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Georges Rapoport - One of the best experts on this subject based on the ideXlab platform.
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ClpP of Bacillus subtilis is required for competence development, motility, Degradative Enzyme synthesis, growth at high temperature and sporulation
Molecular Microbiology, 2002Co-Authors: Tarek Msadek, Frank Kunst, Véronique Dartois, Marie‐laure Herbaud, François Denizot, Georges RapoportAbstract:The nucleotide sequence of the Bacillus subtilis clpP gene was determined. The predicted protein shows very high similarity to members of the ClpP family of proteolytic subunits (68% amino acid sequence identity with that of Escherichia coli). We show that ClpP plays an essential role in stationary phase adaptive responses. Indeed, a delta clpP mutant was constructed and shown to display a pleiotropic phenotype, including a deficiency in both sporulation initiation and competence for DNA uptake. The delta clpP mutant has a highly filamentous morphology and appears to be non-motile, as judged by swarm plate assays. Expression of clpP is strongly induced under heat shock conditions, and ClpP is shown to be essential for growth of B. subtilis at high temperature. The role of ClpP in the sporulation and competence regulatory pathways was investigated. ClpP is required for expression of the spollA and spollG operons, encoding the sigmaF and sigmaE sporulation-specific sigma factors. ClpP is also necessary for the expression of the comK gene, encoding a positive transcriptional regulator of competence genes. ComK-dependent transcription of sacB, encoding the exocellular Degradative Enzyme levansucrase, was found to be abolished in the delta clpP mutant. MecA has been characterized previously as a negative regulator of comK expression, whose overproduction inhibits both sporulation and competence development. Expression of a mecA'-'lacZ translational fusion is shown to be increased in the delta clpP mutant. We suggest that ClpP is involved in controlling MecA levels in the cell through proteolysis. Increased levels of MecA in the absence of ClpP are at least partly responsible for the observed pleiotropic phenotype of the delta clpP mutant.
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clpp of bacillus subtilis is required for competence development motility Degradative Enzyme synthesis growth at high temperature and sporulation
Molecular Microbiology, 1998Co-Authors: Tarek Msadek, Frank Kunst, Véronique Dartois, Marie‐laure Herbaud, François Denizot, Georges RapoportAbstract:The nucleotide sequence of the Bacillus subtilis clpP gene was determined. The predicted protein shows very high similarity to members of the ClpP family of proteolytic subunits (68% amino acid sequence identity with that of Escherichia coli ). We show that ClpP plays an essential role in stationary phase adaptive responses. Indeed, a ΔclpP mutant was constructed and shown to display a pleiotropic phenotype, including a deficiency in both sporulation initiation and competence for DNA uptake. The ΔclpP mutant has a highly filamentous morphology and appears to be non-motile, as judged by swarm plate assays. Expression of clpP is strongly induced under heat shock conditions, and ClpP is shown to be essential for growth of B. subtilis at high temperature. The role of ClpP in the sporulation and competence regulatory pathways was investigated. ClpP is required for expression of the spoIIA and spoIIG operons, encoding the σF andσE sporulation-specific sigma factors. ClpP is also necessary for the expression of the comK gene, encoding a positive transcriptional regulator of competence genes. ComK-dependent transcription of sacB, encoding the exocellular Degradative Enzyme levansucrase, was found to be abolished in the ΔclpP mutant. MecA has been characterized previously as a negative regulator of comK expression, whose overproduction inhibits both sporulation and competence development. Expression of a mecA′–′lacZ translational fusion is shown to be increased in the ΔclpP mutant. We suggest that ClpP is involved in controlling MecA levels in the cell through proteolysis. Increased levels of MecA in the absence of ClpP are at least partly responsible for the observed pleiotropic phenotype of the ΔclpP mutant.
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salt stress is an environmental signal affecting Degradative Enzyme synthesis in bacillus subtilis
Journal of Bacteriology, 1995Co-Authors: Frank Kunst, Georges RapoportAbstract:Growth under conditions of salt stress has important effects on the synthesis of Degradative Enzymes in Bacillus subtilis. Salt stress strongly stimulates the expression of sacB, encoding levansucrase (about ninefold), and downregulates the expression of aprE, encoding alkaline protease (about sixfold). It is suggested that the DegS-DegU two-component system is involved in sensing salt stress. Moreover, it has been shown that the level of sacB expression strongly depends on the growth conditions; its expression level is about eightfold higher in cells grown on agar plates than in cells grown in liquid medium.
