Gene Cloning,Nucleotide Sequencing,and Purification and Characterization of the Low-Specificity l-Threonine Aldolase from Pseudomonas sp. Strain NCIMB 10558

A low-specificity l-threonine aldolase (l-TA) gene from Pseudomonas sp. strain NCIMB 10558 was cloned and sequenced. The gene contains an open reading frame consisting of 1,041 nucleotides corresponding to 346 amino acid residues. The gene was overexpressed in Escherichia coli cells, and the recombinant enzyme was purified and characterized. The enzyme, requiring pyridoxal 5′-phosphate as a coenzyme, is strictly l specific at the α position, whereas it cannot distinguish between threo and erythro forms at the β position. In addition to threonine, the enzyme also acts on various other l-β-hydroxy-α-amino acids, including l-β-3,4-dihydroxyphenylserine, l-β-3,4-methylenedioxyphenylserine, and l-β-phenylserine. The predicted amino acid sequence displayed less than 20% identity with those of low-specificity l-TA from Saccharomyces cerevisiae, l-allo-threonine aldolase from Aeromonas jandaei, and four relevant hypothetical proteins from other microorganisms. However, lysine 207 of low-specificity l-TA from Pseudomonas sp. strain NCIMB 10558 was found to be completely conserved in these proteins. Site-directed mutagenesis experiments showed that substitution of Lys207 with Ala or Arg resulted in a significant loss of enzyme activity, with the corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys207 of the l-TA probably functions as an essential catalytic residue, forming an internal Schiff base with the pyridoxal 5′-phosphate of the enzyme to catalyze the reversible aldol reaction.β-Hydroxy-α-amino acids constitute an important class of compounds. They are natural products in their own right and are components of a range of antibiotics, for example, cyclosporin A, lysobactin, and vancomycin (30) and bouvardin and deoxybouvardin (6). 4-Hydroxy-l-threonine is a precursor of rizobitoxine, a potent inhibitor of pyridoxal 5′-phosphate (PLP)-dependent enzymes (32). 3,4,5-Trihydroxyl-l-aminopentanoic acid is a key component of polyoxins (32). l-threo-3,4-Dihydroxyphenylserine is a new drug for Parkinson’s disease therapy (13). However, the industrial production of β-hydroxy-α-amino acids has been limited to chemical synthesis processes, which need multiple steps to isolate the four isomers (l-threo form, d-threo form, l-erythro form, and d-erythro form). Threonine aldolase (EC 4.1.2.5), which stereospecifically catalyzes the retro-aldol cleavage of threonine, is a potentially useful catalyst for the synthesis of substituted amino acids from aldehyde and glycine (27, 31, 32).Two different types of threonine aldolases are known so far. l-allo-Threonine aldolase (l-allo-TA), isolated and purified from Aeromonas jandaei DK-39 (8), stereospecifically catalyzes the reversible interconversion of l-allo-threonine and glycine. Low-specificity l-threonine aldolase (l-TA) catalyzes the cleavage of both l-threonine and l-allo-threonine to glycine and acetaldehyde, as well as the reverse reaction, aldol condensation. The enzymes have been purified and characterized from Candida humicola (9, 34) and Saccharomyces cerevisiae (12). Low-specificity l-TA activity has also been shown to exist in mammals (7, 23, 26) and a variety of other microbial species (2, 4, 17, 35). The enzyme is physiologically important for the synthesis of cellular glycine in yeast (12, 15, 16). Threonine aldolases with distinct stereospecificities are ideal targets for enzymology studies on structural and functional relationships. However, information on the primary structures of threonine aldolases was limited to our recent studies (11, 12). The construction of an overproduction system for threonine aldolase will be indispensable for the industrial biosyntheses of β-hydroxy-α-amino acids.The present work focuses on the cloning, sequencing, and overexpression in Escherichia coli cells of the low-specificity l-TA gene from Pseudomonas sp. strain NCIMB 10558, the purification and characterization of the recombinant enzyme, and the identification of the active-site lysine residue of the enzyme by site-directed mutagenesis. Evidence is presented that Lys207 of low-specificity l-TA probably functions as a catalytic residue, forming an internal Schiff base with the PLP of the enzyme to catalyze the reversible aldol reaction. This is the first report showing a purified enzyme with l-β-3,4-dihydroxyphenylserine aldolase and l-β-3,4-methylenedioxyphenylserine aldolase activities, providing a new route for the industrial production of these important unnatural amino acids.     

Peptidoglycan Structural Dynamics during Germination of Bacillus subtilis 168 Endospores

Peptidoglycan structural dynamics during endospore germination of Bacillus subtilis 168 have been examined by muropeptide analysis. The first germination-associated peptidoglycan structural changes are detected within 3 min after the addition of the specific germinant l-alanine. We detected in the spore-associated material new muropeptides which, although they have slightly longer retention times by reversed-phase (RP)-high-pressure liquid chromatography (HPLC) than related ones in dormant spores, show the same amino acid composition and molecular mass. Two-dimensional nuclear magnetic resonance (NMR) analysis shows that the chemical changes to the muropeptides on germination are minor and are probably limited to stereochemical inversion. These new muropeptides account for almost 26% of the total muropeptides in spore-associated material after 2 h of germination. The exudate of germinated spores of B. subtilis 168 contains novel muropeptides in addition to those present in spore-associated material. Exudate-specific muropeptides have longer retention times, have no reducing termini, and exhibit a molecular mass 20 Da lower than those of related reduced muropeptides. These new products are anhydro-muropeptides which are generated by a lytic transglycosylase, the first to be identified in a gram-positive bacterium. There is also evidence for the activity of a glucosaminidase during the germination process. Quantification of muropeptides in spore-associated material indicates that there is a heterogeneous distribution of muropeptides in spore peptidoglycan. The spore-specific residue, muramic δ-lactam, is proposed to be a major substrate specificity determinant of germination-specific lytic enzymes, allowing cortex hydrolysis without any effect on the primordial cell wall.The extreme heat resistance of dormant bacterial endospores has made them an important problem in the production of safe foodstuffs (3). The spore cell wall peptidoglycan is considered to play a major role in the maintenance of heat resistance and dormancy (6). Bacillus subtilis spore peptidoglycan is composed of two layers. A thin, inner layer called the primordial cell wall retains the basic vegetative cell peptidoglycan structure. The primordial cell wall represents 2 to 4% of the total endospore peptidoglycan, is not digested during germination, and serves as the initial cell wall during outgrowth (2, 5, 25, 29). The outer thick layer of peptidoglycan, known as the cortex, is characterized by several unique spore-specific features. Approximately 50% of the muramic acid residues in the glycan strands are present in the δ-lactam form (2, 24). Muramic acid side chains are composed of 26 and 23% of tetrapeptide and single l-alanine, respectively (2).Despite their extreme dormancy and thermostability, bacterial endospores retain an alert sensory mechanism enabling them to respond within minutes to the presence of specific germinants. Spores of B. subtilis respond to at least two different types of germinative stimuli: (i) l-alanine and (ii) a combination of l-asparagine, glucose, fructose, and KCl (AGFK) (34). The germination response is initiated by the interaction of a receptor protein with specific germinants which triggers the loss of spore-specific properties and the transformation of a dormant resistant bacterial spore into a metabolically active vegetative cell. The germination process is characterized by sequential, interrelated biochemical events. The specific hydrolysis of peptidoglycan in the spore cortex layer is an essential event in germination (2, 25). Its degradation removes the physical constraints of the cortex and allows core expansion and outgrowth (9, 25). As a consequence of cortex hydrolysis, peptidoglycan fragments can be detected in the germination exudate (13, 33).A number of bacterial spore germination-specific cortex-lytic enzymes (GSLEs) have been reported to be involved in cortex hydrolysis (9, 18–20). A gene homologous to that encoding the GSLE from Bacillus cereus has been identified and inactivated in B. subtilis, and the resulting mutant germinates more slowly than the wild type (22). Recently a germination-specific muramidase isolated from a germination extract of Clostridium perfringens S40 has been purified and characterized (4).GSLEs have a high substrate specificity, requiring intact spore cortex for activity (9, 23). The muramidase from C. perfringens S40, however, hydrolyzes cortical fragments but has a strict requirement for the presence of the muramic δ-lactam residues (4). Thus, the GSLEs are highly specialized and may exist as proforms which are specifically activated during germination (9).Very little is known about the mechanism by which the cortex is hydrolyzed during germination and the autolytic enzymes involved. Muropeptide analysis provides a method for fine chemical structural determination of spore cortex (2, 24, 25). In this paper, we report the use of muropeptide analysis to determine the peptidoglycan structural dynamics which occur during spore germination of B. subtilis 168 and the evidence for a number of different enzyme activities.     

