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...dels used are shown in Table 2. Refinement of the coordinates, TLS parameters and atomic temperature factors (anisotropic in the case of CD1908 and CD1925(17–152)) was carried out using Phenix.refine =-=[60]-=-. Model building was performed using Coot [61]. The secondary structure and stereochemistry of the models was analysed by MolProbity [62]. Sequence alignment was performed using ClustalW [63] and the ...

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...and scaled and merged with Scala [58]. Data collection and refinement statistics are shown in Table 2. Structure solution and analysis All structures were solved by molecular replacement using Phaser =-=[59]-=-, molecular replacement models used are shown in Table 2. Refinement of the coordinates, TLS parameters and atomic temperature factors (anisotropic in the case of CD1908 and CD1925(17–152)) was carrie...

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...0.t003 Structures of C. difficile eut BMC Proteins PLOS ONE | www.plosone.org 12 October 2012 | Volume 7 | Issue 10 | e48360 integrated using iMosflm [56] or XDS [57] and scaled and merged with Scala =-=[58]-=-. Data collection and refinement statistics are shown in Table 2. Structure solution and analysis All structures were solved by molecular replacement using Phaser [59], molecular replacement models us...

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...difficile 630 sequence (Genbank ID: NC_009089) by DNA sequencing (GATC Biotech). All constructs were expressed in E. coli B834 (DE3) cells. A single colony was transferred to 200 ml of Zyp-5052 media =-=[55]-=- supplemented with 50 mg/ml kanamycin and grown at 310 K with shaking for 36 hours. Cells were harvested by centrifugation (4,000 g, 30 min) and washed with buffer A (50 mM Tris.HCl pH 8.0, 200 mM NaC...

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...the coordinates, TLS parameters and atomic temperature factors (anisotropic in the case of CD1908 and CD1925(17–152)) was carried out using Phenix.refine [60]. Model building was performed using Coot =-=[61]-=-. The secondary structure and stereochemistry of the models was analysed by MolProbity [62]. Sequence alignment was performed using ClustalW [63] and the corresponding figures were generated using ESP...

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...acteria, each adopting different strategies to survive within the niches they inhabit [1]. Pathogenic species are usually out-competed by the commensal species that make up the healthy gut microbiota =-=[2,3]-=-, but they often make use of toxins directed towards other bacterial species [4], or the host [5] to enable them to colonise environments that would otherwise be occupied by competitors, or to create ...

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...ment was performed using ClustalW [63] and the corresponding figures were generated using ESPript [64]. Oligomerisation states and values of buried surface areas were calculated using the PISA server =-=[65]-=-. Structural superimpositions were calculated using Coot. Crystallographic figures were generated with PyMOL [66]. Thin-section transmission electron microscopy E. coli B834 cells transformed with the...

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...trostatic surfaces and characteristics of CD1908 and CD1918 hexamers. A. The electrostatic potential of the solvent accessible surface of CD1908 was calculated using the default parameters in PDB2PQR =-=[67]-=- and APBS [68] and mapped to the molecular surface in PyMol and displayed over a cartoon representation of the molecule. Blue indicates regions of positive potential (. +5 kT/e) and red indicates nega...

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...aces and characteristics of CD1908 and CD1918 hexamers. A. The electrostatic potential of the solvent accessible surface of CD1908 was calculated using the default parameters in PDB2PQR [67] and APBS =-=[68]-=- and mapped to the molecular surface in PyMol and displayed over a cartoon representation of the molecule. Blue indicates regions of positive potential (. +5 kT/e) and red indicates negative potential...

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...ich can then be used in various metabolic processes, such as the TCA cycle in those bacteria capable of aerobic respiration, lipid biosynthesis, or for substrate level phosphorylation to generate ATP =-=[22]-=-. The genes associated with the ethanolamine ammonia lyase vary widely between PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e48360 species; some bacteria are only able to utilise ...

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...tion has been extensively studied in Salmonella species [15,21,30–32] However, there remain a number of questions about the roles of some of the enzymes associated with the ethanolamine ammonia lyase =-=[15,18,32,33]-=-. Work on the structure of this BMC is limited to X-ray crystal structures of a number of the shell proteins from the E. coli eut operon [34]. To understand the function of the ethanolamine utilisatio...

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...tion has been extensively studied in Salmonella species [15,21,30–32] However, there remain a number of questions about the roles of some of the enzymes associated with the ethanolamine ammonia lyase =-=[15,18,32,33]-=-. Work on the structure of this BMC is limited to X-ray crystal structures of a number of the shell proteins from the E. coli eut operon [34]. To understand the function of the ethanolamine utilisatio...