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a signal transduction network in bacillus subtilis includes the degs degu and comp coma two component systems
1995Co-Authors: Tarek Msadek, Frank Kunst, Georges RapoportAbstract:Soil bacteria such as Bacillus subtilis are subject to drastic variations of environmental conditions such as temperature, humidity, and nutrient source availability. At the onset of the stationary phase, faced with a depletion of essential nutrients, B. subtilis can adopt several responses, including synthesis of macromolecule-degrading Enzymes, competence for genetic transformation, increased motility and chemotaxis, antibiotic production, and finally, sporulation. Regulation by the B. subtilis two-component systems presents several original features. Some of these original features are discussed, within the framework of the DegS/DegU and ComP/ComA signal transduction network. The chapter describes Degradative Enzyme synthesis. Sequence similarities with two-component systems suggest the conserved His-189 residue of the DegS protein kinase and Asp-56 residue of the DegU response regulator as likely candidates for the respective phosphorylation sites of the two proteins. The chapter discusses competence gene expression, and signal transduction network. Both the ComP/ComA and DegS/DegU two-component systems control the expression of late competence genes; however, they seem to act through two different branches in the competence regulatory pathway that intersect to allow expression of comK. An exciting area of future research will be to identify the types of signals involved in regulation by each of these two-component systems and by the other regulators such as MecB/MecA and the ComQ-ComX-Spo0K pathway, as well as determining how these regulators interact within the signal transduction network.
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the phosphorylation state of the degu response regulator acts as a molecular switch allowing either Degradative Enzyme synthesis or expression of genetic competence in bacillus subtilis
Journal of Biological Chemistry, 1992Co-Authors: M K Dahl, Tarek Msadek, Frank Kunst, Georges RapoportAbstract:Abstract Two classes of mutations were identified in the degS and degU regulatory genes of Bacillus subtilis, leading either to deficiency of Degradative Enzyme synthesis (degS or degU mutations) or to a pleiotropic phenotype which includes overproduction of Degradative Enzymes and the loss of genetic competence (degS(Hy) or degU(Hy) mutations). We have shown previously that the DegS protein kinase and the DegU response regulator form a signal transduction system in B. subtilis. We now demonstrate that the DegS protein kinase also acts as a DegU phosphatase. We present evidence that the DegU response regulator has two active conformations: a phosphorylated form which is necessary for Degradative Enzyme synthesis and a nonphosphorylated form required for expression of genetic competence. The degU146-encoded response regulator, allowing expression of genetic competence, has been purified and seems to be modified within the putative phosphorylation site (D56----N) since it is no longer phosphorylated by DegS. Both the degU146 mutation as well as the degS220 mutation, which essentially abolishes DegS protein kinase activity, lead to deficiency of Degradative Enzyme synthesis, indicating the requirement of phosphorylated DegU for the expression of this phenotype. We also purified the degU32(Hy)-encoded protein and showed that this response regulator is phosphorylated by the DegS protein kinase in vitro. In addition, the phosphorylated form of the degU32(Hy)-encoded protein presented a strongly increased stability as compared with the wild type DegU protein, thus leading to hyperproduction of Degradative Enzymes in vivo.
Frank Kunst - One of the best experts on this subject based on the ideXlab platform.
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ClpP of Bacillus subtilis is required for competence development, motility, Degradative Enzyme synthesis, growth at high temperature and sporulation
Molecular Microbiology, 2002Co-Authors: Tarek Msadek, Frank Kunst, Véronique Dartois, Marie‐laure Herbaud, François Denizot, Georges RapoportAbstract:The nucleotide sequence of the Bacillus subtilis clpP gene was determined. The predicted protein shows very high similarity to members of the ClpP family of proteolytic subunits (68% amino acid sequence identity with that of Escherichia coli). We show that ClpP plays an essential role in stationary phase adaptive responses. Indeed, a delta clpP mutant was constructed and shown to display a pleiotropic phenotype, including a deficiency in both sporulation initiation and competence for DNA uptake. The delta clpP mutant has a highly filamentous morphology and appears to be non-motile, as judged by swarm plate assays. Expression of clpP is strongly induced under heat shock conditions, and ClpP is shown to be essential for growth of B. subtilis at high temperature. The role of ClpP in the sporulation and competence regulatory pathways was investigated. ClpP is required for expression of the spollA and spollG operons, encoding the sigmaF and sigmaE sporulation-specific sigma factors. ClpP is also necessary for the expression of the comK gene, encoding a positive transcriptional regulator of competence genes. ComK-dependent transcription of sacB, encoding the exocellular Degradative Enzyme levansucrase, was found to be abolished in the delta clpP mutant. MecA has been characterized previously as a negative regulator of comK expression, whose overproduction inhibits both sporulation and competence development. Expression of a mecA'-'lacZ translational fusion is shown to be increased in the delta clpP mutant. We suggest that ClpP is involved in controlling MecA levels in the cell through proteolysis. Increased levels of MecA in the absence of ClpP are at least partly responsible for the observed pleiotropic phenotype of the delta clpP mutant.