Identification of Galacturonic Acid-1-phosphate Kinase, a New Member of the GHMP Kinase Superfamily in Plants, and Comparison with Galactose-1-phosphate Kinase

The process of salvaging sugars released from extracellular matrix, during plant cell growth and development, is not well understood, and many molecular components remain to be identified. Here we identify and functionally characterize a unique Arabidopsis gene encoding an α-d-galacturonic acid-1-phosphate kinase (GalAK) and compare it with galactokinase. The GalAK gene appeared to be expressed in all tissues implicating that glycose salvage is a common catabolic pathway. GalAK catalyzes the ATP-dependent conversion of α-d-galacturonic acid (d-GalA) to α-d-galacturonic acid-1-phosphate (GalA-1-P). This sugar phosphate is then converted to UDP-GalA by a UDP-sugar pyrophosphorylase as determined by a real-time ¹H NMR-based assay. GalAK is a distinct member of the GHMP kinase family that includes galactokinase (G), homoserine kinase (H), mevalonate kinase (M), and phosphomevalonate kinase (P). Although these kinases have conserved motifs for sugar binding, nucleotide binding, and catalysis, they do have subtle difference. For example, GalAK has an additional domain near the sugar-binding motif. Using site-directed mutagenesis we established that mutation in A368S reduces phosphorylation activity by 40%; A41E mutation completely abolishes GalAK activity; Y250F alters sugar specificity and allows phosphorylation of d-glucuronic acid, the 4-epimer of GalA. Unlike many plant genes that undergo duplication, GalAK occurs as a single copy gene in vascular plants. We suggest that GalAK generates GalA-1-P from the salvaged GalA that is released during growth-dependent cell wall restructuring, or from storage tissue. The GalA-1-P itself is then available for use in the formation of UDP-GalA required for glycan synthesis.d-Galacturonic acid (d-GalA)³ is a quantitatively major glycose present in numerous plant polysaccharides including pectins and arabinogalactan proteins (1, 2). The synthesis of these polysaccharides requires a large number of glycosyltransferases and diverse nucleotide-sugar (NDP-sugar) donors (1, 3). Some of these NDP-sugars are formed by interconversion of pre-existing NDP-sugars and by salvage pathways. For example, the main pathway for UDP-GalA formation is the 4-epimerization of UDP-GlcA, a reaction catalyzed by UDP-GlcA 4-epimerase (4–6). However, in ripening Fragaria fruit d-GalA is incorporated into pectin (7). It is likely that a sugar kinase converts the d-GalA to GalA-1-P (8), which is then converted to UDP-GalA by a nonspecific UDP-sugar pyrophosphorylase (9). Myo-inositol may also be a source of GalA for polysaccharide biosynthesis (10).Galacturonic acid is likely to be generated by enzyme-catalyzed hydrolysis of pectic polysaccharides in plant tissues. Polysaccharide hydrolase activities are present in germinating seeds (11, 12), in germinating and elongating pollen (13–15), and in ripening fruit (14). Thus, monosaccharide salvage pathways may be required for normal plant growth and development.Numerous sugar-1-P kinases, including d-Gal-1-P kinase (16), l-Ara-1-P kinase (17), and l-Fuc-1-P kinase (18), have been described (19), but no d-GalA-1-P kinase has been identified in any species to account for the hydrolysis and recycle of pectic polymers. The subsequent pyrophosphorylation of UDP-sugars could be carried out by UDP-sugar pyrophosphorylases (20).Here, we report the identification and characterization of a functional galacturonic acid kinase (GalAK). We compared the activity of GalAK with a previously uncharacterized Arabidopsis GalK and discussed the evolution of these sugar kinase members of the GHMP kinase.     

Purification and Substrate Specificities of Two α-l-Arabinofuranosidases from Aspergillus awamori IFO 4033

α-l-Arabinofuranosidases I and II were purified from the culture filtrate of Aspergillus awamori IFO 4033 and had molecular weights of 81,000 and 62,000 and pIs of 3.3 and 3.6, respectively. Both enzymes had an optimum pH of 4.0 and an optimum temperature of 60°C and exhibited stability at pH values from 3 to 7 and at temperatures up to 60°C. The enzymes released arabinose from p-nitrophenyl-α-l-arabinofuranoside, O-α-l-arabinofuranosyl-(1→3)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose, and arabinose-containing polysaccharides but not from O-β-d-xylopyranosyl-(1→2)-O-α-l-arabinofuranosyl-(1→3)-O-β-d-xylopyranosyl-(1→4)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose. α-l-Arabinofuranosidase I also released arabinose from O-β-d-xylopy-ranosyl-(1→4)-[O-α-l-arabinofuranosyl-(1→3)]-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose. However, α-l-arabinofuranosidase II did not readily catalyze this hydrolysis reaction. α-l-Arabinofuranosidase I hydrolyzed all linkages that can occur between two α-l-arabinofuranosyl residues in the following order: (1→5) linkage > (1→3) linkage > (1→2) linkage. α-l-Arabinofuranosidase II hydrolyzed the linkages in the following order: (1→5) linkage > (1→2) linkage > (1→3) linkage. α-l-Arabinofuranosidase I preferentially hydrolyzed the (1→5) linkage of branched arabinotrisaccharide. On the other hand, α-l-arabinofuranosidase II preferentially hydrolyzed the (1→3) linkage in the same substrate. α-l-Arabinofuranosidase I released arabinose from the nonreducing terminus of arabinan, whereas α-l-arabinofuranosidase II preferentially hydrolyzed the arabinosyl side chain linkage of arabinan.Recently, it has been proven that l-arabinose selectively inhibits intestinal sucrase in a noncompetitive manner and reduces the glycemic response after sucrose ingestion in animals (33). Based on this observation, l-arabinose can be used as a physiologically functional sugar that inhibits sucrose digestion. Effective l-arabinose production is therefore important in the food industry. l-Arabinosyl residues are widely distributed in hemicelluloses, such as arabinan, arabinoxylan, gum arabic, and arabinogalactan, and the α-l-arabinofuranosidases (α-l-AFases) (EC 3.2.1.55) have proven to be essential tools for enzymatic degradation of hemicelluloses and structural studies of these compounds.α-l-AFases have been classified into two families of glycanases (families 51 and 54) on the basis of amino acid sequence similarities (11). The two families of α-l-AFases also differ in substrate specificity for arabinose-containing polysaccharides. Beldman et al. summarized the α-l-AFase classification based on substrate specificities (3). One group contains the Arafur A (family 51) enzymes, which exhibit very little or no activity with arabinose-containing polysaccharides. The other group contains the Arafur B (family 54) enzymes, which cleave arabinosyl side chains from polymers. However, this classification is too broad to define the substrate specificities of α-l-AFases. There have been many studies of the α-l-AFases (3, 12), especially the α-l-AFases of Aspergillus species (2–8, 12–15, 17, 22, 23, 28–32, 36–39, 41–43, 46). However, there have been only a few studies of the precise specificities of these α-l-AFases. In previous work, we elucidated the substrate specificities of α-l-AFases from Aspergillus niger 5-16 (17) and Bacillus subtilis 3-6 (16, 18), which should be classified in the Arafur A group and exhibit activity with arabinoxylooligosaccharides, synthetic methyl 2-O-, 3-O-, and 5-O-arabinofuranosyl-α-l-arabinofuranosides (arabinofuranobiosides) (20), and methyl 3,5-di-O-α-l-arabinofuranosyl-α-l-arabinofuranoside (arabinofuranotrioside) (19).In the present work, we purified two α-l-AFases from a culture filtrate of Aspergillus awamori IFO 4033 and determined the substrate specificities of these α-l-AFases by using arabinose-containing polysaccharides and the core oligosaccharides of arabinoxylan and arabinan.     

