Production, Regulation and Transportation of Bacillibactin in Bacillus Subtilis.
Summary: Bacillus subtilis produces a catecholate type siderophore "Bacillibactin." This review focuses on the non-ribosomal synthesis, transport and regulation of bacillibactin. Bacillibactin biosynthetic operon contains five genes (dhbACEBF). The uptake of bacillibactin requires the FeuABC transporter, inner- membrane permease, FepDG and YusV ATPase and an esterase encoding gene, besA and while export required YmfE major facilitator super-family (MFS)-type transporter. Fur is the major iron-controlled transcriptional regulator in B. subtilis, which acts as an iron-dependent repressor of the dhb operon in vivo while an iron-independent repressor in vitro. Knowledge of the Fur regulon will be useful in interpreting other global analysis of transcriptional responses.
Key words: Bacillibactin, Bacillus subtilis, regulation, production.
Bacillus subtilis provides a model system for the exploration of metal ion homeostasis in Gram-positive bacteria . Itoic acid, the first example of a catecholate siderophore, was isolated from B. subtilis in 1958  and bacillibactin (BB) was characterized in 2000 . A monomeric unit of BB (2,3-dihydroxybenzoyl glycylthreonine) was isolated from B. licheniformis . An active transport process in B. subtilis was demonstrated in the early 1970s [5, 6, 7] and the subsequent link between environmental iron concentrations and production of phenolic acids was demonstrated [5, 8]. The only siderophore biosynthetic pathway found to date of Bacillus subtilis is that of BB, which has very high homology to the enterobactin biosynthetic pathway . The Itoic acid was first bacterial siderophore that was structurally characterized. It was isolated from low iron liquid cultures of Bacillus subtilis . Itoic acid (DHBG) is the glycine conjugate of 2,3- dihydroxybenzoic acid (DHBA) [5, 9].
In addition, DHBA and DHBG [collectively referred to as DHB (G)] are precursors of the catecholate siderophore bacillibactin which is the cyclic trimeric lactone of 2,3-dihydroxybenzoyl-Gly-Thr (Fig. 1) [3, 10]. A siderophore of this apparent structure "corynebactin" was produced by Corynebacterium glutamicum earlier , but later genome sequence and biochemical analysis recommended that this organism is unlikely to produce this compound . The bacillibactin was the preferred siderophores name for the B. subtilis (Fig. 1) [3, 13] but several papers referred this siderophore as corynebactin [10, 14]. However, recently B. anthracis secreted two siderophores, petrobactin (PB) and BB. The siderophore PB was secreted first while BB was secreted several hours later. Spores development early in an infection may need PB, while delayed BB production suggested a role for BB in the later stages of infection .
The BB and many of the siderophores are produced non-ribosomally by large, multi-domain enzymes termed as non-ribosomal peptide synthetases (NRPS) that can assemble peptides of wide structural diversity and broad biological activity . Regulation of iron uptake is also necessary to prevent oxidative damage that can be exacerbated by excessive iron in the cell  and most of the bacteria depend on the DNA-binding protein Fur (ferric uptake repressor) to control and regulate expression of genes involved in iron uptake . Bacillus subtilis contains three Fur homologues: a ferric uptake repressor (Fur), a zinc uptake repressor (Zur), and a peroxide regulon repressor (PerR) . Our aim of this review was to give an updated summary about BB, a catecholate type siderophore produced by B. subtilis. This review focuses on the BB non-ribosomal synthesis, regulation by ferric uptake repressor (Fur) and transport via permeases, ATPases, transporters and siderophores binding proteins.
Non-Ribosomal Synthesis of Bacillibactin
The completion of B. subtilis genome sequencing enables a search for siderophore-related genes . Rowland and Taber (21) described dhb operon responsible for 2,3-dihydroxybenzoate biosynthetic enzymes. The dhb gene cluster sequences was corrected and five genes were identified involved in the biosynthetic pathway for BB (dhbACEBF) in B. subtilis . Of the five dhb genes, DhbC is an isochorismate synthetase (ICS) and catalyzes the conversion of chorismate to isochorismate, which is an important siderophore or menaquinone precursor. B. subtilis contains two ISC isoenzymes .
