The genome-scale metabolic network analysis of Zymomonas mobilis ZM4 explains physiological features and suggests ethanol and succinic acid production strategies

<p>Abstract</p> <p>Background</p> <p><it>Zymomonas mobilis </it>ZM4 is a Gram-negative bacterium that can efficiently produce ethanol from various carbon substrates, including glucose, fructose, and sucrose, <it>via </it>the Entner-Doudoroff pathway. However, systems metabolic engineering is required to further enhance its metabolic performance for industrial application. As an important step towards this goal, the genome-scale metabolic model of <it>Z. mobilis </it>is required to systematically analyze <it>in silico </it>the metabolic characteristics of this bacterium under a wide range of genotypic and environmental conditions.</p> <p>Results</p> <p>The genome-scale metabolic model of <it>Z. mobilis </it>ZM4, ZmoMBEL601, was reconstructed based on its annotated genes, literature, physiological and biochemical databases. The metabolic model comprises 579 metabolites and 601 metabolic reactions (571 biochemical conversion and 30 transport reactions), built upon extensive search of existing knowledge. Physiological features of <it>Z. mobilis </it>were then examined using constraints-based flux analysis in detail as follows. First, the physiological changes of <it>Z. mobilis </it>as it shifts from anaerobic to aerobic environments (i.e. aerobic shift) were investigated. Then the intensities of flux-sum, which is the cluster of either all ingoing or outgoing fluxes through a metabolite, and the maximum <it>in silico </it>yields of ethanol for <it>Z. mobilis </it>and <it>Escherichia coli </it>were compared and analyzed. Furthermore, the substrate utilization range of <it>Z. mobilis </it>was expanded to include pentose sugar metabolism by introducing metabolic pathways to allow <it>Z. mobilis </it>to utilize pentose sugars. Finally, double gene knock-out simulations were performed to design a strategy for efficiently producing succinic acid as another example of application of the genome-scale metabolic model of <it>Z. mobilis</it>.</p> <p>Conclusion</p> <p>The genome-scale metabolic model reconstructed in this study was able to successfully represent the metabolic characteristics of <it>Z. mobilis </it>under various conditions as validated by experiments and literature information. This reconstructed metabolic model will allow better understanding of <it>Z. mobilis </it>metabolism and consequently designing metabolic engineering strategies for various biotechnological applications.</p

Active site phosphoryl groups in the biphosphorylated phosphotransferase complex reveal dynamics in a millisecond time scale

AbstractThe N-terminal domain of Enzyme I (EIN) and phosphocarrier HPr can form a biphosphorylated complex when they are both phosphorylated by excess cellular phosphoenolpyruvate. Here we show that the electrostatic repulsion between the phosphoryl groups in the biphosphorylated complex results in characteristic dynamics at the active site in a millisecond time scale. The dynamics is localized to phospho-His15 and the stabilizing backbone amide groups of HPr, and does not impact on the phospho-His189 of EIN. The dynamics occurs with the kex of ∼500s−1 which compares to the phosphoryl transfer rate of ∼850s−1 between EIN and HPr. The conformational dynamics in HPr may be important for its phosphotransfer reactions with multiple partner proteins.Structured summary of protein interactionsEIN and HPr bind by nuclear magnetic resonance (View Interaction)

Proinflammatory T-cell responses to gut microbiota promote experimental autoimmune encephalomyelitis

Although the effects of commensal bacteria on intestinal immune development seem to be profound, it remains speculative whether the gut microbiota influences extraintestinal biological functions. Multiple sclerosis (MS) is a devastating autoimmune disease leading to progressive deterioration of neurological function. Although the cause of MS is unknown, microorganisms seem to be important for the onset and/or progression of disease. However, it is unclear how microbial colonization, either symbiotic or infectious, affects autoimmunity. Herein, we investigate a role for the microbiota during the induction of experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Mice maintained under germ-free conditions develop significantly attenuated EAE compared with conventionally colonized mice. Germ-free animals, induced for EAE, produce lower levels of the proinflammatory cytokines IFN-γ and IL-17A in both the intestine and spinal cord but display a reciprocal increase in CD4^(+)CD25^(+)Foxp3^+ regulatory T cells (Tregs). Mechanistically, we show that gut dendritic cells from germ-free animals are reduced in the ability to stimulate proinflammatory T cell responses. Intestinal colonization with segmented filamentous bacteria (SFB) is known to promote IL-17 production in the gut; here, we show that SFBs also induced IL-17A–producing CD4^+ T cells (Th17) in the CNS. Remarkably, germ-free animals harboring SFBs alone developed EAE, showing that gut bacteria can affect neurologic inflammation. These findings reveal that the intestinal microbiota profoundly impacts the balance between pro- and antiinflammatory immune responses during EAE and suggest that modulation of gut bacteria may provide therapeutic targets for extraintestinal inflammatory diseases such as MS

Cryptococcus neoformans Overcomes Stress of Azole Drugs by Formation of Disomy in Specific Multiple Chromosomes

Cryptococcus neoformans is a haploid environmental organism and the major cause of fungal meningoencephalitis in AIDS patients. Fluconazole (FLC), a triazole, is widely used for the maintenance therapy of cryptococcosis. Heteroresistance to FLC, an adaptive mode of azole resistance, was associated with FLC therapy failure cases but the mechanism underlying the resistance was unknown. We used comparative genome hybridization and quantitative real-time PCR in order to show that C. neoformans adapts to high concentrations of FLC by duplication of multiple chromosomes. Formation of disomic chromosomes in response to FLC stress was observed in both serotype A and D strains. Strains that adapted to FLC concentrations higher than their minimal inhibitory concentration (MIC) contained disomies of chromosome 1 and stepwise exposure to even higher drug concentrations induced additional duplications of several other specific chromosomes. The number of disomic chromosomes in each resistant strain directly correlated with the concentration of FLC tolerated by each strain. Upon removal of the drug pressure, strains that had adapted to high concentrations of FLC returned to their original level of susceptibility by initially losing the extra copy of chromosome 1 followed by loss of the extra copies of the remaining disomic chromosomes. The duplication of chromosome 1 was closely associated with two of its resident genes: ERG11, the target of FLC and AFR1, the major transporter of azoles in C. neoformans. This adaptive mechanism in C. neoformans may play an important role in FLC therapy failure of cryptococcosis leading to relapse during azole maintenance therapy