Requirements for Germination of Clostridium sordellii Spores In Vitro

Clostridium sordellii is a spore-forming, obligately anaerobic, Gram-positive bacterium that can cause toxic shock syndrome after gynecological procedures. Although the incidence of C. sordellii infection is low, it is fatal in most cases. Since spore germination is believed to be the first step in the establishment of Bacilli and Clostridia infections, we analyzed the requirements for C. sordellii spore germination in vitro. Our data showed that C. sordellii spores require three structurally different amino acids and bicarbonate for maximum germination. Unlike the case for Bacilli species, d-alanine had no effect on C. sordellii spore germination. C. sordellii spores germinated only in a narrow pH range between 5.7 and 6.5. In contrast, C. sordellii spore germination was significantly less sensitive to temperature changes than that of the Bacilli. The analysis of the kinetics of C. sordellii spore germination showed strong allosteric behavior in the binding of l-phenylalanine and l-alanine but not in that of bicarbonate or l-arginine. By comparing germinant apparent binding affinities to their known in vivo concentrations, we postulated a mechanism for differential C. sordellii spore activation in the female reproductive tract.Clostridium sordellii is an anaerobic, Gram-positive, spore-forming bacterium that is commonly found in soil and in the intestines of animals (4). Many C. sordellii strains are nonpathogenic; however, virulent strains cause lethal infections in several animal species, such as hemorrhagic enteritis in foals, sheep, and cattle (5, 10, 16, 28), omphalitis in foals (43), and wound infection in humans (4, 35).C. sordellii also can cause life-threatening necrotizing infections after gynecological procedures (4). In addition, fatal cases of C. sordellii endometritis following medical abortion with a mifepristone-misoprostol combination have been reported recently (13, 19, 56). The increased use of mifepristone-misoprostol for medical abortion may result in larger numbers of C. sordellii infections (38, 40).Although C. sordellii rarely has been identified in the genital tract, a correlation between gynecological procedures and C. sordellii-mediated toxic shock syndrome is apparent (19). Pregnancy, childbirth, or abortion may predispose some women to acquire C. sordellii in the vaginal tract (19). Under these conditions, C. sordellii infections result in an almost 100% mortality rate.Since there is no national system for tracking and reporting complications associated with gynecological procedures, the identification of the true rates of reproductive tract infections in women is not readily available (8). Therefore, the number of known C. sordellii-associated infections, although low, may be underreported (19, 29). Furthermore, unsafe abortion practices in developing countries cause large mortality rates due to complicating infections (24, 34). In many cases, however, the causative agent of the abortion-associated sepsis have not been characterized (24). Thus, the worldwide morbidity and mortality associated with C. sordellii infections is not currently known.C. sordellii produces several virulence factors. The two major toxins are the lethal toxin (TcsL) and the hemorrhagic toxin (37, 46). The lethal toxin produced by C. sordellii is causally involved in enteritis of domestic animals and in systemic toxicity following infections of humans (46). Furthermore, TcsL is associated with rapid mortality in C. sordellii endometritis rodent models (26). Interestingly, TcsL cytopathic effects are increased at low pH, a characteristic found in the vaginal tract (48). The hemorrhagic toxin is not well characterized, but it has been reported to cause dermal and intestinal necrosis in guinea pigs (6, 52).C. sordellii, like other Bacilli and Clostridia species, has the ability to form metabolically dormant spores that are extremely resistant to environmental stresses, such as heat, radiation, and toxic chemicals (42, 55). Upon encountering a suitable environment, spores germinate into vegetative cells, the form that is responsible for toxin production and disease onset (39, 54).In most cases, the germination process initially is triggered by the detection of low-molecular-weight germinants by a sensitive biosensor (39, 54). This sensor consists of a proteinaceous germination (Ger) receptor encoded, in general, by a tricistronic operon. Spore germination requirements have been studied most extensively for Bacilli and can be initiated by a variety of factors, including amino acids, sugars, and nucleosides (20, 30).Spore germination in the Clostridia generally requires combinations of multiple germinants. The germination of spores of proteolytic Clostridium botulinum types A and B was triggered by a defined three-component mixture comprised of l-alanine (or l-cysteine), l-lactate (or sodium thioglycolate), and sodium bicarbonate (3). In contrast, the optimum germination of spores of nonproteolytic C. botulinum types B, E, and F required binary combinations of l-alanine-l-lactate, l-cysteine-l-lactate, and l-serine-l-lactate (45).Clostridium difficile is a human pathogen that can cause fulminant colitis (11). Interestingly, C. difficile does not encode any known Ger receptors (53). However, it is likely that germination receptors exist, because C. difficile spores must germinate in order to complete their life cycle. While C. difficile germination receptors remain elusive, the spores of C. difficile germinate in rich medium supplemented with bile salts (62). More recently, taurocholate (a bile salt) and glycine (an amino acid) were shown to act as cogerminants for C. difficile spore germination (57, 61).Clostridium bifermentans is a close relative of C. sordellii (14). The minimum requirement for C. bifermentans spore germination was the presence of l-alanine, l-phenylalanine, and l-lactate (59). In addition, an unknown factor present in yeast extract was suggested to enhance germination (59). However, the Ger receptors involved in C. bifermentans spore germination are not known.Even though many Bacilli and Clostridia species use similar metabolites as germinants, the mechanisms of germinant recognition remain to be elucidated. Unfortunately, the multimeric interactions of Ger receptor complexes and the hydrophobic nature of the Ger receptor subunits have hindered our understanding of the mechanism of germinant recognition.To understand the molecular determinants of germinant recognition, we recently applied kinetic methods to study bacterial spore germination (1, 2, 18). Spore germination can be analyzed quantitatively by fitting optical density (OD) decreases to the Michaelis-Menten equation (2). The kinetic parameters obtained allow the determination of the apparent binding affinity (K_m) of spores for the different cogerminants and the maximum rate of spore germination (V_max). In these instances, K_m refers to the concentration of substrate required to reach half of the maximal germination rate. These parameters can, in turn, be used to determine the mechanism of germination and potential interactions between germination receptors. Furthermore, by comparing apparent K_m values to germinant concentrations in vivo, models for spore-germinant complex distribution can be proposed, and rate-limiting steps for the germination process can be derived. Thus, kinetic analysis can yield information on spore activation even if the identities of the germination receptors are not known.Using this procedure, we were able to determine the mechanism for Bacillus anthracis germination with inosine and l-alanine. In turn, this information was used to design nucleoside analogs that inhibit B. anthracis spore germination in vitro and protect macrophages from anthrax cytotoxicity (2).Since C. sordellii germination receptors have not been identified, we used chemical probes and kinetic methods to investigate the conditions necessary for spore germination. We found that C. sordellii spores germinate better at slightly acidic pH. Furthermore, germination rates varied slightly from 25 to 40°C. We also found that C. sordellii spores have an absolute requirement for a small amino acid, a basic amino acid, an aromatic amino acid, and bicarbonate (NaHCO₃) for efficient germination. Kinetic analysis showed allosteric interaction for the putative l-phenylalanine and l-alanine germination receptors. In contrast, l-arginine or bicarbonate recognition followed typical Michaelis-Menten kinetics. The implication of germinant recognition and host environment is discussed.     

Roles of Low-Molecular-Weight Penicillin-Binding Proteins in Bacillus subtilis Spore Peptidoglycan Synthesis and Spore Properties

The peptidoglycan cortex of endospores of Bacillus species is required for maintenance of spore dehydration and dormancy, and the structure of the cortex may also allow it to function in attainment of spore core dehydration. A significant difference between spore and growing cell peptidoglycan structure is the low degree of peptide cross-linking in cortical peptidoglycan; regulation of the degree of this cross-linking is exerted by d,d-carboxypeptidases. We report here the construction of mutant B. subtilis strains lacking all combinations of two and three of the four apparent d,d-carboxypeptidases encoded within the genome and the analysis of spore phenotypic properties and peptidoglycan structure for these strains. The data indicate that while the dacA and dacC products have no significant role in spore peptidoglycan formation, the dacB and dacF products both function in regulating the degree of cross-linking of spore peptidoglycan. The spore peptidoglycan of a dacB dacF double mutant was very highly cross-linked, and this structural modification resulted in a failure to achieve normal spore core dehydration and a decrease in spore heat resistance. A model for the specific roles of DacB and DacF in spore peptidoglycan synthesis is proposed.Peptidoglycan (PG) is the structural element of the bacterial cell wall which determines cell shape and which resists the turgor pressure within the cell. The bacterial endospores produced by species of Bacillus, Clostridium, and several other bacterial genera are modified cells that are able to survive long periods and extreme conditions in a dormant, relatively dehydrated state. The PG wall within the endospore is required for maintenance of the dehydrated state (10, 11), which is the major determinant of spore heat resistance (2, 17, 22). Spore PG appears to be comprised of two distinct though contiguous layers. The thin inner layer, the germ cell wall, appears to have a structure similar to that of the vegetative wall and serves as the initial cell wall of the germinated spore (1, 20, 21, 31). The thicker outer layer, the spore cortex, has a modified structure which may determine its ability to carry out roles specific to the spore, and is rapidly degraded during spore germination (1, 20, 35, 37). The most dramatic of the cortex structural modifications results in partial cleavage or complete removal of ∼75% of the peptide side chains from the glycan strands. Loss of these peptides limits the cross-linking potential of the PG and results in the formation of only one peptide cross-link per 35 disaccharide units in the spore PG, compared to one peptide cross-link per 2.3 to 2.9 disaccharide units in the vegetative PG (1, 20, 36). This low degree of cross-linking has been predicted to give spore PG a flexibility that allows it to have a role in attainment of spore core dehydration (14, 34) in addition to its clear role in maintenance of dehydration. We are studying the structure and mechanism of synthesis of spore PG in an attempt to discern the roles of this structure and its individual components in determining spore properties.A family of proteins called the penicillin-binding proteins (PBPs) polymerizes PG on the external surface of the cell membrane (reviewed in reference 7). The high-molecular-weight (high-MW) members of this family (generally ≥60 kDa) carry the transglycosylase and transpeptidase activities involved in polymerization and cross-linking of the glycan strands. The low-MW PBPs have commonly been found to possess d,d-carboxypeptidase activity. This activity can remove the terminal d-alanine of the peptide side chains and thereby prevent the side chain from serving as a donor in the formation of a peptide cross-link. Analysis of the B. subtilis genome reveals six low-MW PBP-encoding genes: dacA (33), dacB (4), dacC (19), dacF (38), pbpE (23), and pbpX (accession no. {"type":"entrez-nucleotide","attrs":{"text":"Z99112","term_id":"32468745","term_text":"Z99112"}}Z99112). The four dac gene products exhibit very high sequence similarity to proven d,d-carboxypeptidases, and this activity has been demonstrated in vitro for the dacA and dacB products, PBP5 (12) and PBP5* (32), respectively. The sequences of the pbpE and pbpX products are more distantly related, and no activity has yet been established or ruled out for them.PBP5 is the major penicillin-binding and d,d-carboxypeptidase activity found in vegetative cells (12). Although dacA expression declines significantly during sporulation, a significant amount of PBP5 remains during the time of spore PG synthesis (29). A dacA-null mutation results in no obvious effects on vegetative growth, sporulation, spore characteristics, or spore germination (3, 33). However, loss of PBP5 does result in a reduction of cleavage of peptide side chains from the tetrapeptide to the tripeptide form in the spore PG (20). PBP5* is expressed only during sporulation and only in the mother cell compartment of the sporangium, under the control of the RNA polymerase ς^E subunit (4, 5, 28, 29). A dacB-null mutation leading to loss of this d,d-carboxypeptidase results in a fourfold increase in the effective cross-linking of the spore PG (1, 20, 22). This structural change is accompanied by only slight decreases in spore core dehydration and heat resistance (3, 22). The suspected d,d-carboxypeptidase activities of the products of the dacC and dacF genes have not been demonstrated. The latter two genes are expressed only during the postexponential growth phase: dacC is expressed during early stationary phase under the control of ς^H (19) and dacF is expressed only within the forespore under the control of ς^F (27, 38). Null mutations effecting either gene result in no obvious phenotype and no change in spore PG structure (19, 38).The multiplicity of these proteins in sporulating cells and the lack of effect of loss of some of them suggested redundancy of function among these proteins, a situation observed previously with PBPs of a high-MW class (25, 30, 39). In order to examine this possibility we have constructed mutants lacking multiple low-MW PBPs and have examined their sporulation efficiency, spore PG structure, spore heat resistance and wet density, and spore germination and outgrowth. The present study demonstrates a role for the dacF gene product in synthesis of spore PG, and we also present a model for the roles of the dacB and dacF gene products in spore PG formation.     