The BB is structurally related to enterobactin isolated from the Gram-negative bacterium E. coli. Both siderophores contain three 2,3-dihydroxybenzoate moieties for octahedral iron complexation which are coupled to a cyclic amino acid core synthesized by multimodular nonribosomal peptide synthetases. The three genes entE, entB and entF encoding the EntEBF synthetase complex for enterobactin assembly in E. coli were initially used to identify putative homologues named dhbE, dhbB and dhbF in B. subtilis . The erroneous dhbF sequence was revised and characterized as dimodular NRPS in contrast to the monomodular EntF synthetases . Transcription of the dhb operon was found to be controlled by a single sA-dependent promoter that was comprised of a fur box-binding site, an iron regulatory element while the three dhb genes encoding the synthetases for the assembly of BB were resided in one operon . The first step in nonribosomal peptide synthesis is the adenylation of the cognate substrate (amino) acid.
Determination of the substrate selectivity of the catalyzing, A domain was accomplished either by the ATP-PPi exchange assay or by analysis of the selectivity- conferring residues as guided by the nonribosomal code of A domains from NRPS . In the case of DhbEF, studies independently led to the determination of DHB, Gly and Thr selectivity for DhbE, DhbF1 and DhbF2, respectively . Intriguingly here one notable thing was regarding activation of 2,3-dihydroxybenzoate (DHB) and salicylate by DhbE, the selectivity code cannot distinguish between both aryl substrates, although the various adenylating enzymes (EntE and VibE) reveal a clear preference for one or the other substrate. Likewise, DhbE was selective for DHB, activating salicylate with a 6-fold lower catalytic efficiency. The same kind of analysis revealed Gly and Thr selectivity for the first and second modules of DhbF, respectively. A low but significant activation of the predicted glycine substrate by DhbF1-A-PCP was reported .
However, probably due to the intrinsic betaine character, this activation was observed only at slightly higher pH values. In contrast, the ATP-PPi exchange assay with DhbF2-A-PCP revealed a distinct selectivity for the predicted substrate, L-Thr. These moderately differ from the activation pattern as determined for the homologous VibF  and both enzymes showed a clear stereo specificity for the activation of an L-configured amino acid. The second step in non-ribosomal peptide biosynthesis is the transfer of the activated aminoacyl moiety from the adenylate to the thiol group of the adjacent carrier protein domain.
This so-called thiolation reaction (DHB-S-Ppant-DhbB) represents the initial step in BB biosynthesis. Here, DhbE activates the substrate carboxyl acid DHB as DHB-O- AMP; and subsequently, the aryl moiety is transferred to holo-DhbB, yielding the DHB-S-Ppant thioester. The reaction necessarily relies on the presence of a functional HS-Ppant holo-carrier protein, which is usually generated by the action of a dedicated Ppant-transferase. Sfp, the Ppant-transferase , is capable of converting the aryl carrier protein (ArCP) domain of DhbB in vitro from the inactive apo form to the HS-Ppant holo form. The presumption that Sfp may represent the Ppant-transferase of the BB system is corroborated by the fact that its in vivo biosynthesis in B. subtilis inevitably depends on the presence of a functional Sfp protein . Notably, the genome project of B. subtilis strain 168 carries the complete NRPS gene clusters for the biosynthesis of surfactin and BB, but has a defect in the sfp gene.
Consequently, all carrier protein domains remain in their inactive apo form and the strain produces neither of the two NRPS products, although DhbB could be characterized biochemically. The two carrier protein domains of DhbF were determined by sequence homology and it is worthwhile to notice that carrier proteins can be distinguished not only by the moiety they have to carry (acyl, acyl carrier protein; aryl, ArCP; and peptidyl, PCP), but also by the signature of their highly conserved Ppant-binding site. Until now, the following signature sequences could be observed: acyl carrier proteins, (ED)LGXDSL(DAT); ArCPs, (DN)LXXXGLDSXR ; PCPs preceding a C or Te domain, DXFFXXLGGHSLK; PCPs preceding an epimerization domain, DXFFXXLGGDSIK; and C- terminal PCPs, FF(ED)XGGNSLK. According to this classification, the carrier protein of DhbB is clearly an ArCP, whereas the two carrier proteins of DhbF belong to the first class of PCP domains.