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...CD1908 and CD1925(17–152)) was carried out using Phenix.refine [60]. Model building was performed using Coot [61]. The secondary structure and stereochemistry of the models was analysed by MolProbity =-=[62]-=-. Sequence alignment was performed using ClustalW [63] and the corresponding figures were generated using ESPript [64]. Oligomerisation states and values of buried surface areas were calculated using ...

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... secondary structure and stereochemistry of the models was analysed by MolProbity [62]. Sequence alignment was performed using ClustalW [63] and the corresponding figures were generated using ESPript =-=[64]-=-. Oligomerisation states and values of buried surface areas were calculated using the PISA server [65]. Structural superimpositions were calculated using Coot. Crystallographic figures were generated ...

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...egions in italics. doi:10.1371/journal.pone.0048360.t003 Structures of C. difficile eut BMC Proteins PLOS ONE | www.plosone.org 12 October 2012 | Volume 7 | Issue 10 | e48360 integrated using iMosflm =-=[56]-=- or XDS [57] and scaled and merged with Scala [58]. Data collection and refinement statistics are shown in Table 2. Structure solution and analysis All structures were solved by molecular replacement ...

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...0]. The breakdown of ethanolamine is carried out by a two-subunit adenosylcobalamin (AdoCbl) cofactor-dependent ethanolamine ammonia lyase protein complex, which is encoded by the genes eutB and eutC =-=[21]-=-. These genes are usually associated with a number of accessory proteins that activate the AdoCbl cofactor and allow the efficient conversion of the acetaldehyde produced by this enzyme into acetyl-Co...

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...bit [1]. Pathogenic species are usually out-competed by the commensal species that make up the healthy gut microbiota [2,3], but they often make use of toxins directed towards other bacterial species =-=[4]-=-, or the host [5] to enable them to colonise environments that would otherwise be occupied by competitors, or to create new niches through changes to their host organism. Salmonella enterica and Esche...

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...e liberated from the cell membranes of host epithelial cells and other bacteria [12,13]. Phosphatidylethanolamine is broken down readily by bacterial phosphodiesterases into glycerol and ethanolamine =-=[14]-=-, and a number of enteric pathogens, including S. enterica, Enterococcus faecalis and some species of Clostridia can use ethanolamine as a sole source of nitrogen and carbon [15–18]. Indeed, an associ...

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...ic species are usually out-competed by the commensal species that make up the healthy gut microbiota [2,3], but they often make use of toxins directed towards other bacterial species [4], or the host =-=[5]-=- to enable them to colonise environments that would otherwise be occupied by competitors, or to create new niches through changes to their host organism. Salmonella enterica and Escherichia coli speci...

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...sation states and values of buried surface areas were calculated using the PISA server [65]. Structural superimpositions were calculated using Coot. Crystallographic figures were generated with PyMOL =-=[66]-=-. Thin-section transmission electron microscopy E. coli B834 cells transformed with the plasmids for untagged CD1908, CD1918 and CD1925 were grown to mid-log phase in Luria-Bertani media supplemented ...

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...is and some species of Clostridia can use ethanolamine as a sole source of nitrogen and carbon [15–18]. Indeed, an association between ethanolamine metabolism and virulence in S. enterica is emerging =-=[19,20]-=-. The breakdown of ethanolamine is carried out by a two-subunit adenosylcobalamin (AdoCbl) cofactor-dependent ethanolamine ammonia lyase protein complex, which is encoded by the genes eutB and eutC [2...

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... required for the efficient utilisation of the carbon liberated by the breakdown of ethanolamine; six shell proteins, five with BMC protein domains and one with a carboxysome protein CcmL-like domain =-=[50]-=-; and proteins with putative regulatory functions. The requirements for the growth of C. difficile on ethanolamine as a nitrogen or carbon source have not yet been determined, but it is conceivable th...

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...It has been suggested that there may be a degree of conformational flexibility in the b-barrel neck that allows it to act as a gated channel [40], in a similar manner to mechanosensitive ion channels =-=[52]-=-. The extension to the b-barrel seen in CD1908, which is shared with PduU but not EutS, may play a role in the recognition of substrates, or the recruitment of specific protein partners. If the protei...