Biosynthesis and Structure of the Burkholderia cenocepacia K56-2 Lipopolysaccharide Core Oligosaccharide: TRUNCATION OF THE CORE OLIGOSACCHARIDE LEADS TO INCREASED BINDING AND SENSITIVITY TO POLYMYXIN B*

Burkholderia cenocepacia is an opportunistic pathogen that displays a remarkably high resistance to antimicrobial peptides. We hypothesize that high resistance to antimicrobial peptides in these bacteria is because of the barrier properties of the outer membrane. Here we report the identification of genes for the biosynthesis of the core oligosaccharide (OS) moiety of the B. cenocepacia lipopolysaccharide. We constructed a panel of isogenic mutants with truncated core OS that facilitated functional gene assignments and the elucidation of the core OS structure in the prototypic strain K56-2. The core OS structure consists of three heptoses in the inner core region, 3-deoxy-d-manno-octulosonic acid, d-glycero-d-talo-octulosonic acid, and 4-amino-4-deoxy-l-arabinose linked to d-glycero-d-talo-octulosonic acid. Also, glucose is linked to heptose I, whereas heptose II carries a second glucose and a terminal heptose, which is the site of attachment of the O antigen. We established that the level of core truncation in the mutants was proportional to their increased in vitro sensitivity to polymyxin B (PmB). Binding assays using fluorescent 5-dimethylaminonaphthalene-1-sulfonyl-labeled PmB demonstrated a correlation between sensitivity and increased binding of PmB to intact cells. Also, the mutant producing a heptoseless core OS did not survive in macrophages as compared with the parental K56-2 strain. Together, our results demonstrate that a complete core OS is required for full PmB resistance in B. cenocepacia and that resistance is due, at least in part, to the ability of B. cenocepacia to prevent binding of the peptide to the bacterial cell envelope.Burkholderia cenocepacia is a Gram-negative opportunistic pathogen ubiquitously found in the environment (1, 2). Although generally harmless to healthy individuals, B. cenocepacia affects immunocompromised patients (1) such as those with cystic fibrosis and chronic granulomatous disease. Infected cystic fibrosis patients commonly develop chronic lung infections that are very difficult to treat because these bacteria are intrinsically resistant to virtually all clinically useful antibiotics as well as antimicrobial peptides (APs)⁵ (1, 3).Lipopolysaccharide (LPS) is the major surface component of Gram-negative bacteria and consists of lipid A, core oligosaccharide (OS), and in some bacteria O-specific polysaccharide or O antigen (4, 5). The O antigen acts as a protective barrier against desiccation, phagocytosis, and serum complement-mediated killing, whereas the core OS and the lipid A contribute to maintain the integrity of the outer membrane (4, 5). The lipid A also anchors the LPS molecule to the outer leaflet of the outer membrane and accounts for the endotoxic activity of LPS (4, 6). Lipid A is a bisphosphorylated β-1,6-linked glucosamine disaccharide substituted with fatty acids ester-linked at positions 3 and 3′ and amide-linked at positions 2 and 2′ (4). The core OS can be subdivided into the inner core and outer core. The inner core OS typically consists of one or two 3-deoxy-d-manno-octulosonic acid (Kdo) residues linked to the lipid A and three l-glycero-d-manno-heptose residues linked to the first Kdo (4). The outer core OS in enteric bacteria typically consists of 8–12 branched sugars linked to heptose II of the inner core. As a result of phosphate groups on the lipid A and core OS, the bacterial surface has a net negative charge. This plays an important role in the interaction of the bacterial surface with positively charged compounds such as cationic APs, which are cationic amphipathic molecules that kill bacteria by membrane permeabilization. In response to a series of environmental conditions such as low magnesium or high iron, bacteria can express modified LPS molecules that result in a less negative surface. This reduces the binding of APs and promotes resistance to these compounds. Previous studies have shown that Burkholderia LPS molecules possess unique properties. For example, Kdo cannot be detected by classic colorimetric methods in LPS from Burkholderia pseudomallei and Burkholderia cepacia, and the covalent linkage between Kdo and lipid A is more resistant to acid hydrolysis than in conventional LPS molecules (7). In B. cepacia, 4-amino-4-deoxy-l-arabinose (l-Ara4N) is bound to the lipid A by a phosphodiester linkage at position 4 of the nonreducing glucosamine (GlcN II) (8) and is also present as a component of the core OS. Also, instead of two Kdo molecules, the B. cepacia core OS has only one Kdo and the unusual Kdo analog, d-glycero-d-talo-octulosonic acid (Ko), which is nonstoichiometrically substituted with l-Ara4N forming a 1→8 linkage with α-Ko (7, 9). Although this is also the case for the inner core OS of B. cenocepacia J2315 (10), it is not a common feature for the core OS in all Burkholderia. For example, the inner core of Burkholderia caryophylli consists of two Kdo residues and does not possess l-Ara4N (11).Burkholderia species, including B. cenocepacia, are intrinsically resistant to human and non-human APs such as these produced by airway epithelial cells (12, 13), human β-defensin 3 (14), human neutrophil peptides (15), and polymyxin B (PmB) (15, 16). The minimum inhibitory concentration determined for some of these peptides in several Burkholderia species is greater than 500 μg/ml, which could aid these microorganisms during colonization of the respiratory epithelia (13). It has been proposed that the resistance of B. cepacia to cationic APs stems from ineffective binding to the outer membrane, as a consequence of the low number of phosphate and carboxylate groups in the lipopolysaccharide (17), but a systematic analysis of the molecular basis of AP resistance in B. cenocepacia and other Burkholderia is lacking. We have previously reported that a heptoseless B. cenocepacia mutant (SAL1) is significantly more sensitive than the parental clinical strain K56-2 to APs (15). This mutant has a truncated inner core and lacks the outer core, suggesting that a complete core OS is required for resistance of B. cenocepacia to APs.Apart from heptoses, the role of other sugar moieties of the B. cenocepacia core OS in AP resistance is not known. In this study, we report the structure of the core OS for B. cenocepacia strain K56-2 and its isogenic mutants XOA3, XOA7, and XOA8, which carry various core OS truncations. The structural analysis, combined with mutagenesis, allowed us to assign function to the majority of the genes involved in core OS biosynthesis and ligation of the O antigen and to establish that the degree of truncation of the core OS correlates with increased binding and bacterial sensitivity to PmB in vitro and reduced bacterial intracellular survival in macrophages.     

Crystal Structure of a Homolog of Mammalian Serine Racemase from Schizosaccharomyces pombe