The last steps in nonribosomal peptide biosynthesis consist of substrate condensation, followed by product release catalyzed by the Te domain. The peptide chain is then transferred from the last PCP domain onto a Ser residue within the highly conserved core motif TE (Thiosterase Epimerization) (G(HY)SXG)  (Fig. 2). Two ways of Te mediated product release have been described so far: ester hydrolysis by water or nucleophilic attack by amino or hydroxyl groups of the peptide itself, leading to a cyclic lactame or lactone . In the case of enterobactin biosynthesis, mutational analysis of EntF-Te led to reduced product release and enabled  to identify intermediate reaction products such as linear (DHB-Ser)2 and enzyme-bound species such as (DHB-Ser)-S-PCP, (DHB-Ser)1-O-Te, and (DHB-Ser)2-O-Te by ESI-MS.
These data provide evidence that EntF-Te additionally catalyzes the trimerization of the DHB-Ser units prior to cyclization. Owing to rapid loss of DhbF activity, these last steps for BB biosynthesis in vitro are not proved yet.
A clue in the determination of the catalytic role of DhbF- Te was the product analysis by ESI-MS. The ion at m/z 881.2 [M-H]-1 and daughter ions clearly defined BB as (DHB-Gly-Thr)3 trilactone. The dhb genes and homologues support excellently the idea of horizontal gene transfer between Gram-negative and -positive bacteria and genetic rearrangement of NRPS leading to a variety of structurally related siderophores in different organisms. Although the sequence of the DhbEBF modules is colinear to the sequence of the product even then some domains need further attention. The two carrier protein domains of DhbF were determined by sequence homology. The significance of having different types of carrier protein domains has yet to be established and they might have emerged to mediate specific protein-protein interaction with a certain Ppant-transferase or to facilitate the correct interplay with various upstream and downstream partner domains.
In our opinion, the latter represents the more likely variant since, in the first scenario; one had to suppose the existence of type- specific Ppant-transferases. However, it has been shown that EntD, the Ppant transferase associated with enterobactin biosynthesis, modifies both the ArCP domain of EntB  and the PCP domain of EntF . Likewise, sfp has been noticed to be rather nonselective, modifying all kinds of carrier protein domains with high catalytic efficiency . There is defect in the sfp gene that required revision of sequences. Last steps for BB biosynthesis in vitro are not proved yet. Therefore, scientists should concentrate on these issues to solve them.
Bacillibactin Mediated Transport in Bacillus subtilis
The production of DHB(G) was repressed by iron and related catechol compounds and transport was both temperature and energy dependent [5, 7]. The early studies investigated phenolate mediated iron transport; some experimental issues obscure the significance of the results. First, citrate was present in the culture medium as a buffer . As a-hydroxycarboxylate, citric acid can act as an iron chelator; in fact, YfmCDEF is the ferric citrate receptor in B. subtilis . Second, the ferric complexes were made in situ, which could result in the complicated kinetics since the formation of the ferric complexes would equilibrate over the course of the uptake experiment. However, these early studies are still useful in their descriptions of the generalities of iron transport, such as the energy and temperature dependence of the transport, but are not useful in the determination of the specificity of the permeases.
These physiology studies proposed the presence of either a very broad selectivity transport system or the presence of multiple uptake systems.
Quentin et al. (30) inventoried the ABC transport systems of B. subtilis, while Baichoo et al. (31) used microarrays to determine the operons derepressed by both Fur mutations and iron starvation, suggesting the presence of various iron uptake pathways. Ollinger et al.  produced mutants to correlate Fur regulated genes with a particular siderophore. Ferrichrome and ferriox- amine each have a separate substrate binding protein (FhuD and YxeB, respectively), but require the same membrane permease using an ATPase (FhuBGC) [29, 32]. YfiYZ/YfhA/YusV is critical for growth with schizokinen and arthrobactin as iron sources. Citrate is the substrate for another transporter (YfmCDEF) and growth stimulated by elemental iron requires YwbLMN permeases . In addition to siderophore uptake systems, B. subtilis encodes a homologue (YwbL) of an iron permease described in Saccharomyces cerevisiae .
This system may be functionally analogous to the ferrous iron (feo) uptake system of E. coli. B. subtilis requires FeuABC/YusV for growth when provided with ferric BB and enterobactin .
