RESUMO
The flagellum is the most complex macromolecular structure known in bacteria and comprised of around two dozen distinct proteins. The main building block of the long, external flagellar filament, flagellin, is secreted through the flagellar type-III secretion system at a remarkable rate of several tens of thousands amino acids per second, significantly surpassing the rates achieved by other pore-based protein secretion systems. The evolutionary implications and potential benefits of this high secretion rate for flagellum assembly and function, however, have remained elusive. In this study, we provide both experimental and theoretical evidence that the flagellar secretion rate has been evolutionarily optimized to facilitate rapid and efficient construction of a functional flagellum. By synchronizing flagellar assembly, we found that a minimal filament length of 2.5 µm was required for swimming motility. Biophysical modelling revealed that this minimal filament length threshold resulted from an elasto-hydrodynamic instability of the whole swimming cell, dependent on the filament length. Furthermore, we developed a stepwise filament labeling method combined with electron microscopy visualization to validate predicted flagellin secretion rates of up to 10,000 amino acids per second. A biophysical model of flagellum growth demonstrates that the observed high flagellin secretion rate efficiently balances filament elongation and energy consumption, thereby enabling motility in the shortest amount of time. Taken together, these insights underscore the evolutionary pressures that have shaped the development and optimization of the flagellum and type-III secretion system, illuminating the intricate interplay between functionality and efficiency in assembly of large macromolecular structures. Significance statement: Our study demonstrates how protein secretion of the bacterial flagellum is finely tuned to optimize filament assembly rate and flagellum function while minimizing energy consumption. By measuring flagellar filament lengths and bacterial swimming after initiation of flag-ellum assembly, we were able to establish the minimal filament length necessary for swimming motility, which we rationalized physically as resulting from an elasto-hydrodynamic instability of the swimming cell. Our bio-physical model of flagellum growth further illustrates how the physiological flagellin secretion rate is optimized to maximize filament elongation while conserving energy. These findings illuminate the evolutionary pressures that have shaped the function of the bacterial flagellum and type-III secretion system, driving improvements in bacterial motility and overall fitness.
RESUMO
Standardized and thoroughly characterized genetic tools are a prerequisite for studying cellular processes to ensure the reusability and consistency of experimental results. The discovery of fluorescent proteins (FPs) represents a milestone in the development of genetic reporters for monitoring transcription or protein localization in vivo. FPs have revolutionized our understanding of cellular dynamics by enabling the real-time visualization and tracking of biological processes. Despite these advancements, challenges remain in the appropriate use of FPs, specifically regarding their proper application, protein turnover dynamics, and the undesired disruption of cellular functions. Here, we systematically compared a comprehensive set of 15 FPs and assessed their performance in vivo by focusing on key parameters, such as signal over background ratios and protein stability rates, using the Gram-negative model organism Salmonella enterica as a representative host. We evaluated four protein degradation tags in both plasmid- and genome-based systems and our findings highlight the necessity of introducing degradation tags to analyze time-sensitive cellular processes. We demonstrate that the gain of dynamics mediated by the addition of degradation tags impacts the cell-to-cell heterogeneity of plasmid-based but not genome-based reporters. Finally, we probe the applicability of FPs for protein localization studies in living cells using standard and super-resolution fluorescence microscopy. In summary, our study underscores the importance of careful FP selection and paves the way for the development of improved genetic reporters to enhance the reproducibility and reliability of fluorescence-based research in Gram-negative bacteria and beyond.