RESUMEN

How bacteria sense and respond to nitrogen levels are central questions in microbial physiology. In Gram-positive bacteria, nitrogen homeostasis is controlled by an operon encoding glutamine synthetase (GS), a dodecameric machine that assimilates ammonium into glutamine, and the GlnR repressor. GlnR detects nitrogen excess indirectly by binding glutamine-feedback-inhibited-GS (FBI-GS), which activates its transcription-repression function. The molecular mechanisms behind this regulatory circuitry, however, are unknown. Here we describe biochemical and structural analyses of GS and FBI-GS-GlnR complexes from pathogenic and non-pathogenic Gram-positive bacteria. The structures show FBI-GS binds the GlnR C-terminal domain within its active-site cavity, juxtaposing two GlnR monomers to form a DNA-binding-competent GlnR dimer. The FBI-GS-GlnR interaction stabilizes the inactive GS conformation. Strikingly, this interaction also favors a remarkable dodecamer to tetradecamer transition in some GS, breaking the paradigm that all bacterial GS are dodecamers. These data thus unveil unique structural mechanisms of transcription and enzymatic regulation.

Asunto(s)

Glutamato-Amoníaco Ligasa , Nitrógeno , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Glutamina/metabolismo , Nitrógeno/metabolismo

RESUMEN

Limitations to successful single-particle cryo-electron microscopy (cryo-EM) projects include stable sample generation, production of quality cryo-EM grids with randomly oriented particles embedded in thin vitreous ice and access to microscope time. To address the limitation of microscope time, methodologies to more efficiently collect data on a 200 keV Talos Arctica cryo-transmission electron microscope at speeds as fast as 720 movies per hour (∼17 000 per day) were tested. In this study, key parameters were explored to increase data collection speed including: (1) using the beam-image shift method to acquire multiple images per stage position, (2) employing UltrAufoil TEM grids with R0.6/1 hole spacing, (3) collecting hardware-binned data and (4) adjusting the image shift delay factor in SerialEM. Here, eight EM maps of mouse apoferritin at 1.8-1.9 Šresolution were obtained in the analysis with data collection times for each dataset ranging from 56 min to 2 h. An EM map of mouse apoferritin at 1.78 Šwas obtained from an overnight data collection at a speed of 500 movies per hour and subgroup analysis performed, with no significant variation observed in data quality by image shift distance and image shift delay. The findings and operating procedures detailed herein allow for rapid turnover of single-particle cryo-EM structure determination.