That B. subtilis transports enterobactin through the same permease as BB, is evident from previous studies where an excess of nonradiolabeled ferric enterobactin can block the incorporation of radioactive ferric BB. The presence of an additional catecholate receptor, however, is inferred from the inability of nonradioactive ferric BB to block completely the incorporation of radioactive ferric enterobactin even with increasing concentrations of ferric BB . The different chiralities of enterobactin and BB are not the root cause of the permease discrimination. The larger size of BB, rather than chirality, is the discriminating factor at the second receptor. This result is reminiscent of the Salmonella typhimurium siderophore-mediated pathway, where BB requires the IroN receptor and cannot be incorporated by FepA . Molecular models of BB suggest that a more oblate ferric complex is formed, which may block receptor recognition .
The final entrance of BB appears to be mediated by the inner-membrane permease, FepDG . Ollinger et al.  reported the requirement for two lipoproteins for the hydroxamates ferrichrome and ferrioxamine B in B. subtilis although only one receptor was indicated to be crucial for growth on ferric BB. FeTrentam and Ferrioxamine B (FOB) were incorporated into B. subtilis and did not appear to be transported through the same permease as BB. The addition of nonradioactive FOB does not affect the incorporation of radioactive ferric BB while addition of nonradioactive FeTrentam does seem to affect the transport of 55FeBB. The reduction of intake of 55FeBB, however, is not immediate, suggesting that the Trentam could be recognized by the BB permease, but with a much lower affinity. Growth-promotion experiments are required to determine if B. subtilis can utilize the iron provided by Trentam. Dertz et al.  reported that B. subtilis could incorporate a variety of ferric complexes via several parallel pathways.
Replacement of glycine with other neutral amino acids does not affect the recognition, indicating that the primary recognition point is the triscatecholate ferric center. Replacement of the neutral side chain with charged moiety (glutamic acid and lysine) drastically diminishes the incorporation. All the Tren- based amino acid analogs can block ferric BB incorporation, despite the differences in size and charge of the resulting ferric complexes. This indicates that alteration of size or charge can prevent the incorporation of the ferric complex into the cell, but not the binding of the ferric complex to the permease. The B. subtilis catecholate permease tolerates some slight catecholate ring modification but at a substantially reduced rate, it is in contrast to FepA of E. coli, where a completely un-substituted ring is required for recognition . The size does not matter as much as the charge and shape of the ferric complex.
Bacillus subtilis uses two partially overlapping permeases (1 and 2) to acquire iron from its endogenous siderophores (BB and itoic acid). The results regarding solution thermodynamic stability of ferric BB revealed that the addition of a glycine to the catechol chelating arms causes a destabilization of the ferric complex of BB . Most likely, DHB(G) takes part in the solubilization of trace iron in the medium which may then be transported into the cell using the YwbLMN permease. The role of the YwbLMN system in the uptake of iron from ferric-DHB(G) is supported by the recent transport studies demonstrating that ferric-BB competes poorly with radiolabeled Fe-DHBG for uptake 
B. subtilis was unable to grow in the medium containing the iron chelator ethylenediamine-N,N'-bis, 2- hydroxyphenylacetic acid (EDDHA) unless the ability to synthesize the BB is restored or exogenous siderophores are provided. The sfp+ derivatives of B. subtilis did not require exogenous siderophore for growth even in the presence of up to 50 uM EDDHA, this finding was consistent with their ability to synthesize BB. Mutants faulty either for BB synthesis (dhbA) or for uptake (feuA or yusV) grew poorly under these conditions. Among several Fur-regulated ABC transport mutants, only feuABC- exhibited impaired growth during iron starvation. Quantification of intra- and extracellular (ferri)-BB in iron-depleted feuABC- cultures revealed a fourfold increase of the extracellular siderophore concentration, confirming a blocked ferri-BB uptake in the absence of FeuABC.
These results showed that the BB uptake requires the FeuABC transporter and YusV ATPase regardless of whether the siderophore is provided exogenously or produced by the cells . Ferri-BB was found to bind selectively to the periplasmic binding protein FeuA, proving high-affinity transport of the iron- charged siderophore . The BB binds Fe(III) with extremely high affinity .
