylococcus aureus aconitase inactivation unexpectedly inhibits post-exponential-phase growth and enhances stationary-phase survival. Infect Immun 70: 6373–6382.
- 41. Somerville GA, Cockayne A, Durr M, Peschel A, Otto M, et al. (2003) Synthesis and deformylation of Staphylococcus aureus delta-toxin are linked to tricarboxylic acid cycle activity. J Bacteriol 185: 6686–6694.
- 42. Cohen S, Sweeney HM, Leitner F (1967) Relation between iron uptake, pH of growth medium, and penicillinase formation in Staphylococcus aureus. J Bacteriol 93: 1227–1235.
- 43. Mazmanian SK, Ton-That H, Su K, Schneewind O (2002) An iron-regulated sortase anchors a class of surface protein during Staphylococcus aureus pathogenesis. Proc Natl Acad Sci USA 99: 2293–2298.
- 44. Bae T, Banger AK, Wallace A, Glass EM, Aslund F, et al. (2004) Staphylococcus aureus virulence genes identified by bursa aurealis mutagenesis and nematode killing. Proc Natl Acad Sci U S A 101: 12312–12317.
- 45. Wessel D, Flugge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138: 141–143.
- 46.
Friedman DB (2006) Quantitative proteomics for two-dimensional gels using difference gel electrophoresis (DIGE). In: Matthiesen R, editor. Mass spectrometry data analysis in proteomics. Totowa (New Jersey): Humana Press.
- 47. He QY, Mason AB, Woodworth RC (1997) Iron release from recombinant N-lobe and single point Asp63 mutants of human transferrin by EDTA. Biochem J 328(Pt 2): 439–445.


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Log In / Sign-Up | My Account | Customer Service
Log In / Sign-Up | My Account | Customer Service
sendLog In / Sign-Up |
The Snom D7 Expansion Module is a high-performance, high-resolution, easy-to-use expansion module
Add 42 additional programmable keys and LEDs to your Snom 320, 360 or 370 IP phone.
An announcement via snom PA1 is effected simply by initiating a call from a snom telephone with its handset, headset, or via handsfree mode. An efficient 4 Watt amplifier provides sufficient coverage for the entire room or floor space.
Snom Universal Power Supply for 300/700/800 series phones and PA1.
Snom Wireless Headset Adapter is constructed specifically for the Snom 320, 360, 370, 821, and 870 phones.
The snom klarVOICE can be used with any snom 300 series phone. klarVOICE handset uses wide-band technology which makes it possible to deliver a frequency spectrum of 50 to 7,000 Hz.
Unidata ICW-1000G phone pouch
Unidata Multi charger for up to 3 WPU7800 batteries
The Yealink BT40 is a USB dongle that adds Bluetooth connectivity to your SIP-T46G


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Lauren Ralph Lauren 10-mm.Pearl Necklace
Price $48.00
Smooth 10-mm. pearl accents decorate this classic necklace. Complete with a sleek silver-tone clasp, it's one exceptional accessory for dressing up any look!
- 18-in. L
- Foldover clasp
- Polished finish
- Silver-tone metal
- Imported
Shipping & Returns
Shipping your purchase is easy on belk.com. Depending on the destination of your package, you can expect it to arrive within 4-7 business days. Shipping Information
Returns are easy on belk.com. Easy returns
More from: Lauren Ralph Lauren


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St Mary's Art Center
Class begins June 15th to 20th, 2016
Check in and welcome dinner Tuesday June 14, 2016
Paint, Live, and Eat! That should be the motto for this workshop which has become a nearly two decade tradition in Virginia City. Here we expand our boundaries as artists as we paint in the Victorian studio of this venerable building. Participating artists not only take the workshop but stay in the beautifully restored rooms of the 1870's era brick building.
This year the theme will be Creative Landscape Painting. Demonstrations, talks and slide shows will examine fresh ways to see the landscape as an avenue for expression. Strict adherence to the subject is not always a plus when interpreting the poetry of your subject. Develop this side of your artist's soul.
This year the slide/lecture will be on the theme of Creative Landscape Painting which highlights approaches that various artist's have taken in interpretation of the landscape.
Tuition and lodging are included in the cost: $625. Spouses and partners are welcome. 35. per night extra
For those commuting or staying elsewhere Tuition only/ No Lodging $425.
Note: Class starts on Wednesday the 15th but arrival is on Tuesday the 14th with an orientation dinner in the evening. You can check out on Monday the 30th if you wish.
Class limited to 17 artists so please sign up early it fills fast! But please make sure you can attend before registering, cancellations can be problematic.
$200. Deposit
Registration form contact Dale Laitinen
email: firstname.lastname@example.com


