ABSTRACT
The pyoverdine siderophore is produced by Pseudomonas aeruginosa to access iron. Its synthesis involves the complex coordination of four nonribosomal peptide synthetases (NRPSs), which are responsible for assembling the pyoverdine peptide backbone. The precise cellular organization of these NRPSs and their mechanisms of interaction remain unclear. Here, we used a combination of several single-molecule microscopy techniques to elucidate the spatial arrangement of NRPSs within pyoverdine-producing cells. Our findings reveal that PvdL differs from the three other NRPSs in terms of localization and mobility patterns. PvdL is predominantly located in the inner membrane, while the others also explore the cytoplasmic compartment. Leveraging the power of multicolor single-molecule localization, we further reveal co-localization between PvdL and the other NRPSs, suggesting a pivotal role for PvdL in orchestrating the intricate biosynthetic pathway. Our observations strongly indicates that PvdL serves as a central orchestrator in the assembly of NRPSs involved in pyoverdine biosynthesis, assuming a critical regulatory function.
Subject(s)
Oligopeptides , Peptide Synthases , Pseudomonas aeruginosa , Pseudomonas aeruginosa/metabolism , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/enzymology , Oligopeptides/biosynthesis , Oligopeptides/metabolism , Peptide Synthases/metabolism , Peptide Synthases/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Siderophores/biosynthesis , Siderophores/metabolismABSTRACT
Spectrally-resolved single-molecule localization microscopy (srSMLM) has emerged as a powerful tool for exploring the spectral properties of single emitters in localization microscopy. By simultaneously capturing the spatial positions and spectroscopic signatures of individual fluorescent molecules, srSMLM opens up the possibility of investigating an additional dimension in super-resolution imaging. However, appropriate and dedicated tools are required to fully capitalize on the spectral dimension. Here, we propose the application of the spectral phasor analysis as an effective method for summarizing and analyzing the spectral information obtained from srSMLM experiments. The spectral phasor condenses the complete spectrum of a single emitter into a two-dimensional space, preserving key spectral characteristics for single-molecule spectral exploration. We demonstrate the effectiveness of spectral phasor in efficiently classifying single Nile Red fluorescence emissions from largely overlapping cyanine fluorescence signals in dual-color PAINT experiments. Additionally, we employed spectral phasor with srSMLM to reveal subtle alterations occurring in the membrane of Gram-positive Enterococcus hirae in response to gramicidin exposure, a membrane-perturbing antibiotic treatment. Spectral phasor provides a robust, model-free analytic tool for the detailed analysis of the spectral component of srSMLM, enhancing the capabilities of multi-color spectrally-resolved single-molecule imaging.
Subject(s)
Microscopy, Fluorescence , Single Molecule Imaging , Single Molecule Imaging/methods , Microscopy, Fluorescence/methods , Fluorescent Dyes/chemistry , Gramicidin/chemistry , Oxazines/chemistryABSTRACT
Spectrally-resolved single molecule localization microscopy (srSMLM) is a recent technique enriching single molecule localization microscopy with the simultaneous recording of spectra of the single emitters. srSMLM resolution is limited by the number of photons collected per emitters. Sharing a photon budget to record the localization and the spectroscopic information results in a loss of spatial and spectral resolution-or forces the sacrifice of one at the expense of the other. Here, srUnet-a deep-learning Unet-based image processing routine trained to increase the spectral and spatial signals to compensate for the resolution loss inherent in additionally recording the spectral component is reported. Both localization and spectral precision are improved by srUnet-particularly for the low-emitting species. srUnet increases the fraction of localization whose signal can be both spatially and spectrally characterized. It preserves spectral shifts and the linearity of the dispersion of light. It strongly facilitates wavelength assignment in multicolor experiments. srUnet is a simple post-processing add-on boosting srSMLM performance close to conventional SMLM with the potential to turn srSMLM into the new standard for multicolor single molecule imaging.
ABSTRACT
Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with Förster resonance energy transfer (FRET) provide accurate information about PPIs in live cells. FLIM-FRET relies on measuring the fluorescence lifetime decay of a FRET donor at each pixel of the FLIM image, providing quantitative and accurate information about PPIs and their spatial cellular organizations. We propose here a detailed protocol for FLIM-FRET measurements that we applied to monitor PPIs in live Pseudomonas aeruginosa in the particular case of two interacting proteins expressed with highly different copy numbers to demonstrate the quality and robustness of the technique at revealing critical features of PPIs. This protocol describes in detail all the necessary steps for PPI characterization - starting from bacterial mutant constructions up to the final analysis using recently developed tools providing advanced visualization possibilities for a straightforward interpretation of complex FLIM-FRET data.