Bacillus subtilis also encodes another predicted BB esterase, YuiI. The YuiL (BesA) hydrolyzes BB as well as the corresponding iron complexes. The BB binds iron through three 2,3-catecholamide moieties linked to a trithreonine scaffold through glycine spacers, however, the YuiL is less specific than the enterobactin esterase Fes; YuiL can hydrolyze the trilactones of both siderophores, while only the tri-l-serine trilactone is a substrate of Fes . The YuiL catalyzes the hydrolysis of the trilactone cycle of ferri-BB complex, which leads to the formation of BB monomers and to the cytosolic iron release hence making iron accessible for metabolic use . The YuiI gene is immediately upstream of the BB (dhbACEBF) biosynthetic operon and most closely related to IroE that linearizes apo-enterobactin during export . A mutation in YuiI had only a small effect on the ability of cells to utilize BB for iron uptake .
During iron starvation, a YuiI mutant displayed impaired growth and strong intracellular (30-fold) and extracellular (6.5-fold) (ferri)-BB accumulation. Kinetic studies in vitro revealed that YuiI hydrolyses both BB and ferri-BB. BB hydrolysis led to strong accumulation of the tri- and dimeric reaction intermediates, while ferri-BB hydrolysis yielded exclusively the monomeric reaction product and occurred with a 25-fold higher catalytic efficiency than BB single hydrolysis. Thus, ferri-BB was the preferred substrate of the YuiI esterase whose gene locus was designated besA . The screening of a B. subtilis exporter mutant library led to the identification of the YmfE major facilitator superfamily (MFS)-type transporter as a target for BB export. Analysis of iron-limited ymfE mutant cultures displayed an eightfold reduced BB secretion and, on the other hand, a 25-fold increased secretion of the BB precursor 2,3-dihydroxybenzoate .
Above discussion revealed that the uptake of BB requires the FeuABC transporter, inner-membrane permease, FepDG and YusV ATPase and an esterase encoding gene, besA while export required YmfE major facilitator superfamily (MFS)-type transporter. Further studies are required to describe the roles of YuiI and YbbA in BB processing during export or subsequent import. It is suggested that YbbA, which is downstream of feuC, is part of the feuABC operon as these genes are similarly regulated and there is no Fur box immediately upstream of YbbA. The results suggest a revision to the assigned start codons for both the yuiI and fhuB genes.
Regulation of Siderophore Production by Ferric uptake Repressor (Fur)
Fur is the major iron-controlled transcriptional regulator in B. subtilis and directly controls the transcription of at least 39 genes arranged in 20 operons, implicated in either siderophore synthesis or uptake or other functions likely to be associated with iron transport. Owing to the presence of Fur box sequences in regulatory regions, many of these operons were previously predicted to be members of the Fur regulon. The presence of Fur box-like elements in genome sequence of B. subtilis preceding a metallo-regulated gene (mrgC) and the fhuD operon originally led to the suggestion that a Fur homologue might regulate iron uptake functions in B. subtilis . Later on, this idea was strongly supported when a perfect match to the Fur box consensus was found in the regulatory region of the B. subtilis BB (dhb) siderophore biosynthesis operon .
In the B. subtilis genome, 17 sites met this criterion and 13 of them were associated with Fur-repressed genes. Generally, It is known that in vivo Fur binds to Fe(II) and is thus activated to bind DNA, leading to the suppression of iron uptake functions so Fur in vivo, performs as an iron- dependent repressor of the dhb operon [19, 41]. However, in vitro Fe(II) failed to significantly affect either the ability of Fur to suppress transcription or the affinity of Fur for the dhb operator . Therefore, Fur is an iron- independent repressor of dhb transcription. It is suggested that in vivo there is an inhibitory factor, perhaps a metal ion or a low molecular weight ligand, absent from in vitro system. Interestingly, Fur efficiently represses transcr- iption even if RNA polymerase holoenzyme (RNAP) is incubated with the dhb promoter region before the addition of Fur. This finding suggests that either Fur can displace RNAP or that Fur can bind to the promoter region downstream of prebound RNAP.
Further studies are needed to make a distinction between these possibilities.