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Smart Investments Now Can Lead To A Smart Water Future
According to a recent BofA Merrill Lynch Global Research report, water may be harder to acquire than oil by 2030, and the demand could be 40 percent greater than the water available. The report goes on to say that this would affect nearly half of the world's population. One way to reduce the likelihood of this happening is by implementing sustainable water management practices.
Similar to setting up a budget at home to manage your money more efficiently, it's important in the water industry to constantly keeping track of ways to optimize operations that will ensure water needs are being met now and in the future.
If the analogy between your finances and water running through your hands resonates with you, then you know it's very easy to spend money if you aren't keeping track of what's coming in and what's going out. We've all suffered from buyer's remorse, overspending on something we didn't really want or need – it's not a good feeling. That's why it is important to keep track of the water that comes in and out of our treatment plants.
As the research report points out, the demand for water is going to increase in the years to come and we need to be prepared for this. That's why it's necessary to implement innovative measures and utilize new forms of technology to track consumer water use. We've just completed some research that will allow us to do exactly that. Being able to accurately monitor water demand provides the information required to adjust and enhance internal operations, and ultimately increase the quality and the reliability of the water being delivered to each customer.
There's an obvious price to assuring such quality and dependability. It can cost three times more than other utilities (like electric or gas) to run the facilities and systems that provide water services to customers, yet water is typically the least expensive household utility bill. Water and wastewater service are usually the lowest percentage utility cost per household, at an average of 12 percent, compared to gas/oil at 18 percent, telephone at 33 percent and electricity at 37 percent, not to mention what we pay for cell phone and Internet service! At the cost of about a penny per gallon, water is a tremendous value, especially considering that the average price of a gallon of milk or gas today is creeping toward $4.
While it may not be common knowledge, leaking pipes are a major cause of water loss today in the U.S. In fact, the water industry collectively loses about 7 billion gallons of drinking water each day, enough to fill 10,000 Olympic-sized swimming pools, as water moves through the vast 700,000 miles of pipes used to deliver it to the nation's water customers. This is why investing in the maintenance and upgrading of our nation's pipes and other water infrastructure is critical. This investment also includes leak detection technology which has the potential to save up to $4.6 billion a year. Investing now will keep the water flowing in the future.
Frank Sinatra once sang, "Who knows where the road will lead us?" While none of us knows for sure, we should all be prepared. Whether you're saving up to buy a home or to pay for your children's education, budgeting for these demands is essential to your financial stability. The same holds true for the water industry. Water is a precious resource that must be treated and delivered with quality and efficiency.
Authored by:
Mark LeChevallier
Dr. Mark LeChevallier is the Director of Innovation & Environmental Stewardship for American Water in Voorhees, NJ. He received his Bachelor of Science and Masters degrees in Microbiology from Oregon State University in 1978 and 1980 and his Ph.D. in Microbiology in 1985 from Montana State University. Dr. LeChevallier is also known around the industry for his talent for making complex ...
Other Posts by Mark LeChevallier
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McKennon Farell says:
It's going to take a lot more effort to repair the damage that humans have done to the water table and all the natural water that is left on our planet. Honestly, even with the advances in plumbing and sanitisation, every time a company unethically dumps their waste into a stream or somewhere else without taking note of proper disposal methods, we're thrown back into the stone ages for all the advancement we've come up with..


