Cho Ewing (trampant6)

Bacterial cell wall synthesis is an essential process in bacteria and one of the best targets for antibiotics. A critical step on this pathway is the export of the lipid-linked cell wall monomer, Lipid II, by its transporter MurJ. The mechanism by which MurJ mediates the transbilayer movement of Lipid II is not understood because intermediate states of this process have not been observed. Here we demonstrate a method to capture and detect interactions between MurJ and its substrate Lipid II by photo-cross-linking and subsequent biotin-tagging. We show that this method can be used to covalently capture intermediate transport states of Lipid II on MurJ in living cells. Using this strategy we probed several lethal arginine mutants and found that they retain appreciable substrate-binding ability despite being defective in Lipid II transport. We propose that Lipid II binding to these residues during transport induces a conformational change in MurJ required to proceed through the Lipid II transport cycle. The methods described to detect intermediate transport states of MurJ will be useful for characterizing mechanisms of inhibitors.Nitrogen (N) losses from agricultural production contribute to detrimental impacts on water, soil, air, and human health. However, it is still lacking in evaluating global N budgets in agricultural systems. Hence, we conducted a global analysis on the current status of the N flows in the agricultural systems, explored the possible mitigation measures and challenges, and investigated the existing regulations on controlling N pollution. Globally, agricultural soils received a total of 73 kg of N ha-1 year-1 on average, including N fertilizer plus manure (61%), atmospheric N deposition (10%), and N litters and fixation (29%). The estimated global NH3 loss to total N inputs was 17%, which led to a loss of $15 billion year-1. The N use efficiency (NUE) in Eastern China (33%) was much lower than that in the Eastern United States (65%) or Western Europe (61%), leaving much room to enhance the NUE to increase agricultural food production. Meanwhile, higher NH3 losses from N fertilizers and manure were found in Eastern China (22%) than the Eastern United States (17%) and Western Europe (17%). We highlight the urgency to improve the NUE and decrease NH3 loss with lower environmental consequences. Our results showed high potentials to mitigate NH3 volatilization and enhance the NUE by various measures, such as substituting manure N for chemical fertilizer N, applying controlled release fertilizers, and urease inhibitors. These measures should be implemented in combination with the transfer of knowledge to farmers with new technologies and increasing the farm size to enhance the efficiency of agricultural production in the future.Biological nitrogen fixation is catalyzed by the enzyme nitrogenase, which facilitates the cleavage of the relatively inert triple bond of N2. Nitrogenase is most commonly associated with the molybdenum-iron cofactor called FeMoco or the M-cluster, and it has been the subject of extensive structural and spectroscopic characterization over the past 60 years. In the late 1980s and early 1990s, two "alternative nitrogenase" systems were discovered, isolated, and found to incorporate V or Fe in place of Mo. These systems are regulated by separate gene clusters; however, there is a high degree of structural and functional similarity between each nitrogenase. Limited studies with the V- and Fe-nitrogenases initially demonstrated that these enzymes were analogously active as the Mo-nitrogenase, but more recent investigations have found capabilities that are unique to the alternative systems. In this review, we will discuss the reactivity, biosynthetic, and mechanistic proposals for the alternative nitrogenases as well as their electronic and structural properties in comparison to the well-characterized Mo-dependent system. Studies over the past 10 years have been particularly fruitful, though ke