The Fur regulon includes, at least four different transmembrane and ATPase components containing ABC transporters, together with five substrate-binding lipoproteins . The fur mutation displayed elevated levels of the Dhb proteins involved in the synthesis of BB [14, 43], YwbM and four predicted siderophore-binding proteins (FeuA, YfiY, YxeB, and YclQ) . The Dhb biosynthetic enzymes was derepressed under salt  and oxidative stress conditions  and many of the substrate-binding proteins have been detected in the analysis of membrane-associated  or secreted proteins . Extracellular proteome analysis also revealed derepression of the Fur-regulated YoaJ protein, a predicted endoglucanase. In addition, the fur mutation displayed an increased synthesis of PBSX defective prophage encoded proteins. It is not clear whether this is a direct or an indirect effect of the fur deletion. In several cases, groups of Fur-regulated transcription units are clustered.
The YuiI gene, just upstream of the dhb operon, is a member of the Fur regulon. The fhuBGC-fhuD and the yfiY-yfiZyfhA divergons are also Fur regulated. It was suggested that ybbA, which is downstream of feuC, is part of the feuABC operon as these genes are similarly regulated and there is no Fur box immediately upstream of ybbA . The results suggest a revision to the assigned start codons for both the yuiI and fhuB genes. In addition to the Fur regulon, many other genes are affected by mutation of fur, depletion of iron, or both. In most cases, the corresponding regulatory pathways are not well defined. It is possible that Fur regulates other genes in B. subtilis, either as an activator or as an iron-sensitive repressor. A fur mutation slows down the normal growth of the strain. Together, these facts likely account for many of the other changes in gene expression noted in the fur mutant.
Apart from the artifactual expression of the spoIIM gene, the most dramatically induced genes in the fur mutant are the lic operon encoding enzymes for lichenan utilization . The significance of this finding is not yet clear. Conversely, the most dramatically repressed genes in the fur mutant encode ribosomal proteins, presumably a result of growth rate control. On the other hand, an investigation of the regulatory features revealed that BB secretion is independent of the Fur that is in contrast to the other components of the pathway. In fact, the MerR-type transcriptional regulator Mta was found to activate BB secretion and ymfE gene expression, revealing Mta as an additional regulatory member of the BB pathway , however, more research is required to confirm the role of Mta in the regulation of BB and there is certain need to differentiate the role of Fur and Mta in BB production and transport across bacterial cell membrane.
This review gives an updated summary about synthesis, regulation and transport of BB, a catecholate type siderophore produced by Bacillus subtilis. The dhb genes and homologues support excellently the idea of horizontal gene transfer between Gram-negative and Gram-positive bacteria and genetic rearrangement of NRPS leading to a variety of structurally related siderophores in different organisms. We interpreted multiple siderophores uptake systems of Bacillus subtilis that varies with the type of iron or siderophores that are being transported and these systems may include permeases, binding proteins, transporters, and ATPases. Additional studies will be required to determine the specificity of the putative ferri-siderophore uptake systems in the Fur regulon. The uptake of BB requires the FeuABC transporter, inner-membrane permease, FepDG and YusV ATPase and an esterase encoding gene, YuiI that hydrolyses both BB and ferri-BB.
The iron regulation in B. subtilis is controlled by a Fur homolog that binds directly to a Fur box, as inferred from previous genetic analysis. However, the protein binds tightly to DNA in the absence of added iron so the scientists are not yet able to demonstrate iron responsive DNA binding in vitro. Fur functions primarily as an iron-activated repressor; there is no evidence to support a role for Fur as an activator of transcription in B. subtilis, nor do evidence for genes that are repressed by Fur and activated by iron, as has been documented in Helicobacter pylori [49-52]. In addition, another BB pathway regulator Mta has been reported but more research is required to explore its role in BB regulatory pathway especially in BB transport across cell membrane. The interaction of Fur with metal ions is not well understood and further studies should be conducted to evaluate the relationship of Fur regulation of siderophore biosynthesis and transport with different metal ions other than iron.
In addition, scientists can concentrate on the contributions of Fur-regulated operons to cell physiology that have not been systematically assessed. Knowledge of the Fur regulon will be useful in interpreting other global analysis of transcriptional responses.
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College of Resource and Environmental Sciences, Nanjing Agriculture University, No.1, Weigang Road, Nanjing, 210095, Jiangsu Province, P. R. China., firstname.lastname@example.org
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|Author:||Raza, Waseem; Hussain, Qaisar; Qirong Shen|
|Publication:||Journal of the Chemical Society of Pakistan|
|Date:||Aug 31, 2012|
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