d-Serine is an endogenous coagonist for the N-methyl-d-aspartate receptor and is involved in excitatory neurotransmission in the brain. Mammalian pyridoxal 5′-phosphate-dependent serine racemase, which is localized in the mammalian brain, catalyzes the racemization of l-serine to yield d-serine and vice versa. The enzyme also catalyzes the dehydration of d- and l-serine. Both reactions are enhanced by Mg·ATP in vivo. We have determined the structures of the following three forms of the mammalian enzyme homolog from Schizosaccharomyces pombe: the wild-type enzyme, the wild-type enzyme in the complex with an ATP analog, and the modified enzyme in the complex with serine at 1.7, 1.9, and 2.2 Å resolution, respectively. On binding of the substrate, the small domain rotates toward the large domain to close the active site. The ATP binding site was identified at the domain and the subunit interface. Computer graphics models of the wild-type enzyme complexed with l-serine and d-serine provided an insight into the catalytic mechanisms of both reactions. Lys-57 and Ser-82 located on the protein and solvent sides, respectively, with respect to the cofactor plane, are acid-base catalysts that shuttle protons to the substrate. The modified enzyme, which has a unique “lysino-d-alanyl” residue at the active site, also exhibits catalytic activities. The crystal-soaking experiment showed that the substrate serine was actually trapped in the active site of the modified enzyme, suggesting that the lysino-d-alanyl residue acts as a catalytic base in the same manner as inherent Lys-57 of the wild-type enzyme.d-Serine, which is present at a high level in the mammalian brain, serves as an endogenous coagonist for the N-methyl-d-aspartate (NMDA)⁵ receptor selectively localized on the postsynaptic membrane of the excitatory synapse (1–5) and is involved in excitatory neurotransmission and higher brain functions such as learning and memory (3, 6, 7). Stimulation of the NMDA receptor requires the binding of d-serine as well as the agonist l-glutamate. The major enzyme for d-serine synthesis from l-serine in the brain is considered to be pyridoxal 5′-phosphate (PLP)-dependent serine racemase (SR) (8–10). d-Serine and SR are localized on protoplasmic astrocytes that have the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor. Glutamate released from presynaptic neurons approaches and activates the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor, which in turn induces SR to produce d-serine and is followed by d-serine release from astrocytes that act on the NMDA receptor. Recently, it was shown that not only glia but also neurons synthesize and release d-serine involved in signaling (11). SR also catalyzes α,β-elimination of water from d- or l-serine to form pyruvate and ammonia as well as the conversion of l-serine into d-serine and vice versa and is presumed to link d-serine synthesis and energy metabolism of astrocytes (12) and to control the d-serine level (13). Mg·ATP, which is fully bound to SR under physiological conditions, stimulates racemization and the α,β-elimination reaction catalyzed by SR (12, 14).SR was first discovered in pupae of the silkworm Bombyx mori (15), which was followed by purification of the enzyme from a rat brain and cloning of the mouse and human genes (8, 9). The primary structure of mammalian SR is distinct from those of racemases from prokaryotes but is similar to those of fold-type II PLP-dependent enzymes (16–18). We have cloned and expressed the Schizosaccharomyces pombe gene homologous to human and mouse SRs, the sequence identities being 35.1 and 37.4%, respectively, in Escherichia coli. The protein product is a bifunctional enzyme that catalyzes racemization and the α,β-elimination reaction of D, l-serine as mammalian SR does. SR from S. pombe (spSR) comprises 322 residues (the N-terminal Met is removed in the purified enzyme) and one PLP per subunit, the subunit molecular weight being 34,917. The mammalian SR homolog, spSR, is an interesting target enzyme for the development of a novel therapeutic compound controlling the d-serine level because d-serine is the product of an SR-catalyzed reaction. In our recent report, the active site of spSR was shown to be modified with its natural substrate serine by mass spectroscopic and x-ray studies (19). Interestingly, the catalytic lysine, which originally forms a Schiff base with PLP, is converted to a lysino-d-alanyl residue through the reaction with the substrate, serine (Fig. 1). The modified enzyme exhibits racemase (54% of the wild-type enzyme) and α,β-elimination (68% of the wild-type enzyme) activities with the amino group of the d-alanyl moiety of the lysinoalanyl residue forming a Schiff base with PLP in place of the lysine (19). In addition, the mammalian SR seems to be possibly modified to have a lysinoalanyl residue at the active site, as observed in spSR (20).Open in a separate windowFIGURE 1.Covalent modification of the active site. The catalytic Lys-57 in spSRw is converted to lysino-d-alanyl residue. The α-amino group (indicated with “α”) of the d-alanyl moiety in the residue acts as a catalytic base in spSRm. The circled P is a phosphate group.Although the structure of modified spSR (spSRm) has been determined (19), the structure-function relationship of essential wild-type spSR (spSRw), the binding mode of activator Mg·ATP, the catalytic base to shuttle protons to the substrate d-serine, and the substrate recognition of the modified enzyme have not yet been uncovered. We now report the three-dimensional structures of unliganded spSRw in the open form, spSRw·AMP-PCP in the open form, and spSRm·serine in the closed form.     

Only the N-Terminal Domain of FtsK Functions in Cell Division

Deletion of ftsK results in the inhibition of cell division, but this inhibition can be reversed by a plasmid carrying only the first ∼17% of ftsK. The division block can be suppressed in most mutants by deletion of dacA, which codes for the d-alanine:d-alanine carboxypeptidase PBP5, or in all mutants by overexpression of ftsN. Overexpression of ftsK inhibits cell division and the formation of FtsZ rings. This division block is not due to the induction of either the SOS or the heat shock regulons.The ftsK gene of Escherichia coli encodes a large protein (1,329 amino acids) which belongs to a family of bacterium- and plasmid-encoded proteins (2), at least some of which are required for DNA transfer between cells (12, 14, 17, 23) or between a mother cell and a spore compartment (24, 25). The FtsK protein is predicted to have an N-terminal domain (of about 200 amino acids) with several (four or five) membrane-spanning α helices, a proline-glutamine-rich region (∼660 amino acids), and a cytoplasmic domain (∼469 amino acids) with a consensus nucleotide-binding pocket (2). Two independent missense mutations (ftsK44 and ftsK3531) cause different single-amino-acid substitutions in the N-terminal domain, resulting in a temperature-dependent block of cell division (2; unpublished data). The division defect in both of these mutants can be suppressed by deletion of the dacA gene, coding for a d-alanine:d-alanine carboxypeptidase (PBP5), and can be complemented by cloned fragments of the wild-type ftsK gene that contain only the first 600 to 700 bp (2; unpublished data). The temperature sensitivity of cells carrying the ftsK44 or ftsK3531 allele is suppressed by plasmids carrying ftsN (17a).The present report describes the effects of disruption, deletion, and overexpression of the ftsK gene. We conclude that only the N-terminal ∼200-amino-acid domain of FtsK is required for cell division and that deletion of the remainder of the protein is not lethal. The ftsK gene is preceded by an SOS-inducible promoter, P_dinH (19, 20), and we show here that overexpression of FtsK blocks cell division in an SfiA-independent manner. It is therefore possible that ftsK overexpression forms part of an SfiA-independent, SOS-inducible division block.     

Kinetic and Functional Analysis of l-Threonine Kinase, the PduX Enzyme of Salmonella enterica

The PduX enzyme of Salmonella enterica is an l-threonine kinase used for the de novo synthesis of coenzyme B₁₂ and the assimilation of cobyric acid. PduX with an N-terminal histidine tag (His₈-PduX) was produced in Esch e richia coli and purified. The recombinant enzyme was soluble and active. Kinetic analysis indicated a steady-state Ordered Bi Bi complex mechanism in which ATP is the first substrate to bind. Based on a multiple sequence alignment of PduX homologues and other GHMP (galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase) family members, 14 PduX variants having changes at 10 conserved serine/threonine and aspartate/glutamate sites were constructed by site-directed mutagenesis. Each variant was produced in E. coli and purified. Comparison of the circular dichroism spectra and kinetic properties of the PduX variants with those of the wild-type enzyme indicated that Glu-24 and Asp-135 are needed for proper folding, Ser-99 and Glu-132 are used for ATP binding, and Ser-253 and Ser-255 are critical to l-threonine binding whereas Ser-100 is essential to catalysis, but its precise role is uncertain. The studies reported here are the first to investigate the kinetic and catalytic mechanisms of l-threonine kinase from any organism.The B₁₂ coenzymes (adenosylcobalamin (AdoCbl)² and methylcobalamin) are the largest cofactors known in biology. They are essential for human health and have important metabolic roles in many microbes (1, 2). The B₁₂ coenzymes are synthesized de novo only by certain prokaryotes and from corrinoid precursors by a broader range of organisms (1, 2). De novo synthesis by prokaryotes is the ultimate source of B₁₂, and this process has been extensively studied because of its importance to diverse biological forms and to the commercial production of B₁₂ as a dietary supplement (1). Salmonella enterica is an important model organism for studies of B₁₂ synthesis (3, 4). This organism carries out de novo synthesis under anaerobic conditions and assimilates corrinoids such as cobinamide and cobyric acid (Cby) under both aerobic and anaerobic conditions (3, 5). We recently showed that the PduX enzyme of S. enterica is an l-threonine (l-Thr) kinase, which is required for the de novo synthesis of AdoCbl and methylcobalamin and the assimilation of Cby (6). PduX catalyzes the conversion of l-Thr and ATP to ADP and l-threonine-O-3-phosphate (l-Thr-P), a required building block for the de novo synthesis of B₁₂ (Fig. 1) (6). By sequence similarity, the PduX enzyme of S. enterica belongs to the galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase (GHMP) family (7), which is a novel family of kinases found in metabolic pathways of small molecules. Although there have been no three-dimensional structures of PduX or other l-Thr kinases reported to date, the crystal structures of several GHMP kinases have been solved (8–22). These structures reveal a unique kinase fold and a novel nucleotide-binding mode that are conserved among members of the GHMP kinase family. Members also contain three common sequence motifs consisting of about 10–15 contiguous amino acids (9, 23). Motif I is at the N-terminal region, and its function is uncertain. Motif II is the most conserved with a typical sequence of Pro-X-X-X-Gly-Leu-X-Ser-Ser-Ala. This region is involved in nucleotide binding and distinguishes the GHMP family from other protein kinase families. Motif III is near the C terminus and contains a small glycine-rich loop that appears well positioned to interact with substrate that is phosphorylated by ATP (24). However, there are highly divergent sequences outside these three motifs, and modes of oligomerization, substrate binding, and catalytic mechanism vary among different GHMP family members (14).Open in a separate windowFIGURE 1.Roles of PduX in B₁₂ synthesis in S. enterica. PduX is required for both the de novo synthesis of AdoCbl and methylcobalamin (MeCbl) and the assimilation of cobyric acid. AdoCbl is required for growth of S. enterica on 1,2-PD and ethanolamine as well for the activity of the MetH methionine synthase that converts homocysteine to methionine. Btu, B₁₂ uptake system; Cbi, cobinamide; AdoCbi, adenosylcobinamide; AdoCby, adenosylcobyric acid; AdoCbi-P, adenosylcobinamide phosphate; AP-P, (R)-1-amino-2-propanol-O-2-phosphate.Based on structural and biochemical data, members of the GHMP family are subdivided into two groups depending on their catalytic mechanisms. In the first group, which includes rat mevalonate kinase, the crystal structure shows an aspartate residue in the active site positioned to act as a catalytic base that abstracts a proton from the substrate hydroxyl group. In addition, there is a lysine residue that is thought to reduce the pK_a of the substrate hydroxyl group and to stabilize the pentacoordinated γ-phosphoryl group of ATP (18). In the second group, such as Escherichia coli homoserine kinase, there appears to be no suitable residue positioned to act as a catalytic base. Consequently it has been hypothesized that homoserine kinase achieves catalysis through transition state stabilization alone (14).In the present study we investigated the catalytic mechanism of PduX by a series of kinetic studies and mutational analyses of 10 invariant serine/threonine and aspartate/glutamate residues. Results indicate a steady-state Ordered Bi Bi mechanism in which ATP binds first. We also identified several amino acids critical to substrate binding and catalysis. To our knowledge, these are the first reported studies on the catalytic mechanism of l-Thr kinase from any organism.     

