Transcriptional fusion mutants and Tn5-B20 transposon mutants were isolated where the only genes affected are believed to either be involved in the hypothetical GABA shunt or code for subunits of the alpha-ketoglutarate dehydrogenase enzyme complex of Sinorhizobium meliloti. The growth phenotypes of Rm30222 (gabT) and eight mutants in gabD1, 2, 3, and 4 on minimal media were comparable to that of the wild-type. Compared to wild-type, Rm30222 (gabT) lacked alpha-ketoglutarate-dependent gamma-aminobutyrate transaminase activity showed high induction of gabT on GABA but produced green plants indicative of being Fix+. Mutants in gabD alleles maintained wild-type levels of succinic semialdehyde dehydrogenase activity and could fix nitrogen as well as the wild-type in symbiosis. / Mutation of sucB encoding a subunit of a-ketoglutarate dehydrogenase produced a mutant, Rm30230, that initially had difficulty growing on minimal media supplemented with either arabinose or glutamate. In symbiosis with alfalfa, Rm30230 had a fix- phenotype and was also devoid of alpha-ketoglutarate dehydrogenase activity. The ability of Rm30230 to grow on arabinose or glutamate, without alpha-ketoglutarate dehydrogenase activity, strengthens the hypothesis that S. meliloti has a functional GABA shunt allowing it to circumvent the forward-direction TCA cycle from alpha-ketoglutarate to succinate. Mutation of the second potential dihydrolipoamide succinyltransferase component (E2) of alpha-ketoglutarate dehydrogenase yielded Rm30267 (SMb20019) with wild-type growth on minimal media and a Fix+ phenotype in plants. The introduction or a sucB mutation into the SMb20019 mutant background (Rm30275) was comparable to the sole sucB mutation. This finding shows that the locus SMb20019 cannot be substituted for sucB in the alpha-ketoglutarate dehydrogenase enzyme complex.

No description available.

Succinic semialdehyde dehydrogenase (ALDH5A1) deficiency (SSADH-d) is an autosomal recessive, inborn error of gamma-aminobutyric acid (GABA) metabolism that results in psychomotor retardation, ataxia and seizures. A mouse model of SSADH-d (the Aldh5a1-/- mouse) was created to study the pathophysiology and treatment of SSADH-d. Aldh5a1-/- mice have psychomotor retardation and a progressive seizure phenotype results in death around P25. The present experiments tested the effects of a ketogenic diet in the treatment of Aldh5a1-/- mice. The KD was found to prolong the lives of Aldh5a1-/- mice by >300% while significantly delaying the onset the ataxia and preventing weight loss that is seen in untreated Aldh5a1-/- mice. Electrophysiological recordings revealed a corresponding decrease in seizures in KD fed mutants, as compared to control diet (CD) fed mutants. We assessed spontaneous miniature postsynaptic currents (mPSC) in CD and KD fed mutants. We found that CD fed mutants had significantly decreased inhibitory mPSC (mIPSC) activity compared to CD fed wildtype controls. mIPSC activity was restored in KD fed Aldh5a1-/- mice. A similar effect was found in [35S]TBPS binding experiments. TBPS binding was significantly reduced in CD fed Aldh5a1-/- mice, but restored in KD fed mutants. Plasma analysis revealed that an elevation of serum beta-hydroxybutyrate may play a role in the KD’s effects. The KD led to a significant elevation in the number of hippocampal mitochondria in mutant mice. Further, the KD was able to normalize the deficiencies in the hippocampal ATP levels seen in the Aldh5a1-/- mice. The present data suggest that the KD is able to significantly improve the Aldh5a1-/- phenotype. The effect of the KD on mIPSC activity is novel and furthers our understanding of how the KD may exert its effects. The mitochondrial studies confirm the findings of others, that the KD elevates the number of mitochondria. The KD also restores ATP deficiencies in Aldh5a1-/- mice, which is a novel finding. Together, these show that the KD may be an effective treatment for SSADH-d in humans. These data also further our understanding of the KD’s mechanisms of action.

Succinic semialdehyde dehydrogenase (ALDH5A1) deficiency (SSADH-d) is an autosomal recessive, inborn error of gamma-aminobutyric acid (GABA) metabolism that results in psychomotor retardation, ataxia and seizures. A mouse model of SSADH-d (the Aldh5a1-/- mouse) was created to study the pathophysiology and treatment of SSADH-d. Aldh5a1-/- mice have psychomotor retardation and a progressive seizure phenotype results in death around P25. The present experiments tested the effects of a ketogenic diet in the treatment of Aldh5a1-/- mice. The KD was found to prolong the lives of Aldh5a1-/- mice by >300% while significantly delaying the onset the ataxia and preventing weight loss that is seen in untreated Aldh5a1-/- mice. Electrophysiological recordings revealed a corresponding decrease in seizures in KD fed mutants, as compared to control diet (CD) fed mutants. We assessed spontaneous miniature postsynaptic currents (mPSC) in CD and KD fed mutants. We found that CD fed mutants had significantly decreased inhibitory mPSC (mIPSC) activity compared to CD fed wildtype controls. mIPSC activity was restored in KD fed Aldh5a1-/- mice. A similar effect was found in [35S]TBPS binding experiments. TBPS binding was significantly reduced in CD fed Aldh5a1-/- mice, but restored in KD fed mutants. Plasma analysis revealed that an elevation of serum beta-hydroxybutyrate may play a role in the KD’s effects. The KD led to a significant elevation in the number of hippocampal mitochondria in mutant mice. Further, the KD was able to normalize the deficiencies in the hippocampal ATP levels seen in the Aldh5a1-/- mice. The present data suggest that the KD is able to significantly improve the Aldh5a1-/- phenotype. The effect of the KD on mIPSC activity is novel and furthers our understanding of how the KD may exert its effects. The mitochondrial studies confirm the findings of others, that the KD elevates the number of mitochondria. The KD also restores ATP deficiencies in Aldh5a1-/- mice, which is a novel finding. Together, these show that the KD may be an effective treatment for SSADH-d in humans. These data also further our understanding of the KD’s mechanisms of action.