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clpp of bacillus subtilis is required for competence development motility Degradative Enzyme synthesis growth at high temperature and sporulation
Molecular Microbiology, 1998Co-Authors: Tarek Msadek, Frank Kunst, Véronique Dartois, Marie‐laure Herbaud, François Denizot, Georges RapoportAbstract:The nucleotide sequence of the Bacillus subtilis clpP gene was determined. The predicted protein shows very high similarity to members of the ClpP family of proteolytic subunits (68% amino acid sequence identity with that of Escherichia coli ). We show that ClpP plays an essential role in stationary phase adaptive responses. Indeed, a ΔclpP mutant was constructed and shown to display a pleiotropic phenotype, including a deficiency in both sporulation initiation and competence for DNA uptake. The ΔclpP mutant has a highly filamentous morphology and appears to be non-motile, as judged by swarm plate assays. Expression of clpP is strongly induced under heat shock conditions, and ClpP is shown to be essential for growth of B. subtilis at high temperature. The role of ClpP in the sporulation and competence regulatory pathways was investigated. ClpP is required for expression of the spoIIA and spoIIG operons, encoding the σF andσE sporulation-specific sigma factors. ClpP is also necessary for the expression of the comK gene, encoding a positive transcriptional regulator of competence genes. ComK-dependent transcription of sacB, encoding the exocellular Degradative Enzyme levansucrase, was found to be abolished in the ΔclpP mutant. MecA has been characterized previously as a negative regulator of comK expression, whose overproduction inhibits both sporulation and competence development. Expression of a mecA′–′lacZ translational fusion is shown to be increased in the ΔclpP mutant. We suggest that ClpP is involved in controlling MecA levels in the cell through proteolysis. Increased levels of MecA in the absence of ClpP are at least partly responsible for the observed pleiotropic phenotype of the ΔclpP mutant.
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salt stress is an environmental signal affecting Degradative Enzyme synthesis in bacillus subtilis
Journal of Bacteriology, 1995Co-Authors: Frank Kunst, Georges RapoportAbstract:Growth under conditions of salt stress has important effects on the synthesis of Degradative Enzymes in Bacillus subtilis. Salt stress strongly stimulates the expression of sacB, encoding levansucrase (about ninefold), and downregulates the expression of aprE, encoding alkaline protease (about sixfold). It is suggested that the DegS-DegU two-component system is involved in sensing salt stress. Moreover, it has been shown that the level of sacB expression strongly depends on the growth conditions; its expression level is about eightfold higher in cells grown on agar plates than in cells grown in liquid medium.
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a signal transduction network in bacillus subtilis includes the degs degu and comp coma two component systems
1995Co-Authors: Tarek Msadek, Frank Kunst, Georges RapoportAbstract:Soil bacteria such as Bacillus subtilis are subject to drastic variations of environmental conditions such as temperature, humidity, and nutrient source availability. At the onset of the stationary phase, faced with a depletion of essential nutrients, B. subtilis can adopt several responses, including synthesis of macromolecule-degrading Enzymes, competence for genetic transformation, increased motility and chemotaxis, antibiotic production, and finally, sporulation. Regulation by the B. subtilis two-component systems presents several original features. Some of these original features are discussed, within the framework of the DegS/DegU and ComP/ComA signal transduction network. The chapter describes Degradative Enzyme synthesis. Sequence similarities with two-component systems suggest the conserved His-189 residue of the DegS protein kinase and Asp-56 residue of the DegU response regulator as likely candidates for the respective phosphorylation sites of the two proteins. The chapter discusses competence gene expression, and signal transduction network. Both the ComP/ComA and DegS/DegU two-component systems control the expression of late competence genes; however, they seem to act through two different branches in the competence regulatory pathway that intersect to allow expression of comK. An exciting area of future research will be to identify the types of signals involved in regulation by each of these two-component systems and by the other regulators such as MecB/MecA and the ComQ-ComX-Spo0K pathway, as well as determining how these regulators interact within the signal transduction network.