Regulation and Characterization of a Newly Deduced Cell Wall Hydrolase Gene (cwlJ) Which Affects Germination of Bacillus subtilis Spores

The predicted amino acid sequence of Bacillus subtilis ycbQ (renamed cwlJ) exhibits high similarity to those of the deduced C-terminal catalytic domain of SleBs, the specific cortex-hydrolyzing enzyme of B. cereus and the deduced one of B. subtilis. We constructed a cwlJ::lacZ fusion in the B. subtilis chromosome. The β-galactosidase activity and results of Northern hybridization and primer extension analyses of the cwlJ gene indicated that it is transcribed by Eς^E RNA polymerase. cwlJ-deficient spores responded to both l-alanine and AGFK, the A₅₈₀ values of spore suspensions decreased more slowly than in the case of the wild-type strain, and the mutant spores released less dipicolinic acid than did those of the wild-type strain during germination. However, the mutant spores released only slightly less hexosamine than did the wild-type spores. In contrast, B. subtilis sleB spores did not release hexosamine at a significant level. While cwlJ and sleB spores were able to germinate, CJSB (cwlJ sleB) spores could not germinate but exhibited initial germination reactions, e.g., partial decrease in A₅₈₀ and slow release of dipicolinic acid. CJSB spores became slightly gray after 6 h in the germinant, but their refractility was much greater than that of sleB mutant spores. The roles of the sleB and cwlJ mutations in germination and spore maturation are also discussed.During sporulation and germination of Bacillus subtilis, the action of autolysins is assumed to be required for asymmetric septum peptidoglycan hydrolysis, engulfment, cortex maturation, mother cell lysis, and cortex hydrolysis during germination (28, 33). Mother cell lysis depends on the compensatory effect of cell wall hydrolases CwlB (LytC) and CwlC (11, 13, 34). For cortex maturation, a defect in the cwlD gene leads to a lack of germination and blocking of the formation of muramic acid lactam structure in the cortex (2, 26, 31). Recently, Makino and colleagues reported that the B. cereus sleB gene encodes a 24-kDa mature germination-specific N-acetylmuramoyl-l-alanine amidase which degrades decoated spores from various organisms (18, 22). B. subtilis sleB is homologous to B. cereus sleB, and B. subtilis sleB mutant spores are able to germinate and form colonies. However, B. subtilis SleB showed no activity against degraded decoated spores or other substrates (21).Our work on the B. subtilis genome sequencing project has revealed the ycbQ gene, which is homologous with the cortex-hydrolyzing sleB genes (22, 25). In this study, we describe the regulation and function of the cwlJ (ycbQ) gene and the compensatory effect of the CwlJ and B. subtilis SleB proteins on germination.     

Zif,the Zoocin A Immunity Factor,Is a FemABX-Like Immunity Protein with a Novel Mode of Action

Producer cell immunity to the streptococcolytic enzyme zoocin A, which is a d-alanyl-l-alanine endopeptidase, is due to Zif, the zoocin A immunity factor. Zif has high degrees of similarity to MurM and MurN (members of the FemABX family of proteins), which are responsible for the addition of amino acids to cross bridges during peptidoglycan synthesis in streptococci. In this study, purified peptidoglycans from strains with and without zif were compared to determine how Zif modifies the peptidoglycan layer to cause resistance to zoocin A. The peptidoglycan from each strain was hydrolyzed using the streptococcolytic phage lysin B30, and the resulting muropeptides were separated by reverse-phase high-pressure liquid chromatography, labeled with 4-sulfophenyl isothiocyanate, and analyzed by tandem mass spectrometry in the negative-ion mode. It was determined that Zif alters the peptidoglycan by increasing the proportion of cross bridges containing three l-alanines instead of two. This modification decreased binding of the recombinant target recognition domain of zoocin A to peptidoglycan. Zif-modified peptidoglycan also was less susceptible to hydrolysis by the recombinant catalytic domain of zoocin A. Thus, Zif is a novel FemABX-like immunity factor because it provides resistance to a bacteriolytic endopeptidase by lengthening the peptidoglycan cross bridge rather than by causing an amino acid substitution.During streptococcal peptidoglycan synthesis, monomer subunits are generated inside the cell, with nonribosomal peptidyl transferases responsible for the addition of amino acids onto the epsilon amino group of lysine in the subunits. These nonribosomal peptidyl transferases are part of the FemABX family of proteins, some of which have been implicated in penicillin resistance (5, 26). In Streptococcus pneumoniae peptidoglycan synthesis, MurM attaches either an l-alanine or an l-serine to the epsilon amino group of lysine, and MurN then adds an l-alanine (11, 26).Zoocin A is a d-alanyl-l-alanine endopeptidase produced by Streptococcus equi subsp. zooepidemicus 4881 that hydrolyzes peptidoglycan cross bridges of susceptible streptococci (12). Zoocin A has two functional domains (18). The N-terminal catalytic domain (CAT) has high degrees of similarity to several other bacteriolytic endopeptidases, including the staphylolytic enzyme lysostaphin. The C-terminal target recognition domain (TRD), which facilitates binding of the enzyme to peptidoglycan (1), has very little similarity to any characterized conserved domain.Producer cell immunity to zoocin A is due to zif (zoocin A immunity factor), which is adjacent to zooA on the chromosome and is transcribed divergently (4). Zif has high degrees of similarity to MurM and MurN and also to the lysostaphin resistance protein and other FemABX-like immunity proteins (23). Previously characterized FemABX-like immunity proteins provide resistance to peptidoglycan cross-bridge hydrolases by inserting an amino acid different from those specified by the normal FemABX-like proteins (6, 9, 15, 25), whereas Zif does not (4). It has been shown previously that Zif-specified resistance to zoocin A is an intrinsic characteristic of the peptidoglycan layer (12). Therefore, Zif must modify the peptidoglycan layer in a novel way that provides resistance to zoocin A. In the present study, Zif was shown to insert an additional l-alanine into the peptidoglycan cross bridges, which inhibited both binding of the zoocin A TRD and the ability of the zoocin A CAT to hydrolyze the cross bridge.     

The Elucidation of the Structure of Thermotoga maritima Peptidoglycan Reveals Two Novel Types of Cross-link

