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ABSTRACT Quinones play a central role in maintaining redox balance and conserving energy but can trigger oxidative stress at high levels. However, the mechanisms by which microbes regulate quinone levels remain poorly understood, hindering effective metabolic engineering to modulate microbes for quinone production. Here, we show that the biosynthesis of the menaquinone precursor 1,4-dihydroxy-2-naphthoic acid (DHNA) in the lactic acid bacteriumLactococcus lactisis regulated by a combined genetic, enzymatic, and metabolic mechanism. Using synthetic biology approaches, we found that enzymes MenF and MenD both contribute to DHNA regulation, with MenD playing a more prominent role in controlling DHNA concentrations. A mathematical model elucidates a two-phase regulatory pattern resulting from the interplay of reversible flux and allosteric feedback inhibition, where either MenF or MenD can serve as the regulatory enzyme, depending on their relative expression ratio. In addition, the overproduction of DHNA is constrained by substrate availability, ensuring a sufficient but not excessive DHNA level to benefit cell growth while mitigating cytotoxicity. Collectively, these mechanisms maintain a fine-tuned physiological quinone level and suggest that modulating substrate supplement and MenF-to-MenD ratio could be keys for engineering DHNA production. IMPORTANCEQuinones are crucial molecules in cellular respiration, helping cells produce energy and maintain balance in their redox state. However, excessive quinone levels can be toxic, making it vital for microbes to tightly regulate their production. Our study uncovers howLactococcus lactis, a key food fermenting bacterium, uses a multi-layer mechanism to maintain optimal levels of the menaquinone precursor 1,4-dihydroxy-2-naphthoic acid (DHNA). By combining biosensors, genetic perturbations, and modeling, we show how cells balance the benefits and toxicity of quinones. These findings not only reveal fundamental microbial physiology but also provide strategies to engineer microbes for improved quinone production. 

Abstract Under starvation conditions, a spot of a few millionMyxococcus xanthuscells on agar will migrate inward to form aggregates that mature into dome-shaped fruiting bodies. This migration is thought to occur within structures called ‘streams,’ which are considered crucial for initiating aggregation. The prevailing traffic jam model hypothesizes that intersections of streams cause cell crowding and ‘jamming,’ thereby initiating the process of aggregate formation. However, this hypothesis has not been rigorously tested, in part due to the lack of a standardized, quantifiable definition of streams. To address this gap, we captured time-lapse movies and conducted fluorescent cell tracking experiments using wild-type and two motility-deficient mutantM. xanthusstrains. By quantitatively defining streams and developing a novel stream detection mask, we show that streams are not essential for nascent aggregate formation, though they may accelerate the process. Moreover, our results indicate that streaming has a genetic component: disrupting only one of the twoM. xanthusmotility systems hinders stream formation. Together, these findings challenge the idea that stream intersections are required to drive aggregate formation and suggest thatM. xanthusaggregation may be driven by mechanisms independent of streaming, highlighting the need for alternative models to fully explain aggregation dynamics. 

ABSTRACT A wide range of biological systems, from microbial swarms to bird flocks, display emergent behaviors driven by coordinated movement of individuals. To this end, individual organisms interact by recognizing their kin and adjusting their motility based on others around them. However, even in the best-studied systems, the mechanistic basis of the interplay between kin recognition and motility coordination is not understood. Here, using a combination of experiments and mathematical modeling, we uncover the mechanism of an emergent social behavior in Myxococcus xanthus . By overexpressing the cell surface adhesins TraA and TraB, which are involved in kin recognition, large numbers of cells adhere to one another and form organized macroscopic circular aggregates that spin clockwise or counterclockwise. Mechanistically, TraAB adhesion results in sustained cell-cell contacts that trigger cells to suppress cell reversals, and circular aggregates form as the result of cells’ ability to follow their own cellular slime trails. Furthermore, our in silico simulations demonstrate a remarkable ability to predict self-organization patterns when phenotypically distinct strains are mixed. For example, defying naive expectations, both models and experiments found that strains engineered to overexpress different and incompatible TraAB adhesins nevertheless form mixed circular aggregates. Therefore, this work provides key mechanistic insights into M. xanthus social interactions and demonstrates how local cell contacts induce emergent collective behaviors by millions of cells. IMPORTANCE In many species, large populations exhibit emergent behaviors whereby all related individuals move in unison. For example, fish in schools can all dart in one direction simultaneously to avoid a predator. Currently, it is impossible to explain how such animals recognize kin through brain cognition and elicit such behaviors at a molecular level. However, microbes also recognize kin and exhibit emergent collective behaviors that are experimentally tractable. Here, using a model social bacterium, we engineer dispersed individuals to organize into synchronized collectives that create emergent patterns. With experimental and mathematical approaches, we explain how this occurs at both molecular and population levels. The results demonstrate how the combination of local physical interactions triggers intracellular signaling, which in turn leads to emergent behaviors on a population scale. 

ABSTRACT Single mutations frequently alter several aspects of cell behavior but rarely reveal whether a particular statistically significant change is biologically significant. To determine which behavioral changes are most important for multicellular self-organization, we devised a new methodology using Myxococcus xanthus as a model system. During development, myxobacteria coordinate their movement to aggregate into spore-filled fruiting bodies. We investigate how aggregation is restored in two mutants, csgA and pilC , that cannot aggregate unless mixed with wild-type (WT) cells. To this end, we use cell tracking to follow the movement of fluorescently labeled cells in combination with data-driven agent-based modeling. The results indicate that just like WT cells, both mutants bias their movement toward aggregates and reduce motility inside aggregates. However, several aspects of mutant behavior remain uncorrected by WT, demonstrating that perfect recreation of WT behavior is unnecessary. In fact, synergies between errant behaviors can make aggregation robust. IMPORTANCE Self-organization into spatial patterns is evident in many multicellular phenomena. Even for the best-studied systems, our ability to dissect the mechanisms driving coordinated cell movement is limited. While genetic approaches can identify mutations perturbing multicellular patterns, the diverse nature of the signaling cues coupled to significant heterogeneity of individual cell behavior impedes our ability to mechanistically connect genes with phenotype. Small differences in the behaviors of mutant strains could be irrelevant or could sometimes lead to large differences in the emergent patterns. Here, we investigate rescue of multicellular aggregation in two mutant strains of Myxococcus xanthus mixed with wild-type cells. The results demonstrate how careful quantification of cell behavior coupled to data-driven modeling can identify specific motility features responsible for cell aggregation and thereby reveal important synergies and compensatory mechanisms. Notably, mutant cells do not need to precisely recreate wild-type behaviors to achieve complete aggregation.