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the phosphorylation state of the degu response regulator acts as a molecular switch allowing either Degradative Enzyme synthesis or expression of genetic competence in bacillus subtilis
Journal of Biological Chemistry, 1992Co-Authors: M K Dahl, Tarek Msadek, Frank Kunst, Georges RapoportAbstract:Abstract Two classes of mutations were identified in the degS and degU regulatory genes of Bacillus subtilis, leading either to deficiency of Degradative Enzyme synthesis (degS or degU mutations) or to a pleiotropic phenotype which includes overproduction of Degradative Enzymes and the loss of genetic competence (degS(Hy) or degU(Hy) mutations). We have shown previously that the DegS protein kinase and the DegU response regulator form a signal transduction system in B. subtilis. We now demonstrate that the DegS protein kinase also acts as a DegU phosphatase. We present evidence that the DegU response regulator has two active conformations: a phosphorylated form which is necessary for Degradative Enzyme synthesis and a nonphosphorylated form required for expression of genetic competence. The degU146-encoded response regulator, allowing expression of genetic competence, has been purified and seems to be modified within the putative phosphorylation site (D56----N) since it is no longer phosphorylated by DegS. Both the degU146 mutation as well as the degS220 mutation, which essentially abolishes DegS protein kinase activity, lead to deficiency of Degradative Enzyme synthesis, indicating the requirement of phosphorylated DegU for the expression of this phenotype. We also purified the degU32(Hy)-encoded protein and showed that this response regulator is phosphorylated by the DegS protein kinase in vitro. In addition, the phosphorylated form of the degU32(Hy)-encoded protein presented a strongly increased stability as compared with the wild type DegU protein, thus leading to hyperproduction of Degradative Enzymes in vivo.
Tarek Msadek - One of the best experts on this subject based on the ideXlab platform.
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the degu orphan response regulator of listeria monocytogenes autorepresses its own synthesis and is required for bacterial motility virulence and biofilm formation
Microbiology, 2008Co-Authors: Ibtissem Gueriri, Sarah Dubrac, Camille Cyncynatus, Alejandro Toledo Arana, Olivier Dussurget, Tarek MsadekAbstract:The Gram-positive intracellular pathogen Listeria monocytogenes is endowed with 17 sets of genes encoding two-component systems. L. monocytogenes is closely related to the Gram-positive model bacterium Bacillus subtilis, in which we have shown previously that the DegS/DegU system plays a central role in controlling stationary phase adaptive responses, including Degradative Enzyme synthesis and competence. Although an orthologue of the DegU response regulator is present in L. monocytogenes, the gene encoding the cognate DegS kinase is conspicuously absent. We have inactivated the degU gene of L. monocytogenes and shown that DegU negatively regulates its own synthesis. Direct binding of L. monocytogenes DegU to its own promoter region was shown in vitro by gel mobility shift and DNase I footprinting experiments. DegU was also shown to bind upstream from the motB operon, which also encodes the GmaR anti-repressor of flagellar synthesis. In contrast to the situation in B. subtilis, DegU was shown to be essential for flagellar synthesis and bacterial motility in L. monocytogenes and is cotranscribed with the yviA gene located downstream. We also show that DegU is required for growth at high temperatures, adherence to plastic surfaces and the formation of efficient biofilms by L. monocytogenes. DegU plays a role in virulence of L. monocytogenes as well: in a murine intravenous infection model, an 11-fold increase in LD50 was observed for the degU mutant. Taken together, our results indicate that despite the lack of the DegS kinase, DegU is fully functional as an orphan response regulator, and plays a central role in controlling several crucial adaptive responses in L. monocytogenes.
