Biofilms are structured communities of tightly associated cells that constitute the predominant state of bacterial growth in natural and human-made environments. Although the core genetic circuitry that controls biofilm formation in model bacteria such as Bacillus subtilis has been well characterized, little is known about the role that metabolism plays in this complex developmental process. Here, we performed a time-resolved analysis of the metabolic changes associated with pellicle biofilm formation and development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. We report surprisingly widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. Most of these metabolic alterations were hitherto unrecognized as biofilm associated. For example, we observed increased activity of the tricarboxylic acid (TCA) cycle during early biofilm growth, a shift from fatty acid biosynthesis to fatty acid degradation, reorganization of iron metabolism and transport, and a switch from acetate to acetoin fermentation. Close agreement between metabolomic, transcriptomic, and proteomic measurements indicated that remodeling of metabolism during biofilm development was largely controlled at the transcriptional level. Our results also provide insights into the transcription factors and regulatory networks involved in this complex metabolic remodeling. Following upon these results, we demonstrated that acetoin production via acetolactate synthase is essential for robust biofilm growth and has the dual role of conserving redox balance and maintaining extracellular pH. This report represents a comprehensive systems-level investigation of the metabolic remodeling occurring during B. subtilis biofilm development that will serve as a useful road map for future studies on biofilm physiology. IMPORTANCE Bacterial biofilms are ubiquitous in natural environments and play an important role in many clinical, industrial, and ecological settings. Although much is known about the transcriptional regulatory networks that control biofilm formation in model bacteria such as Bacillus subtilis, very little is known about the role of metabolism in this complex developmental process. To address this important knowledge gap, we performed a time-resolved analysis of the metabolic changes associated with bacterial biofilm development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. Here, we report a widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. This report serves as a unique hypothesis-generating resource for future studies on bacterial biofilm physiology. Outside the biofilm research area, this work should also prove relevant to any investigators interested in microbial physiology and metabolism.

Mutant prion protein particles form axonal aggregates inside endolysosomes via axonal transport and endosomal fusion.

Highlights d STK4-mediated LC3B phosphorylation lowers LC3B binding to the transport protein FYCO1 d LC3B phosphorylation inhibition decreases retrograde transport of autophagosomes d Block of LC3B phosphorylation also compromises autophagosome-lysosome association d The STK4-LC3B-FYCO1 axis is a nutrient-sensitive, autophagy-regulatory pathway

Intra-axonal misfolded protein aggregates are a pathological feature of neurodegenerative diseases. How aggregates are formed and cleared is key to maintaining proteostasis. By systematically analyzing the trafficking itinerary of a misfolded GPI-anchored prion protein (PrP) mutant, we unveil endocytic pathways that drive its immediate degradation in the soma, versus its aggregation in axons inside endosomal structures we termed endoggresomes. Axonal sorting occurs post-Golgi, by association of mutant PrP vesicles with Arl8b/kinesin-1/HOPS, a complex that earmarks them for axonal entry, fusion, and aggregation via a mechanism of axonal rapid endosomal sorting and transport-dependent aggregation (ARESTA). Endoggresomes persist in axons due to transport and lysosomal deficits, impairing calcium dynamics and accelerating neuronal death. Reducing ARESTA inhibits endoggresome formation and circumvents these defects. These data identify the endo-lysosomal system as critical for the sorting of misfolded PrP, and ARESTA as an actionable anti-aggregation target that can ameliorate axonal dysfunction in the prionopathies.

Dystrophic axons comprising misfolded mutant prion protein (PrP) aggregates are a characteristic pathological feature in the prionopathies. These aggregates form inside endolysosomes -called endoggresomes-, within swellings that line up the length of axons of degenerating neurons. The pathways impaired by endoggresomes that result in failed axonal and consequently neuronal health, remain undefined. Here, we dissect the local subcellular impairments that occur within individual mutant PrP endoggresome swelling sites in axons. Quantitative high-resolution light and electron microscopy revealed the selective impairment of the acetylated vs tyrosinated microtubule cytoskeleton, while micro-domain image analysis of live organelle dynamics within swelling sites revealed deficits uniquely to the MT-based active transport system that translocates mitochondria and endosomes toward the synapse. Cytoskeletal and defective transport results in the retention of mitochondria, endosomes, and molecular motors at swelling sites, enhancing mitochondria-Rab7 late endosome contacts that induce mitochondrial fission via the activity of Rab7, and render mitochondria dysfunctional. Our findings point to mutant Pr Pendoggresome swelling sites as selective hubs of cytoskeletal deficits and organelle retention that drive the remodeling of organelles along axons. We propose that the dysfunction imparted locally within these axonal micro-domains spreads throughout the axon over time, leading to axonal dysfunction in prionopathies.