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More Videos...
OLDSMOBILE - The 1st Generation Rocket V8s Part 1 of 3
THE FIRST GENERATION ROCKET V8 BLOCK was produced from 1949-64. It started as a 303 in 1949, then grew to a 324, then a 371--(which also came in a special J2 Rocket version that came out in 1957), and finished off with it's largest displacement---a 394 that came out in 1959 (the 394 was produced until 1964). But after the 394 in 1959, the lastly developed engine of this "first generation era" was a special smaller lighter 215v8 with a turbo charger produced in the early 60s. The first intermediate sized musclecar came in 1964 "The Pontiac GTO". But prior to the Intermediate sized musclecars, the early 50s OLDSMOBILE ROCKETS can strongly be argued as being THE FIRST MUSCLE CAR (especially the 88's because they were a few hundred pounds lighter than a 98). My dad was blessed with getting a 303v8 1950 Olds 4dr 88 handed down to him in 1955 when my Grandpa got a new 55 Olds.
1957 Oldsmobile Super 88 Test drive
Here is an awesome 1957 Oldsmobile Deluxe Super 88 Holiday Coupe (style code 3637SD) 2 door hardtop. 2 local owners- Last owner who worked at a local Olds dealer and maintained vehicle with NOS parts. Original 371ci 4 barrel 277HP V8 rebuilt and runs perfect. Factory Power steering and Power brakes. 1 repaint in the mid 1980's, Beautiful body, chrome, stainless, glass and moldings. West coast No salt body, trunk and underbody - No rust! Look and underbody pictures original GM red oxide primer. Extensive books and records, title and registrations since 1959. WA 1963 license plates included. Check out the outstanding original interior, Original Seats, door panels, headliner, trim etc. No pitting on chrome bezels and trim. Doors shut like new. Everything works - Radio, horn, blinkers, gauges even back up lights. All 5 matching original wheels, Jack and lug wrench. Original cloth trunk liner. Data plate - Style - 3637SD, Body BK 223, Trim 373, Paint 40. Stock Exhaust thru rear bumper in show quality chrome - See close up pictures. This is a car that our pictures don't do justice! Any questions? Call Mark at 425-876-5553
1957 Olds Deluxe 88 Custom
1957 Oldsmobile Deluxe 88, 3,958 odometer mileage, VIN# 577C14171, 371 Olds Rocket with 4-Barrel Carb Engine, Automatic Trans, Dual Exhaust Chrome Tips, Fender Skirts, Front End Custom Painted Flames, RH & LH Mirrors, Chrome Air Cleaner, Deluxe AM Radio, Custom Interior, Heater-Clock, Under hood area is clean and kept in good running condition, Body is very good, paint shows well, the frame & trunk appear clean and solid. Interior is in excellent condition that has been professionally redone custom with high-grade vinyl trim. Chrome and weather-strips look good, Wide whitewall tires on original rims with deluxe wheel covers, very little use since completed, Overall Clean Condition. VEHICLE WILL BE AVAILABLE FOR VIEWING IN AUGUST. Titled Vehicle Location: Repocast.com, 601 Gordon Industrial Ct., Byron Center, MI 49315 (OFF-SITE)
OLDSMOBILE - The 442 & Hurst Olds Story Part 1 of 3


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Hey hoping some of the gurus here can give some advice.
I have being diagnosed with borderline hypothyroidism tsh ranging between 4-7 and t3 ranging from below the range to just in it 58nmol/l upwards.
I have being told that treatment is "not necessary" until TSH hits 10. In my opinion the range is too broad as it is besides extending it.
Anyway for a person who wants to gain muscle and obviously not fat am i at a disadvantage. My metabolism is already quite high.
I know T3 causes increased nutrient uptake by the muscles but also increases SHBG and energy utilisation.
What do you guys think.
Should I do anything.
Thanks a lot