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...tion has been extensively studied in Salmonella species [15,21,30–32] However, there remain a number of questions about the roles of some of the enzymes associated with the ethanolamine ammonia lyase =-=[15,18,32,33]-=-. Work on the structure of this BMC is limited to X-ray crystal structures of a number of the shell proteins from the E. coli eut operon [34]. To understand the function of the ethanolamine utilisatio...

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...is and some species of Clostridia can use ethanolamine as a sole source of nitrogen and carbon [15–18]. Indeed, an association between ethanolamine metabolism and virulence in S. enterica is emerging =-=[19,20]-=-. The breakdown of ethanolamine is carried out by a two-subunit adenosylcobalamin (AdoCbl) cofactor-dependent ethanolamine ammonia lyase protein complex, which is encoded by the genes eutB and eutC [2...

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...mmation caused by these toxins large quantities of phospholipids, particularly the abundant phosphatidylethanolamine, are liberated from the cell membranes of host epithelial cells and other bacteria =-=[12,13]-=-. Phosphatidylethanolamine is broken down readily by bacterial phosphodiesterases into glycerol and ethanolamine [14], and a number of enteric pathogens, including S. enterica, Enterococcus faecalis a...

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... widespread strategy among enteric pathogens [18] and shares a number of common features with the compartmentalisation of carbon fixation and the utilisation of propanediol by other bacterial species =-=[26]-=-. The shells of the BMCs must all act in the same way, to encapsulate enzymes and to provide a semiFigure 4. Crystal structure of CD1918. A. Cartoon representation of the CD1918 monomer with secondary...

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...arrel (Fig. 6B white oval), this cleft corresponds to the region that coordinates divalent cations and acts as the active site in the metal binding cupins, such as those with sugar isomerase activity =-=[43]-=- (Fig 7A/B). CD1925 shares 30% sequence identity with EutQ from S. typhimurium (PDBID: 2PYT, unpublished structural genomics output) and superimposes with an rms Ca deviation of 1.25 Å over 116 amino...

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...ucts, while preventing the escape of metabolic intermediates. Transmission electron microscopy of thin sections of E. coli cells overexpressing carboxysome proteins from Halothiobacillus neapolitanus =-=[35]-=-, Citrobacter freundii propanediol utilisation BMC proteins [48], Clostridium kluyveri ethanol utilisation BMC [49] and S. enterica ethanolamine utilisation proteins [37], have revealed the presence o...

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...he microcompartment, or is found within the cytosol. The presence of an unstructured N-terminal region is however consistent with the possibility that this protein is localised within the compartment =-=[54]-=-. The function of the protein as either a transporter, a partner in a signalling cascade, or an enzyme remains to be determined. Nevertheless, the amenability of this protein to high-resolution struct...

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... associated accessory proteins; enzymes required for the utilisation of the organic carbon liberated by the lyase; regulatory proteins and six proteins with homology to the carboxysome shell proteins =-=[38]-=- (Fig. 1, Table 1 and Supporting Information S1for detailed description of the operon). To understand the structure and function of the C. difficile eut bacterial microcompartment we have determined t...

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..., or to create new niches through changes to their host organism. Salmonella enterica and Escherichia coli species are common causes of gastroenteritis and diarrheal illness in the healthy population =-=[6,7]-=-, while Clostridium difficile is a major cause of hospital acquired diarrhoea and has significant risks of morbidity and mortality in the elderly and immune compromised patients [8,9]. With an ageing ...

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... 2.3 (2.0) Completeness (%) 99.7 (99.9) 98.3 (97.9) 97.9 (99.4) Mean I/sigma I 12.4 (2.5) 12 (3.5) 11.5 (2.4) Rsym 0.058 (0.495) 0.063 (0.356) 0.058 (0.356) Molecular Replacement Model 3CGI [40] 2EWH =-=[69]-=- 2PYT Refinement Number of reflections 34,282 30,452 14,8298 Rcryst 0.140 0.166 0.136 Rfree 0.180 0.196 0.147 Number of atoms (no-H) 1,930 2,062 2,648 rmsd bond lengths (Å) 0.016 0.015 0.014 bond ang...

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...en or carbon source have not yet been determined, but it is conceivable that the organism will grow on ethanolamine under fermentative conditions similar to those seen for S. enterica, or E. faecalis =-=[15,16]-=-, with the production of a functional BMC. The sequestration of ethanolamine utilisation within a BMC is a widespread strategy among enteric pathogens [18] and shares a number of common features with ...

