Tapping into the native Pseudomonas bacterial biofilm structure by high-resolution multidimensional solid-state NMR.
Chang-Hyeock Byeon1, Ted Kinney1, Hakan Saricayir1
1Department of Structural Biology, School of Medicine, University of Pittsburgh, Biomedical Science Tower 3, 3501 Fifth Avenue, Pittsburgh, PA 15261, United States.
Journal of Magnetic Resonance (San Diego, Calif. : 1997)
|November 20, 2023
Summary
This study used advanced solid-state NMR to analyze native bacterial biofilms without labeling. Researchers identified numerous chemical sites, differentiating protein and polysaccharide components in Pseudomonas biofilms.
Area of Science:
- Biophysics
- Microbiology
- Analytical Chemistry
Background:
- Bacterial biofilms are complex microbial communities crucial in infections and industrial settings.
- Understanding biofilm structure and composition is vital for developing effective antimicrobial strategies.
- Current methods often require isotope labeling or struggle to analyze native, complex biofilm structures.
Purpose of the Study:
- To develop and apply a high-resolution multidimensional magic-angle spinning (MAS) solid-state NMR (ssNMR) technique for characterizing native bacterial biofilms.
- To identify and differentiate molecular components, including proteins and polysaccharides, within Pseudomonas fluorescens biofilms at natural abundance.
- To establish a pipeline for analyzing native biofilms that can be applied to other microbial systems.
Main Methods:
- Utilized multidimensional magic-angle spinning (MAS) solid-state NMR (ssNMR) spectroscopy.
- Employed INEPT-based 2D 1H-13C ssNMR and 1D ssNMR with peak deconvolution.
- Compared Carr-Purcell-Meiboom-Gill (CP) and Insensitive Nuclei Enhancement (INEPT) ssNMR spectra to assess molecular dynamics.
- Used FapC protein signals as a reference for identifying biofilm components.
Main Results:
- Successfully characterized native Pseudomonas fluorescens colony biofilms without isotope labeling.
- Identified approximately 80 distinct chemical sites in 1D spectra and 134 in 2D spectra.
- Differentiated and identified signals from biofilm proteins and various polysaccharide species (glucose, mannan, galactose, heptose, rhamnan, fucose, N-acylated mannuronic acid).
- Assessed molecular dynamics by comparing CP buildup behaviors, distinguishing mobile and rigid biofilm fractions.
Conclusions:
- This study represents the first high-resolution multidimensional ssNMR characterization of a native bacterial biofilm.
- The developed experimental pipeline is versatile and applicable to diverse in vitro and natural biofilm systems.
- This approach offers significant potential for advancing biofilm research and combating biofilm-associated infections.