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PHP changes and LESSONS on my project
This project was awarded to onedeepen for $10 USD / hour.Get free quotes for a project like this
Project Budget$8 - $15 USD / hour
Total Bids13
Project Description
I want to do further development for my PHP project.
i know PHP and MYSQL but not enough to take it for the next level.
the project is built in PHP in OOP programing from scratch.
* Seeking a patient person with strong skills in PHP to teach me to do changes to the website.
the work will be on my computer (localhost), using Teamviewer and skype for the auadio chat.
at the beggining i need 2 hours for working together.
i belive that i need 2 hours on daily basic to work together.
i will pay 12$ per hour.
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Just when Teresa Giudice thinks she's free of her enemy, Danielle Staub (who has reportedly been fired), sources say she'll have a new rival on Season 3 of "The Real Housewives of New Jersey": Melissa Gorga, her sister-in-law.
"I believe Melissa is coming on the show because they're at her house filming," a Giudice family friend tells Radaroline.com. "They've been filming for weeks… at first [Gorga] wasn't sure she wanted to go on the show, but she decided to accept it."
According to the site's source, Bravo has added Gorga to the Jersey cast and Teresa is not happy about it. The source says that Teresa is holding out on signing on for next season, because of it.
"Teresa and Melissa don't get along…They don't speak," the source says. "She doesn't want to be on the show with her…"
Gorga is married to Teresa's brother and it sounds like there's some competition going on. He owns a real estate company and met Gorga when she was his secretary. Hm, could Teresa think her sister-in-law is a gold digger?
"Their house is actually twice the size as Joe and Teresa's," says the family friend. "It's enormous! She has two live-in nannies and lives in a $4 million mansion on an exclusive lot in Montville. Teresa and Joe's house looks like a garage compared to theirs."
Bravo has not confirmed that Gorga has joined the cast.
Gorga or not, we're not sure Teresa can turn down Bravo's paycheck with her finances the way they are, right?
Follow Zap2it and TheRealJethro on Twitter and Zap2it on Facebook for the latest celebrity news and buzz.


text: LG MFJM53 8GB Music PlayerJason Chen5/22/06 5:45pmFiled to: Portable MediaLgMfjm53Mp3Mpeg4Ogg0EditPromoteShare to KinjaToggle Conversation toolsGo to permalink This image was lost some time after publication, but you can still view it here. LG shows that black is the new black with the MFJM53 portable music player. Shipping with an 8GB hard drive—like the iPod mini that Apple forgot—the MFJM53 has a slick finish but substandard features. Advertisement MP3, WAV, WMA and OGG support for audio, and MPEG4 support for video makes this passable. It does get 30 hours of music playback and 4 hours of video playback, which is more than Apple's newest video iPods.No pricing information yet. Advertisement LG quietly launches 8GB music player [Reg Hardware]ReplyLeave a reply


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Interior Pvc Foam Door With Glass - Chinaprice: contact company for price
scientific formula, producing unique pvc low foam door with such
advantages as no formaldehyde, moisture and waterproof, fireproof, heat
and sound insulation, easy fixing and unique design.
2. Standard size: Width700/800/900mm, height unlimited (door panel)
3. Door panel thickness: 40mm
4. Wall thickness: ≤ 400mm
5. Inner structure: Central hollow grid
6. Colour: Black walnut, red cherry, light cherry, white wood, cedar, maple, oak.
7. Accessories: High quality of hinges and locks
Company Contact:
- Posted By: Canyo Group
- Phone: 862022382752538007
- Address: No.2308,#West Tower, Huapu Plaza, Huaming Road,Tia, Canton, China
- Email:
- Website: http://www.canyogroup.com
Published date: December 24, 2012 -
- Business Description: Canyo is specialized in pvc hollow foam door product manufacturing
located in Dalian, China, including pvc door panel, door frame, pvc
hollow foam panel, pvc windowsill board, pvc skirting, pvc decorative
board and so on.
We use European advanced extruding and hot stamping technique with scientific formula, producing unique pvc low foam door with such advantages as no formaldehyde, moisture and waterproof, fireproof, heat and sound insulation, easy fixing and unique design.
Here are most competitive advantages of CANYO products:
*Non toxic & no formaldehyde release
*Waterproof&Anti corrosion
*Fireproof(Automatically extinguish itself and meet the standard of GB/B1 class)
*Heat&Sound Insulation for its central hollow inside and close bubble hole
*Easy to assemble and dismount, door panel and frame can be cut,sawed, planed,adhesive and drilled in a nail directly like natural wood( Grip:1110N)
*Available for various sizes&different color styles
*Life span at least 30 years,etc.
Any comments/enquiry, feel free to contact us!
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Eating at the Tuscan Cooking Class
Eugene V, June 2013
After we cooked, we were able to eat what we cooked. These are some of the people we met and cooked with including one couple who we met on the Chianti Wine tour.
Chicken & Mushroom Rolls
Amy T, September 2010
Dinner Time!
Amy T, September 2010
Florence Cooking Class Italy
Pam V, USA, August 2010
Tuscan cooking class: Preparations
Anonymous, USA, November 2009
another cellar shot
Steve N, November 2015
truly magnificent place to eat a meal you just cooked!
wider shot of cellar
Steve N, November 2015
My wife is there as well at our table as well!