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...ins that are homologous to carboxysome shell proteins [23–27]. These metabolic compartments allow the efficient utilisation of various carbon sources and are termed bacterial microcompartments (BMCs) =-=[28]-=-. The sequestration of ethanolamine metabolism within a BMC is thought to protect the cell from the acetaldehyde produced as an intermediate in its breakdown and to prevent the loss of this volatile c...

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...acteria, each adopting different strategies to survive within the niches they inhabit [1]. Pathogenic species are usually out-competed by the commensal species that make up the healthy gut microbiota =-=[2,3]-=-, but they often make use of toxins directed towards other bacterial species [4], or the host [5] to enable them to colonise environments that would otherwise be occupied by competitors, or to create ...

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...althy population [6,7], while Clostridium difficile is a major cause of hospital acquired diarrhoea and has significant risks of morbidity and mortality in the elderly and immune compromised patients =-=[8,9]-=-. With an ageing population that is becoming increasingly reliant on hospital care, there is much interest in understanding the molecular basis of the metabolism of C. difficile and its role in intest...

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..., comprising the complete native protein sequence, less the N-terminal methionine, with a single amino acid visible from the C-terminal His6 tag in chain A. CD1908 displays a permuted BMC domain fold =-=[39]-=-, with a four-stranded antiparallel b-sheet flanked by two a-helices on one face and one helix on the other with an N-terminal b-strand extension (Fig. 2A). The protein forms a hexamer with pairs of m...

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...compartment of S. enterica [40]. The role of these proteins in the microcompartment shell and possible function in substrate transport, or recruitment of enzymes to the lumen, is yet to be determined =-=[51]-=-. However, the common structures these proteins share, despite the different substrate specificities of the microcompartments they belong to, suggests a shared function independent of substrate. It ha...

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...f C. difficile and its role in intestinal colonisation and virulence. Nutritional stress induces the expression of the C. difficile toxins, which act on host cells and induce an inflammatory response =-=[10,11]-=-. As a consequence of the cellular damage and ensuing inflammation caused by these toxins large quantities of phospholipids, particularly the abundant phosphatidylethanolamine, are liberated from the ...

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...f C. difficile and its role in intestinal colonisation and virulence. Nutritional stress induces the expression of the C. difficile toxins, which act on host cells and induce an inflammatory response =-=[10,11]-=-. As a consequence of the cellular damage and ensuing inflammation caused by these toxins large quantities of phospholipids, particularly the abundant phosphatidylethanolamine, are liberated from the ...

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...tion has been extensively studied in Salmonella species [15,21,30–32] However, there remain a number of questions about the roles of some of the enzymes associated with the ethanolamine ammonia lyase =-=[15,18,32,33]-=-. Work on the structure of this BMC is limited to X-ray crystal structures of a number of the shell proteins from the E. coli eut operon [34]. To understand the function of the ethanolamine utilisatio...

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..., or to create new niches through changes to their host organism. Salmonella enterica and Escherichia coli species are common causes of gastroenteritis and diarrheal illness in the healthy population =-=[6,7]-=-, while Clostridium difficile is a major cause of hospital acquired diarrhoea and has significant risks of morbidity and mortality in the elderly and immune compromised patients [8,9]. With an ageing ...

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...etabolism within a BMC is thought to protect the cell from the acetaldehyde produced as an intermediate in its breakdown and to prevent the loss of this volatile compound and its carbon from the cell =-=[29]-=-. The biochemistry of the ethanolamine utilisation has been extensively studied in Salmonella species [15,21,30–32] However, there remain a number of questions about the roles of some of the enzymes a...

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...althy population [6,7], while Clostridium difficile is a major cause of hospital acquired diarrhoea and has significant risks of morbidity and mortality in the elderly and immune compromised patients =-=[8,9]-=-. With an ageing population that is becoming increasingly reliant on hospital care, there is much interest in understanding the molecular basis of the metabolism of C. difficile and its role in intest...

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...es associated with the ethanolamine ammonia lyase [15,18,32,33]. Work on the structure of this BMC is limited to X-ray crystal structures of a number of the shell proteins from the E. coli eut operon =-=[34]-=-. To understand the function of the ethanolamine utilisation BMC, it is necessary to understand its architecture, including the features that are unique to this particular class of BMC and those that ...

