Redox Imbalance Underlies the Fitness Defect Associated with Inactivation of the Pta-AckA Pathway in Staphylococcus aureus.

The phosphotransacetylase-acetate kinase (Pta-AckA) pathway is thought to be a vital ATP generating pathway for Staphylococcus aureus. Disruption of the Pta-AckA pathway during overflow metabolism causes significant reduction in growth rate and viability, albeit not due to intracellular ATP depletion. Here, we demonstrate that toxicity associated with inactivation of the Pta-AckA pathway resulted from an altered intracellular redox environment. Growth of the pta and ackA mutants under anaerobic conditions partially restored cell viability. NMR metabolomics analyses and (13)C6-glucose metabolism tracing experiments revealed the activity of multiple pathways that promote redox (NADH/NAD(+)) turnover to be enhanced in the pta and ackA mutants during anaerobic growth. Restoration of redox homeostasis in the pta mutant by overexpressing l- lactate dehydrogenase partially restored its viability under aerobic conditions. Together, our findings suggest that during overflow metabolism, the Pta-AckA pathway plays a critical role in preventing cell viability defects by promoting intracellular redox homeostasis.

Chemostat cultivation and transcriptional analyses of Clostridium acetobutylicum mutants with defects in the acid and acetone biosynthetic pathways.

Clostridium acetobutylicum is a model organism for the biotechnologically important acetone-butanol-ethanol (ABE) fermentation. With the objective to rationally develop strains with improved butanol production, detailed insights into the physiological and genetic mechanisms of solvent production are required. Therefore, pH-controlled phosphate-limited chemostat cultivation and DNA microarray technology were employed for an in-depth analysis of knockout mutants with defects in the central fermentative metabolism. The set of studied mutants included strains with inactivated phosphotransacetylase (pta), phosphotransbutyrylase (ptb), and acetoacetate decarboxylase (adc) encoding genes, as well as an adc/pta double knockout mutant. A comprehensive physiological characterization of the mutants was performed by continuous cultivation, allowing for a well-defined separation of acidogenic and solventogenic growth, combined with the advantage of the high reproducibility of steady-state conditions. The ptb-negative strain C. acetobutylicum ptb::int(87) exhibited the most striking metabolite profile: Sizable amounts of butanol (29 ± 1.3 mM) were already produced during acidogenic growth. The product patterns of the mutants as well as accompanying transcriptomic data are presented and discussed.

Preliminary structural studies on the MtxX protein from Methanococcus jannaschii.

Methanococcus jannaschii has an mtr gene cluster expressing N(5)-methyltetrahydromethanopterin:coenzyme M methyltransferase, which generates methane by reducing CO(2) with H(2) with concomitant energy production under strictly anaerobic conditions. Some methanogenic archaea also have an mtr gene-cluster homologue, the mtxXAH gene cluster. M. jannaschii has both an entire mtr gene cluster and a single mtxX gene instead of the whole mtxXAH gene cluster. A PSI-BLAST search, secondary-structure prediction and the absence of phosphotransacetylase activity in M. jannaschii strongly support the possibility that the MtxX protein constitutes a unique methyltransferase family. In this study, the MtxX protein from M. jannaschii has been cloned, expressed, purified and crystallized. Synchrotron data were collected to 2.9 A from a crystal of selenomethionine-substituted MtxX protein. The crystal belonged to the primitive hexagonal space group P6(1)22, with unit-cell parameters a = 54.9, b = 54.9, c = 341.1 A, beta = 120.0 degrees . A full structure determination is under way in order to provide insight into the structure-function relationship of this protein.

Escherichia coli derivatives lacking both alcohol dehydrogenase and phosphotransacetylase grow anaerobically by lactate fermentation.

Escherichia coli mutants lacking alcohol dehydrogenase (adh mutants) cannot synthesize the fermentation product ethanol and are unable to grow anaerobically on glucose and other hexoses. Similarly, phosphotransacetylase-negative mutants (pta mutants) neither excrete acetate nor grow anaerobically. However, when a strain carrying an adh deletion was selected for anaerobic growth on glucose, spontaneous pta mutants were isolated. Strains carrying both adh and pta mutations were observed by in vivo nuclear magnetic resonance and shown to produce lactic acid as the major fermentation product. Various combinations of adh pta double mutants regained the ability to grow anaerobically on hexoses, by what amounts to a homolactic fermentation. Unlike wild-type strains, such adh pta double mutants were unable to grow anaerobically on sorbitol or on glucuronic acid. The growth properties of strains carrying various mutations affecting the enzymes of fermentation are discussed in terms of redox balance.