Thermotoga maritima is a Gram-negative, hyperthermophilic bacterium whose peptidoglycan contains comparable amounts of l- and d-lysine. We have determined the fine structure of this cell-wall polymer. The muropeptides resulting from the digestion of peptidoglycan by mutanolysin were separated by high-performance liquid chromatography and identified by amino acid analysis after acid hydrolysis, dinitrophenylation, enzymatic determination of the configuration of the chiral amino acids, and mass spectrometry. The high-performance liquid chromatography profile contained four main peaks, two monomers, and two dimers, plus a few minor peaks corresponding to anhydro forms. The first monomer was the d-lysine-containing disaccharide-tripeptide in which the d-Glu-d-Lys bond had the unusual γ→ϵ arrangement (GlcNAc-MurNAc-l-Ala-γ-d-Glu-ϵ-d-Lys). The second monomer was the conventional disaccharide-tetrapeptide (GlcNAc-MurNAc-l-Ala-γ-d-Glu-l-Lys-d-Ala). The first dimer contained a disaccharide-l-Ala as the acyl donor cross-linked to the α-amine of d-Lys in a tripeptide acceptor stem with the sequence of the first monomer. In the second dimer, donor and acceptor stems with the sequences of the second and first monomers, respectively, were connected by a d-Ala⁴-α-d-Lys³ cross-link. The cross-linking index was 10 with an average chain length of 30 disaccharide units. The structure of the peptidoglycan of T. maritima revealed for the first time the key role of d-Lys in peptidoglycan synthesis, both as a surrogate of l-Lys or meso-diaminopimelic acid at the third position of peptide stems and in the formation of novel cross-links of the l-Ala¹(α→α)d-Lys³ and d-Ala⁴(α→α)d-Lys³ types.Peptidoglycan (or murein) is a giant macromolecule whose main function is the protection of the cytoplasmic membrane against the internal osmotic pressure. It is composed of alternating residues of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc)² cross-linked by short peptides (1). The composition of the peptide stem in nascent peptidoglycan is l-Ala¹-γ-d-Glu²-X³-d-Ala⁴-d-Ala⁵, where X is most often meso-diaminopimelic acid (meso-A₂pm) or l-lysine in Gram-negative and Gram-positive species, respectively (2, 3). In the mature macromolecule, the last d-Ala residue is removed. Cross-linking of the glycan chains generally occurs between the carboxyl group of d-Ala at position 4 of a donor peptide stem and the side-chain amino group of the diamino acid at position 3 of an acceptor peptide stem (4→3 cross-links). Cross-linking is either direct or through a short peptide bridge such as pentaglycine in Staphylococcus aureus (2, 3). The enzymes for the formation of the 4→3 cross-links are active-site serine dd- transpeptidases that belong to the penicillin-binding protein (PBP) family and are the essential targets of β-lactam antibiotics in pathogenic bacteria (4). Catalysis involves the cleavage of the d-Ala⁴-d-Ala⁵ bond of a donor peptide stem and the formation of an amide bond between the carboxyl of d-Ala⁴ and the side chain amine at the third position of an acceptor stem. Transpeptidases of the ld specificity are active-site cysteine enzymes that were shown to act as surrogates of the PBPs in mutants of Enterococcus faecium resistant to β-lactam antibiotics (5). They cleave the X³-d-Ala⁴ bond of a donor stem peptide to form 3→3 cross-links. This alternate mode of cross-linking is usually marginal, although it has recently been shown to predominate in non-replicative “dormant” forms of Mycobacterium tuberculosis (6).Thermotoga maritima is a Gram-negative, extremely thermophilic bacterium isolated from geothermally heated sea floors by Huber et al. (7). A morphological characteristic is the presence of an outer sheath-like envelope called “toga.” Although the organism has received considerable attention for its biotechnological potential, studies about its peptidoglycan are scarce (8–11), and in particular the fine structure of the macromolecule is still unknown. In their initial work, Huber et al. (7) showed that the composition of its peptidoglycan was unusual for a Gram-negative species, because it contained both isomers of lysine and no A₂pm. Recently, we purified and studied the properties of T. maritima MurE (12); this enzyme is responsible for the addition of the amino acid residue at position 3 of the peptide stem (13, 14). We demonstrated that T. maritima MurE added in vitro l- and d-Lys to UDP-MurNAc-l-Ala-d-Glu. Although l-Lys was added in the usual way, yielding the conventional nucleotide UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys containing a d-Glu(γ→α)l-Lys amide bond, the d-isomer was added in an “upside-down” manner, yielding the novel nucleotide UDP-MurNAc-l-Ala-d-Glu(γ→ϵ)d-Lys. We also showed that the d-Lys-containing nucleotide was not a substrate for T. maritima MurF, the subsequent enzyme in the biosynthetic pathway, whereas this ligase catalyzed the addition of dipeptide d-Ala-d-Ala to the l-Lys-containing tripeptide, yielding the conventional UDP-MurNAc-pentapeptide (12).However, both the l-Lys-containing UDP-MurNAc-pentapeptide and d-Lys-containing UDP-MurNAc-tripeptide were used as substrates by T. maritima MraY with comparable efficiencies in vitro (12). This observation implies that the unusual d-Lys-containing peptide stems are likely to be translocated to the periplasmic face of the cytoplasmic membrane and to participate in peptidoglycan polymerization. Therefore, we have determined here the fine structure of T. maritima peptidoglycan and we have shown that l-Lys- and d-Lys-containing peptide stems are both present in the polymer, the latter being involved in the formation of two novel types of peptidoglycan cross-link.     

Two d-2-Hydroxy-acid Dehydrogenases in Arabidopsis thaliana with Catalytic Capacities to Participate in the Last Reactions of the Methylglyoxal and ��-Oxidation Pathways

The Arabidopsis thaliana locus At5g06580 encodes an ortholog to Saccharomyces cerevisiae d-lactate dehydrogenase (AtD-LDH). The recombinant protein is a homodimer of 59-kDa subunits with one FAD per monomer. A substrate screen indicated that AtD-LDH catalyzes the oxidation of d- and l-lactate, d-2-hydroxybutyrate, glycerate, and glycolate using cytochrome c as an electron acceptor. AtD-LDH shows a clear preference for d-lactate, with a catalytic efficiency 200- and 2000-fold higher than that for l-lactate and glycolate, respectively, and a K_m value for d-lactate of ∼160 μm. Knock-out mutants showed impaired growth in the presence of d-lactate or methylglyoxal. Collectively, the data indicated that the protein is a d-LDH that participates in planta in the methylglyoxal pathway. Web-based bioinformatic tools revealed the existence of a paralogous protein encoded by locus At4g36400. The recombinant protein is a homodimer of 61-kDa subunits with one FAD per monomer. A substrate screening revealed highly specific d-2-hydroxyglutarate (d-2HG) conversion in the presence of an organic cofactor with a K_m value of ∼580 μm. Thus, the enzyme was characterized as a d-2HG dehydrogenase (AtD-2HGDH). Analysis of knock-out mutants demonstrated that AtD-2HGDH is responsible for the total d-2HGDH activity present in A. thaliana. Gene coexpression analysis indicated that AtD-2HGDH is in the same network as several genes involved in β-oxidation and degradation of branched-chain amino acids and chlorophyll. It is proposed that AtD-2HGDH participates in the catabolism of d-2HG most probably during the mobilization of alternative substrates from proteolysis and/or lipid degradation.l- and d-lactate dehydrogenases belong to evolutionarily unrelated enzyme families (1). l-Lactate is oxidized by l-lactate:NAD oxidoreductase (EC 1.1.1.27), which catalyzes the reaction l-lactate + NAD → pyruvate + NADH, and by l-lactate cytochrome c oxidoreductase (l-lactate cytochrome c oxidoreductase, EC 1.1.2.3), which catalyzes the reaction l-lactate + 2 cytochrome c (oxidized) → pyruvate + 2 cytochrome c (reduced). Both groups are found in eubacteria, archebacteria, and eukaryotes. All known plant sequences belong to the EC 1.1.1.27 group (1). On the other hand, d-lactate is oxidized by d-lactate:NAD oxidoreductase (d-lactate:NAD oxidoreductase, EC 1.1.1.28), which catalyzes the reaction d-lactate + NAD → pyruvate + NADH, and by d-lactate cytochrome c oxidoreductase (d-lactate cytochrome c oxidoreductase, EC 1.1.2.4), which catalyzes the reaction d-lactate + 2 cytochrome c (oxidized) → pyruvate + 2 cytochrome c (reduced).Although l-lactate dehydrogenase belongs to the most intensely studied enzyme families (2, 3), our knowledge about the structure, kinetics, and biological function of d-LDH³ is limited. d-LDHs have mainly been identified in prokaryotes and fungi where they play an important role in anaerobic energy metabolism (4–10). In Saccharomyces cerevisiae and Kluyveromyces lactis, a mitochondrial flavoprotein d-lactate ferricytochrome c oxidoreductase (d-lactate cytochrome c oxidoreductase), catalyzing the oxidation of d-lactate to pyruvate, is required for the utilization of d-lactate (8, 11). In S. cerevisiae it was suggested that d-LDH is involved in the metabolism of methylglyoxal (MG) (12).In eukaryotic cells, d-lactate results from the glyoxalase system (13, 14). This system is the main MG catabolic pathway, comprising the enzymes glyoxalase I (lactoylglutathione lyase, EC 4.4.1.5) and glyoxalase II (hydroxyacylglutathione hydrolase, EC 3.1.2.6). MG (CH₃-CO-CHO; see structure in Fig. 4) is a cytotoxic compound formed primarily as a by-product of glycolysis through nonenzymatic phosphate elimination from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (15), and its production in various plants is enhanced under stress conditions such as salt, drought, cold, and heavy metal stress (16, 17). Moreover, the overexpression of glyoxalase I or II was shown to confer resistance to salt stress in tobacco and rice (17, 18). It is assumed that the role of the MG pathway, from MG synthase to d-lactate cytochrome c oxidoreductase in the extant metabolism, is to detoxify MG, whereas in the early state of metabolic development it might function as an anaplerotic route for the tricarboxylic acid cycle (15).Open in a separate windowFIGURE 4.Scheme showing the involvement of AtD-LDH in the methylglyoxal pathway and of AtD-2HGDH in the respiration of substrates from proteolysis and/or lipid degradation. d-Lactate resulting from the glyoxalase system is converted to pyruvate by AtD-LDH. The electrons originated may be transferred to the respiratory chain through cytochrome c in the intermembrane space. d-2-HG produced in the peroxisomes (as shown in supplemental Fig. S3) is transported to the mitochondria and converted to 2-ketoglutarate by AtD-2HGDH. Electrons are donated to the electron transport chain through the ETF/ETFQO system. Dotted files represent possible transport processes. 2-KG, 2-ketoglutarate. CIII, complex III. CIV, complex IV. e⁻, electron. ETF, electron transfer protein. ETFQO, ETF-ubiquinone oxidoreductase. GSH, glutathione. Pyr, pyruvate. TCA cycle, tricarboxylic acid cycle; UQ, ubiquinone.Glyoxalase I catalyzes the formation of S-d-lactoylglutathione from the hemithioacetal formed nonenzymatically from MG and glutathione, although glyoxalase II catalyzes the hydrolysis of S-d-lactoylglutathione to regenerate glutathione and liberate d-lactate. Glyoxalase I and II activities are present in all tissues of eukaryotic organisms. Glyoxalase I is found in the cytosol, whereas glyoxalase II localizes to the cytosol and mitochondria (13, 19, 20). Although glyoxalase I and II were extensively characterized, there are only few reports on the characterization of d-LDH. Recently, Atlante et al. (13) showed that externally added d-lactate caused oxygen consumption by mitochondria and that this metabolite was oxidized by a mitochondrial flavoprotein in Helianthus tuberosus.The complete sequence of Arabidopsis thaliana opened the way to search for genes encoding d-LDHs. Based on similarity with the d-LDH from S. cerevisiae (DLD1), an A. thaliana ortholog was identified. In this study, the isolation and structural and biochemical characterization of the recombinant mature d-LDH from A. thaliana (AtD-LDH) and its paralog, which was found to be a d-2-hydroxyglutarate dehydrogenase (AtD-2HGDH), is described. Whereas AtD-LDH has a narrow substrate specificity and the preferred substrates are d-lactate and d-2-hydroxybutyrate, AtD-2HGDH showed activity exclusively with d-2-hydroxyglutarate. Based on gene coexpression analysis and analysis of corresponding knock-out mutants, the participation of these previously unrecognized mitochondrial activities in plant metabolism is discussed.     