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... final refined model had an Rcryst of 0.140 and Rfree of 0.180 (see Table 2 for data collection and refinement statistics). A single CD1908 monomer superimposes on PduU from S. enterica (PDBID: 3CGI) =-=[40]-=- and EutS from E. coli (PDBID: 3IA0) [34] with root mean square Ca deviations of 0.9 Å and 1.1 Å, respectively, over 111 aligned residues. The CD1908 hexamer adopts a conformation almost identical t...

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...s opening. doi:10.1371/journal.pone.0048360.g005 Structures of C. difficile eut BMC Proteins PLOS ONE | www.plosone.org 8 October 2012 | Volume 7 | Issue 10 | e48360 CD1918 is closely related to PduA =-=[41]-=- from S. enterica, EutM [42] from E. coli and EtuA from C. kluyveri [49]. The work of Parsons [48] and Heldt [49] indicates that these proteins are likely to play a central role in the organisation of...

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...ansmission electron microscopy of thin sections of E. coli cells overexpressing carboxysome proteins from Halothiobacillus neapolitanus [35], Citrobacter freundii propanediol utilisation BMC proteins =-=[48]-=-, Clostridium kluyveri ethanol utilisation BMC [49] and S. enterica ethanolamine utilisation proteins [37], have revealed the presence of higher-order protein structures. To understand the possible ro...

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... is lined with aromatic and hydrophobic residues. A pair of acidic residues (Glu100 and Asp102) lie within this pocket in an arrangement that is consistent with a role in ligand binding, or catalysis =-=[53]-=- (Fig. 5B/ D) and identical to the arrangement found in the sugar epimerase cupins [45]. Isothermal titration calorimetry, co-crystallisation and soaking experiments were performed with substrates and...

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...Halothiobacillus neapolitanus [35], Citrobacter freundii propanediol utilisation BMC proteins [48], Clostridium kluyveri ethanol utilisation BMC [49] and S. enterica ethanolamine utilisation proteins =-=[37]-=-, have revealed the presence of higher-order protein structures. To understand the possible roles that the proteins in this study may Figure 3. Alignment of CD1908 and its homologues. A. Ribbon view o...

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...A from S. enterica (PDBID:3NGK) Structures of C. difficile eut BMC Proteins PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e48360 [41], and 72% with EutM from E. coli (PDBID: 3MPW) =-=[42]-=-. It superimposes on these proteins with root mean square Ca deviations of 0.5 and 0.7 Å respectively over 86 aligned residues, forming identical hexameric arrangements to these proteins. Functional ...

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...nomics output) and superimposes with an rms Ca deviation of 1.25 Å over 116 amino acids. Despite low sequence identity with metal binding cupins, such as those from Thermotoga maritima (PDBID: 1VJ2) =-=[44]-=- (14%), and Bacillus subtilis (2Y0O) [45] (12%), these structures superimpose with rms Ca deviations of 1.88 and 1.74 Å respectively, over 98 residues in both cases. The conservation of the cupin cor...

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... core (Fig. 7A). In contrast to the metal-binding catalytic cupins, CD1925 and the EutQ family, do not possess the histidine residues that are responsible for metal coordination in the oxidoreductase =-=[46]-=- and epimerase [47] classes of cupins. In the place of the histidine residues are aromatic (Trp94) and hydrophobic residues (Leu 96, Ile134) and in the fourth position a glutamic acid residue (Glu100)...

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...mmation caused by these toxins large quantities of phospholipids, particularly the abundant phosphatidylethanolamine, are liberated from the cell membranes of host epithelial cells and other bacteria =-=[12,13]-=-. Phosphatidylethanolamine is broken down readily by bacterial phosphodiesterases into glycerol and ethanolamine [14], and a number of enteric pathogens, including S. enterica, Enterococcus faecalis a...

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...ms Ca deviation of 1.25 Å over 116 amino acids. Despite low sequence identity with metal binding cupins, such as those from Thermotoga maritima (PDBID: 1VJ2) [44] (14%), and Bacillus subtilis (2Y0O) =-=[45]-=- (12%), these structures superimpose with rms Ca deviations of 1.88 and 1.74 Å respectively, over 98 residues in both cases. The conservation of the cupin core is evident in these structures, with th...

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... contrast to the metal-binding catalytic cupins, CD1925 and the EutQ family, do not possess the histidine residues that are responsible for metal coordination in the oxidoreductase [46] and epimerase =-=[47]-=- classes of cupins. In the place of the histidine residues are aromatic (Trp94) and hydrophobic residues (Leu 96, Ile134) and in the fourth position a glutamic acid residue (Glu100) (Fig. 7B/C), which...