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ClpP of Bacillus subtilis is required for competence development, motility, Degradative Enzyme synthesis, growth at high temperature and sporulation
Molecular Microbiology, 2002Co-Authors: Tarek Msadek, Frank Kunst, Véronique Dartois, Marie‐laure Herbaud, François Denizot, Georges RapoportAbstract:The nucleotide sequence of the Bacillus subtilis clpP gene was determined. The predicted protein shows very high similarity to members of the ClpP family of proteolytic subunits (68% amino acid sequence identity with that of Escherichia coli). We show that ClpP plays an essential role in stationary phase adaptive responses. Indeed, a delta clpP mutant was constructed and shown to display a pleiotropic phenotype, including a deficiency in both sporulation initiation and competence for DNA uptake. The delta clpP mutant has a highly filamentous morphology and appears to be non-motile, as judged by swarm plate assays. Expression of clpP is strongly induced under heat shock conditions, and ClpP is shown to be essential for growth of B. subtilis at high temperature. The role of ClpP in the sporulation and competence regulatory pathways was investigated. ClpP is required for expression of the spollA and spollG operons, encoding the sigmaF and sigmaE sporulation-specific sigma factors. ClpP is also necessary for the expression of the comK gene, encoding a positive transcriptional regulator of competence genes. ComK-dependent transcription of sacB, encoding the exocellular Degradative Enzyme levansucrase, was found to be abolished in the delta clpP mutant. MecA has been characterized previously as a negative regulator of comK expression, whose overproduction inhibits both sporulation and competence development. Expression of a mecA'-'lacZ translational fusion is shown to be increased in the delta clpP mutant. We suggest that ClpP is involved in controlling MecA levels in the cell through proteolysis. Increased levels of MecA in the absence of ClpP are at least partly responsible for the observed pleiotropic phenotype of the delta clpP mutant.
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When the going gets tough: survival strategies and environmental signaling networks in Bacillus subtilis
Trends in Microbiology, 1999Co-Authors: Tarek MsadekAbstract:Regulatory pathways involving two-component histidine kinase/response regulator proteins of Bacillus subtilis are highly interconnected and form a signal transduction network controlling stationary-phase adaptive responses. These include chemotaxis and motility, Degradative Enzyme synthesis, antibiotic production, natural competence for DNA uptake, and sporulation. Many of these responses are mutually exclusive, with different control levels involving protein-environment, protein-protein and protein-DNA interactions, allowing the bacteria to adapt rapidly to environmental changes.
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clpp of bacillus subtilis is required for competence development motility Degradative Enzyme synthesis growth at high temperature and sporulation
Molecular Microbiology, 1998Co-Authors: Tarek Msadek, Frank Kunst, Véronique Dartois, Marie‐laure Herbaud, François Denizot, Georges RapoportAbstract:The nucleotide sequence of the Bacillus subtilis clpP gene was determined. The predicted protein shows very high similarity to members of the ClpP family of proteolytic subunits (68% amino acid sequence identity with that of Escherichia coli ). We show that ClpP plays an essential role in stationary phase adaptive responses. Indeed, a ΔclpP mutant was constructed and shown to display a pleiotropic phenotype, including a deficiency in both sporulation initiation and competence for DNA uptake. The ΔclpP mutant has a highly filamentous morphology and appears to be non-motile, as judged by swarm plate assays. Expression of clpP is strongly induced under heat shock conditions, and ClpP is shown to be essential for growth of B. subtilis at high temperature. The role of ClpP in the sporulation and competence regulatory pathways was investigated. ClpP is required for expression of the spoIIA and spoIIG operons, encoding the σF andσE sporulation-specific sigma factors. ClpP is also necessary for the expression of the comK gene, encoding a positive transcriptional regulator of competence genes. ComK-dependent transcription of sacB, encoding the exocellular Degradative Enzyme levansucrase, was found to be abolished in the ΔclpP mutant. MecA has been characterized previously as a negative regulator of comK expression, whose overproduction inhibits both sporulation and competence development. Expression of a mecA′–′lacZ translational fusion is shown to be increased in the ΔclpP mutant. We suggest that ClpP is involved in controlling MecA levels in the cell through proteolysis. Increased levels of MecA in the absence of ClpP are at least partly responsible for the observed pleiotropic phenotype of the ΔclpP mutant.
