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The major human pathogen Staphylococcus aureus forms biofilms comprising of a fibrin network that increases attachment to surfaces and shields bacteria from the immune system. It secretes two coagulases, Coagulase (Coa) and von Willebrand factor binding protein (vWbp), which hijack the host coagulation cascade and trigger the formation of this fibrin clot. However, it is unclear how Coa and vWbp contribute differently to the localisation and dynamics of clot assembly in growing biofilms.Here, we address this question using high-precision time-resolved confocal microscopy of fluorescent fibrin to establish the spatiotemporal dynamics of fibrin clot formation in functional biofilms. We also use fluorescent fusion proteins to visualise the locations of Coa and vWbp in biofilms using both confocal laser scanning and high resolution highly inclined and laminated optical sheet microscopy. We visualise and quantify the spatiotemporal dynamics of fibrin production during initiation of biofilms in plasma amended with fluorescently labelled fibrinogen.We find that human serum stimulates coagulase production, and that Coa and vWbp loosely associate to the bacterial cell surface. Coa localises to cell surfaces to produce a surface-attached fibrin pseudocapsule but can diffuse from cells to produce matrix-associated fibrin. vWbp produces matrix-associated fibrin in the absence of Coa, and furthermore accelerates pseudocapsule production when Coa is present. Finally, we observe that fibrin production varies across the biofilm. A sub-population of non-dividing cells does not produce any pseudocapsule but remains within the protective extended fibrin network, which could be important for the persistence of S. aureus biofilm infections as antibiotics are more effective against actively growing cells.Our findings indicate a more cooperative role between Coa and vWbp in building fibrin networks than previously thought, and a bet-hedging cell strategy where some cells produce biofilm matrix while others do not, but instead assume a dormant phenotype that could be associated with antibiotic tolerance.

As camera pixel arrays have grown larger and faster, and optical microscopy techniques ever more refined, there has been an explosion in the quantity of data acquired during routine light microscopy. At the single-molecule level, analysis involves multiple steps and can rapidly become computationally expensive, in some cases intractable on office workstations. Complex bespoke software can present high activation barriers to entry for new users. Here, we redevelop our quantitative single-molecule analysis routines into an optimized and extensible Python program, with GUI and command-line implementations to facilitate use on local machines and remote clusters, by beginners and advanced users alike. We demonstrate that its performance is on par with previous MATLAB implementations but runs an order of magnitude faster. We tested it against challenge data and demonstrate its performance is comparable to state-of-the-art analysis platforms. We show the code can extract fluorescence intensity values for single reporter dye molecules and, using these, estimate molecular stoichiometries and cellular copy numbers of fluorescently-labeled biomolecules. It can evaluate 2D diffusion coefficients for the characteristically short single-particle tracking data. To facilitate benchmarking we include data simulation routines to compare different analysis programs. Finally, we show that it works with 2-color data and enables colocalization analysis based on overlap integration, to infer interactions between differently labelled biomolecules. By making this freely available we aim to make complex light microscopy single-molecule analysis more democratized.

Here we use singe-molecule optical proteomics and computational analysis of live cell bacterial images, using millisecond super-resolved tracking and quantification of fluorescently labelled protein SpoIIE in single live Bacillus subtilis bacteria to understand its crucial role in cell development. Asymmetric cell division during sporulation in Bacillus subtilis presents a model system for studying cell development. SpoIIE is a key integral membrane protein phosphatase that couples morphological development to differential gene expression. However, the basic mechanisms behind its operation remain unclear due to limitations of traditional tools and technologies. We instead used advanced single-molecule imaging of fluorescently tagged SpoIIE in real time on living cells to reveal vital changes to the patterns of expression, localization, mobility and stoichiometry as cells undergo asymmetric cell division then engulfment of the smaller forespore by the larger mother cell. We find, unexpectedly, that SpoIIE forms tetramers capable of cell- and stage-dependent clustering, its copy number rising to ~ 700 molecules as sporulation progresses. We observed that slow moving SpoIIE clusters initially located at septa are released as mobile clusters at the forespore pole as phosphatase activity is manifested and compartment-specific RNA polymerase sigma factor, σF, becomes active. Our findings reveal that information captured in its quaternary organization enables one protein to perform multiple functions, extending an important paradigm for regulatory proteins in cells. Our findings more generally demonstrate the utility of rapid live cell single-molecule optical proteomics for enabling mechanistic insight into the complex processes of cell development during the cell cycle.