Glutamate mediated metabolic neutralization mitigates propionate toxicity in intracellular Mycobacterium tuberculosis

Metabolic networks in biological systems are interconnected, such that malfunctioning parts can be corrected by other parts within the network, a process termed adaptive metabolism. Unlike Bacillus Calmette-Guérin (BCG), Mycobacterium tuberculosis (Mtb) better manages its intracellular lifestyle by executing adaptive metabolism. Here, we used metabolomics and identified glutamate synthase (GltB/D) that converts glutamine to glutamate (Q → E) as a metabolic effort used to neutralize cytoplasmic pH that is acidified while consuming host propionate carbon through the methylcitrate cycle (MCC). Methylisocitrate lyase, the last step of the MCC, is intrinsically downregulated in BCG, leading to obstruction of carbon flux toward central carbon metabolism, accumulation of MCC intermediates, and interference with GltB/D mediated neutralizing activity against propionate toxicity. Indeed, vitamin B12 mediated bypass MCC and additional supplement of glutamate led to selectively correct the phenotypic attenuation in BCG and restore the adaptive capacity of BCG to the similar level of Mtb phenotype. Collectively, a defective crosstalk between MCC and Q → E contributes to attenuation of intracellular BCG. Furthermore, GltB/D inhibition enhances the level of propionate toxicity in Mtb. Thus, these findings revealed a new adaptive metabolism and propose GltB/D as a synergistic target to improve the antimicrobial outcomes of MCC inhibition in Mtb

Activation and inhibition of pyruvate carboxylase from Rhizobium etli

While crystallographic structures of the R. etli pyruvate carboxylase (PC) holoenzyme revealed the location and probable positioning of the essential activator, Mg(2+), and nonessential activator, acetyl-CoA, an understanding of how they affect catalysis remains unclear. The current steady-state kinetic investigation indicates that both acetyl-CoA and Mg(2+) assist in coupling the MgATP-dependent carboxylation of biotin in the biotin carboxylase (BC) domain with pyruvate carboxylation in the carboxyl transferase (CT) domain. Initial velocity plots of free Mg(2+) vs pyruvate were nonlinear at low concentrations of Mg(2+) and a nearly complete loss of coupling between the BC and CT domain reactions was observed in the absence of acetyl-CoA. Increasing concentrations of free Mg(2+) also resulted in a decrease in the K(a) for acetyl-CoA. Acetyl phosphate was determined to be a suitable phosphoryl donor for the catalytic phosphorylation of MgADP, while phosphonoacetate inhibited both the phosphorylation of MgADP by carbamoyl phosphate (K(i) = 0.026 mM) and pyruvate carboxylation (K(i) = 2.5 mM). In conjunction with crystal structures of T882A R. etli PC mutant cocrystallized with phosphonoacetate and MgADP, computational docking studies suggest that phosphonoacetate could coordinate to one of two Mg(2+) metal centers in the BC domain active site. Based on the pH profiles, inhibition studies, and initial velocity patterns, possible mechanisms for the activation, regulation, and coordination of catalysis between the two spatially distinct active sites in pyruvate carboxylase from R. etli by acetyl-CoA and Mg(2+) are described.Tonya N. Zeczycki, Ann L. Menefee, Sarawut Jitrapakdee, John C. Wallace, Paul V. Attwood, Martin St. Maurice, and W. Wallace Clelan

Distribution and diversity of phytate-mineralizing bacteria

Phytate, the most abundant organic phosphorus compound in soil, dominates the biotic phosphorus input from terrestrial runoffs into aquatic systems. Microbial mineralization of phytate by phytases is a key process for recycling phosphorus in the biosphere. Bioinformatic studies were carried out on microbial genomes and environmental metagenomes in the NCBI and the CAMERA databases to determine the distribution of the four known classes of phytase in the microbial world. The Β-propeller phytase is the only phytase family that can be found in aquatic environments and it is also distributed in soil and plant bacteria. The Β-propeller phytase-like genes can be classified into several subgroups based on their domain structure and the positions of their conserved cysteine residues. Analysis of the genetic contexts of these subgroups showed that Β-propeller phytase genes exist either as an independent gene or are closely associated with a TonB-dependent receptor-like gene in operons, suggesting that these two genes are functionally linked and thus may play an important role in the cycles of phosphorus and iron. Our work suggests that Β-propeller phytases play a major role in phytate-phosphorus cycling in both soil and aquatic microbial communities. © 2007 International Society for Microbial Ecology All rights reserved.link_to_subscribed_fulltex