Crystal Structure of Bacillus anthracis Transpeptidase Enzyme CapD

Bacillus anthracis elaborates a poly-γ-d-glutamic acid capsule that protects bacilli from phagocytic killing during infection. The enzyme CapD generates amide bonds with peptidoglycan cross-bridges to anchor capsular material within the cell wall envelope of B. anthracis. The capsular biosynthetic pathway is essential for virulence during anthrax infections and can be targeted for anti-infective inhibition with small molecules. Here, we present the crystal structures of the γ-glutamyltranspeptidase CapD with and without α-l-Glu-l-Glu dipeptide, a non-hydrolyzable analog of poly-γ-d-glutamic acid, in the active site. Purified CapD displays transpeptidation activity in vitro, and its structure reveals an active site broadly accessible for poly-γ-glutamate binding and processing. Using structural and biochemical information, we derive a mechanistic model for CapD catalysis whereby Pro⁴²⁷, Gly⁴²⁸, and Gly⁴²⁹ activate the catalytic residue of the enzyme, Thr³⁵², and stabilize an oxyanion hole via main chain amide hydrogen bonds.Spores of Bacillus anthracis are the causative agents of anthrax disease (1). Upon entry into their hosts, spores germinate and replicate as vegetative bacilli (1). The formation of a thick capsule encasing vegetative forms enables bacilli to escape granulocyte0 and macrophage-mediated phagocytosis, and the pathogen eventually disseminates throughout all tissues of an infected host (2, 3). Bacilli secrete lethal and edema toxins, which cause macrophage necrosis and precipitate anthrax death (4–7). The genes providing for toxin and capsule formation are carried on two large virulence plasmids, pXO1 and pXO2, respectively (8, 9). Loss of any one plasmid leads to virulence attenuation, a feature that has been exploited for the generation of vaccine-type strains (10–14).Unlike polysaccharide-based capsules that are commonly found in bacterial pathogens, the capsular material of B. anthracis is composed of poly-γ-d-glutamic acid (PDGA)³ (3). All the genes necessary for capsule biogenesis are located in the capBCADE gene cluster on plasmid pXO2 (15–19). CapD is the only protein of this cluster that is located on the bacterial surface (16). CapD shares sequence similarity with bacterial and mammalian γ-glutamyl transpeptidases (GGTs; EC 2.3.2.2) (17). GGTs belong to the N-terminal nucleophile hydrolases (Ntn) family (Protein Structure Classification (Class (C), Architecture (A), Topology (T) and Homologous superfamily (H)) (CATH) id 3.60.60.10) (20). These enzymes assemble as a single polypeptide chain and acquire activity by undergoing autocatalytic processing to heterodimer.Bacterial GGTs catalyze the first step in glutathione degradation. For example, Helicobacter pylori GGT removes glutamate from glutathione tripeptide via the formation of a γ-glutamyl acyl enzyme. This intermediate is resolved by the nucleophilic attack of a water molecule, causing the release of γ-glutamate (21, 22). Mammalian enzymes transfer the γ-glutamyl intermediate to the amino group of a peptide, thereby completing a transpeptidation reaction (23). The B. anthracis CapD precursor is also programmed for autocatalytic cleavage (17). Similar to mammalian GGTs, CapD also catalyzes a transpeptidation reaction; however, this reaction promotes the covalent linkage of PDGA to the bacterial envelope (16, 24). We have recently demonstrated the cell wall anchor structure of capsule filaments in the envelope of B. anthracis, identifying an amide bond between the terminal carboxyl group of PDGA and the side amino group of m-diaminopimelic acid cross-bridges within muropeptides (24). The CapD-catalyzed transpeptidation reaction could be recapitulated in vitro using purified recombinant CapD, γ-d-Glu_n peptide, and muropeptide substrates (24). In the absence of the physiological nucleophile (muropeptides), CapD acyl intermediates can be resolved by the nucleophilic attack of water to generate hydrolysis products.Here, we report the high resolution crystal structure of CapD in the absence and presence of a glutamate dipeptide and compare it with the known structures of H. pylori and Escherichia coli GGTs. By combining structural, genetic, and biochemical approaches, we identify the unique features of CapD that distinguish the protein from GGTs and detect several residues that are important for CapD autocatalytic cleavage and PDGA processing. This structural information will further the development of small molecule inhibitors that disrupt CapD activity and that may be useful as anti-infective therapies for anthrax.     

Studies of the Commitment Step in the Germination of Spores of Bacillus Species

Spores of Bacillus species are said to be committed when they continue through nutrient germination even when germinants are removed or their binding to spores'' nutrient germinant receptors (GRs) is both reversed and inhibited. Measurement of commitment and the subsequent release of dipicolinic acid (DPA) during nutrient germination of spores of Bacillus cereus and Bacillus subtilis showed that heat activation, increased nutrient germinant concentrations, and higher average levels of GRs/spore significantly decreased the times needed for commitment, as well as lag times between commitment and DPA release. These lag times were also decreased dramatically by the action of one of the spores'' two redundant cortex lytic enzymes (CLEs), CwlJ, but not by the other CLE, SleB, and CwlJ action did not affect the timing of commitment. The timing of commitment and the lag time between commitment and DPA release were also dependent on the specific GR activated to cause spore germination. For spore populations, the lag times between commitment and DPA release were increased significantly in spores that germinated late compared to those that germinated early, and individual spores that germinated late may have had lower appropriate GR levels/spore than spores that germinated early. These findings together provide new insight into the commitment step in spore germination and suggest several factors that may contribute to the large heterogeneity among the timings of various events in the germination of individual spores in spore populations.Spores of Bacillus species can remain dormant for long times and are extremely resistant to a variety of environmental stresses (26). However, under appropriate conditions, normally upon the binding of specific nutrients to spores'' nutrient germinant receptors (GRs), spores can come back to active growth through a process called germination followed by outgrowth (19, 20, 25, 26). Germination of Bacillus subtilis spores can be triggered by l-alanine or l-valine or a combination of l-asparagine, d-glucose, d-fructose, and K⁺ (AGFK). These nutrient germinants trigger germination by binding to and interacting with GRs that have been localized to the spore''s inner membrane (12, 20). l-Alanine and l-valine bind to the GerA GR, while the AGFK mixture triggers germination by interacting with both the GerB and GerK GRs (25). Normally, l-asparagine alone does not trigger B. subtilis spore germination. However, a mutant form of the GerB GR, termed GerB*, displays altered germinant specificity such that l-asparagine alone will trigger the germination of gerB* mutant spores (1, 18).A number of events occur in a defined sequence during spore germination. Initially, exposure of spores to nutrient germinants causes a reaction that commits spores to germinate, even if the germinant is removed or displaced from its cognate GR (7, 10, 21, 27, 28). This commitment step is followed by release of monovalent cations, as well as the spore core''s large pool of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) along with divalent cations, predominantly Ca²⁺, that are chelated with DPA (Ca-DPA). In Bacillus spores, the release of Ca-DPA triggers the hydrolysis of spores'' peptidoglycan cortex by either of two cortex lytic enzymes (CLEs), CwlJ and SleB (11, 16, 23). CwlJ is activated during germination by Ca-DPA as it is being released from individual spores, while SleB activation requires that most Ca-DPA be released (14, 16, 17). Cortex hydrolysis, in turn, allows the spore core to expand and fully hydrate, which leads to activation of enzymes and initiation of metabolism in the spore core (21, 25).As noted above, commitment is the first event that can be assessed during spore germination, although the precise mechanism of commitment is not known. Since much has been learned about proteins important in spore germination in the many years since commitment was last studied (25, 26), it seemed worth reexamining commitment, with the goal of determining those factors that influence this step in the germination process. Knowledge of factors important in determining kinetics of commitment could then lead to an understanding of what is involved in this reaction.Kinetic analysis of spore germination, as well as commitment, has mostly been based on the decrease in optical density at 600 nm (OD₆₀₀) of spore suspensions, which monitors a combination of events that occur well after commitment, including DPA release, cortex hydrolysis, and core swelling (25-27). In the current work, we have used a germination assay that measures DPA release, an early event in spore germination, and have automated this assay to allow routine measurement of commitment, as well as DPA release from large numbers of spore samples simultaneously. This assay has allowed comparison of the kinetics of DPA release and commitment during germination and study of the effects of heat activation, germinant concentration, GR levels, and CLEs on commitment.