FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.

Manko, Hanna; Normant, Vincent; Perraud, Quentin; Steffan, Tania; Gasser, Véronique; Boutant, Emmanuel; Réal, Éléonore; Schalk, Isabelle J.; Godet, Julien

doi:10.3791/61602

Cited by 5 publications

(7 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We used Pseudomonas aeruginosa PAO1 strains in which either PvdL, PvdI, PvdJ, or PvdD was fused to eGFP at the chromosomal level. The expression of the fluorescent enzymes was induced by iron-deficient growing conditions similarly to the wild-type PAO1 [ 24 ]. In fixed cells, the eGFP moiety served as the target for immunostaining, where an anti-eGFP primary antibody was subsequently recognized by a secondary antibody containing a DNA-PAINT docking strand.…”

Section: Resultsmentioning

confidence: 99%

PvdL Orchestrates the Assembly of the Nonribosomal Peptide Synthetases Involved in Pyoverdine Biosynthesis in Pseudomonas aeruginosa

Manko,

Steffan,

Gasser

et al. 2024

IJMS

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 99%

PvdL Orchestrates the Assembly of the Nonribosomal Peptide Synthetases Involved in Pyoverdine Biosynthesis in Pseudomonas aeruginosa

Manko,

Steffan,

Gasser

et al. 2024

IJMS

Self Cite

View full text Add to dashboard Cite

show abstract

“…The Pseudomonas aeruginosa strains used in this study are listed in supplementary materials (table S1). Mutants construction was performed as described in detail in (27). For imaging, cells were grown in lysogeny broth (LB) (L3152 Sigma Aldrich) at 30 °C under 200 rpm orbital shaking for 24 hours.…”

Section: Methodsmentioning

confidence: 99%

“…Ti:Sapphire oscillator (Mai Tai DeepSee, Spectra Physics -80 MHz repetition rate, ≈ 70 fs pulse width) at 10-20 mW was used for two-photon excitation at 930 nm. Emitted photons were collected through a 680 nm short pass filter (F75-680, AHF, Germany) and a 525/50 nm band-pass filter (F37-516, AHF, Germany) and directed to a fibre-coupled avalanche photo-diode (SPCM-AQR-14-FC, Perkin Elmer) connected to a time-correlated single photon counting (TCSPC) module (SPC830, Becker & Hickl, Ger-More details on the FLIM FRET setup are given in references (27,50). The data analysis was performed using a commercial software (SPCImage, V8.1, Becker & Hickl, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…We used Pseudomonas aeruginosa PAO1 strains in which either PvdL, PvdI, PvdJ, or PvdD were fused to eGFP at the chromosomal level. The expression of the fluorescent enzymes was induced by iron-deficient growing conditions similarly to the wild-type PAO1 (27). In fixed cells, the eGFP moiety served as the target for immunostaining, where an anti-eGFP primary antibody was subsequently recognized by a secondary antibody containing a DNA-PAINT docking strand.…”

Section: Localizations Of Nrpss In Cellsmentioning

confidence: 99%

See 1 more Smart Citation

PvdL Orchestrates the Assembly of the Nonribosomal Peptide Synthetases Involved in Pyoverdine Biosynthesis inPseudomonas aeruginosa

Manko,

Steffan,

Gasser

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

The pyoverdine siderophore is produced byPseudomonas aeruginosato access iron. Its synthesis involves the complex co-ordination of four nonribosomal peptide synthetases (NRPSs), 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 to elucidate the spatial arrangement of NRPSs within pyoverdine-producing cells. Our findings revealed that PvdL differs from the three other NRPS in term of localization and mobility patterns. PvdL is predominantly located at the inner membrane, while the other 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.

show abstract

“…Cells were finally washed three times with PBS, permeabilized with lysosyme and stored at +4 °C. The detailed protocol can be found elsewhere (35).…”

Section: Dna Origami Nanorulersmentioning

confidence: 99%

Advancing Spectrally-Resolved Single Molecule Localization Microscopy using Deep Learning

Manko

Godet

2022

Preprint

Self Cite

View full text Add to dashboard Cite

Spectrally-Resolved Single Molecule Localization Microscopy (srSMLM) is a recent multidimensional technique enriching single molecule localization imaging by the simultaneous recording of single emitters spectra. As for SMLM, the localization precision is fundamentally limited by the number of photons collected per emitters . But srSMLM is more impacted because splitting the emission light from single emitters into a spatial and a spectral channel further reduces the number of photons available for each channel and impairs both spatial and spectral precision - or forces the sacrifice of one or the other. Here, we explored the potential of deep learning to overcome this limitation. We report srUnet - a Unet-based image processing that enhances the spectral and spatial signals and compensates for the signal loss inherent in recording the spectral component. We showed that localization and spectral precision of low-emitting species remain as good as those obtained with a high photons budget together with improving the fraction of localizations whose signal is both spatially and spectrally interpretable. srUnet is able to deal with spectral shift and its application to multicolour imaging in biological sample is straightforward. srUnet advances spectrally resolved single molecule localization microscopy to achieve performance close to conventional SMLM without complicating its use.

show abstract

FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.

Cited by 5 publications

References 29 publications

PvdL Orchestrates the Assembly of the Nonribosomal Peptide Synthetases Involved in Pyoverdine Biosynthesis in Pseudomonas aeruginosa

PvdL Orchestrates the Assembly of the Nonribosomal Peptide Synthetases Involved in Pyoverdine Biosynthesis in Pseudomonas aeruginosa

PvdL Orchestrates the Assembly of the Nonribosomal Peptide Synthetases Involved in Pyoverdine Biosynthesis inPseudomonas aeruginosa

Advancing Spectrally-Resolved Single Molecule Localization Microscopy using Deep Learning

Contact Info

Product

Resources

About