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a signal transduction network in bacillus subtilis includes the degs degu and comp coma two component systems
1995Co-Authors: Tarek Msadek, Frank Kunst, Georges RapoportAbstract:Soil bacteria such as Bacillus subtilis are subject to drastic variations of environmental conditions such as temperature, humidity, and nutrient source availability. At the onset of the stationary phase, faced with a depletion of essential nutrients, B. subtilis can adopt several responses, including synthesis of macromolecule-degrading Enzymes, competence for genetic transformation, increased motility and chemotaxis, antibiotic production, and finally, sporulation. Regulation by the B. subtilis two-component systems presents several original features. Some of these original features are discussed, within the framework of the DegS/DegU and ComP/ComA signal transduction network. The chapter describes Degradative Enzyme synthesis. Sequence similarities with two-component systems suggest the conserved His-189 residue of the DegS protein kinase and Asp-56 residue of the DegU response regulator as likely candidates for the respective phosphorylation sites of the two proteins. The chapter discusses competence gene expression, and signal transduction network. Both the ComP/ComA and DegS/DegU two-component systems control the expression of late competence genes; however, they seem to act through two different branches in the competence regulatory pathway that intersect to allow expression of comK. An exciting area of future research will be to identify the types of signals involved in regulation by each of these two-component systems and by the other regulators such as MecB/MecA and the ComQ-ComX-Spo0K pathway, as well as determining how these regulators interact within the signal transduction network.
S E Ealick - One of the best experts on this subject based on the ideXlab platform.
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structure determination and characterization of the vitamin b6 Degradative Enzyme e 2 acetamidomethylene succinate hydrolase
Biochemistry, 2010Co-Authors: Kathryn M Mcculloch, Tathagata Mukherjee, Tadhg P. Begley, S E EalickAbstract:The gene identification and kinetic characterization of (E)-2-(acetamidomethylene)succinate (E-2AMS) hydrolase has recently been described. This Enzyme catalyzes the final reaction in the degradation of vitamin B(6) and produces succinic semialdehyde, acetate, ammonia, and carbon dioxide from E-2AMS. The structure of E-2AMS hydrolase was determined to 2.3 A using SAD phasing. E-2AMS hydrolase is a member of the alpha/beta hydrolase superfamily and utilizes a serine/histidine/aspartic acid catalytic triad. Mutation of either the nucleophilic serine or the aspartate resulted in inactive Enzyme. Mutation of an additional serine residue in the active site causes the Enzyme to be unstable and is likely structurally important. The structure also provides insight into the mechanism of hydrolysis of E-2AMS and identifies several potential catalytically important residues.
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structure of the plp Degradative Enzyme 2 methyl 3 hydroxypyridine 5 carboxylic acid oxygenase from mesorhizobium loti maff303099 and its mechanistic implications
Biochemistry, 2009Co-Authors: K M Mcculloch, Tathagata Mukherjee, Tadhg P. Begley, S E EalickAbstract:A vitamin B6 Degradative pathway has recently been identified and characterized in Mesorhizobium loti MAFF303099. One of the Enzymes on this pathway, 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO), is a flavin-dependent Enzyme and catalyzes the oxidative ring-opening of 2-methyl-3-hydroxypyridine-5-carboxylic acid to form E-2-(acetamino-methylene)succinate. The gene for this Enzyme has been cloned, and the corresponding protein has been overexpressed in Escherichia coli and purified. The crystal structure of MHPCO has been solved to 2.1 A using SAD phasing with and without the substrate MHPC bound. These crystal structures provide insight into the reaction mechanism and suggest roles for active site residues in the catalysis of a novel oxidative ring-opening reaction.
K. Michael Gibson - One of the best experts on this subject based on the ideXlab platform.
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Increased guanidino species in murine and human succinate semialdehyde dehydrogenase (SSADH) deficiency.
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 2006Co-Authors: Erwin E.w. Jansen, Maneesh Gupta, Nanda M. Verhoeven, Cornelis Jakobs, Andreas Schulze, Henry Senephansiri, O. Carter Snead, K. Michael GibsonAbstract:Abstract Mice with targeted deletion of the GABA-Degradative Enzyme succinate semialdehyde dehydrogenase (SSADH; Aldh5a1 ; OMIM 271980) manifest globally elevated GABA and regionally decreased arginine in brain extracts. We examined the hypothesis that arginine–glycine amidinotransferase catalyzed the formation of guanidinobutyrate (GB) from increased GABA by quantifying guanidinoacetate (GA), guanidinopropionate (GP) and GB in brain extracts employing stable isotope dilution gas chromatographic-mass spectrometry. GA and GB were up to 4- and 22-fold elevated, respectively, in total and regional (cerebellum, hippocampus, cortex) brain extracts derived from SSADH −/− mice. Corresponding analyses of urine and cerebrospinal fluid derived from SSADH-deficient patients revealed significant ( P