Tapping into the native Pseudomonas bacterial biofilm structure by high-resolution multidimensional solid-state NMR.

We present a multidimensional magic-angle spinning (MAS) solid-state NMR (ssNMR) study to characterize native Pseudomonas fluorescens colony biofilms at natural abundance without isotope-labelling. By using a high-resolution INEPT-based 2D 1H-13C ssNMR spectrum and thorough peak deconvolution at the 1D ssNMR spectra, approximately 80/134 (in 1D/2D) distinct biofilm chemical sites were identified. We compared CP and INEPT 13C ssNMR spectra to differentiate signals originating from the mobile and rigid fractions of the biofilm, and qualitatively determined dynamical changes by comparing CP buildup behaviors. Protein and polysaccharide signals were differentiated and identified by utilizing FapC protein signals as a template, a biofilm forming functional amyloid from Pseudomonas. We identified several biofilm polysaccharide species such as glucose, mannan, galactose, heptose, rhamnan, fucose and N-acylated mannuronic acid by using 1H and 13C chemical shifts obtained from the 2D spectrum. To our knowledge, this study marks the first high-resolution multidimensional ssNMR characterization of a native bacterial biofilm. Our experimental pipeline can be readily applied to other in vitro biofilm model systems and natural biofilms and holds the promise of making a substantial impact on biofilm research, fostering new ideas and breakthroughs to aid in the development of strategic approaches to combat infections caused by biofilm-forming bacteria.

Tapping into the native Pseudomonas Bacterial Biofilm Structure by High-Resolution 1D and 2D MAS solid-state NMR.

We present a high-resolution 1D and 2D magic-angle spinning (MAS) solid-state NMR (ssNMR) study to characterize native Pseudomonas fluorescens colony biofilms at natural abundance without isotope-labelling. By using a high-resolution INEPT-based 2D 1 H- 13 C ssNMR spectrum and thorough peak deconvolution approach at the 1D ssNMR spectra, approximately 80/134 (in 1D/2D) distinct biofilm chemical sites were identified. We compared CP and INEPT 13 C ssNMR spectra to different signals originating from the mobile and rigid fractions of the biofilm, and qualitative determined dynamical changes by comparing CP buildup behaviors. Protein and polysaccharide signals were differentiated and identified by utilizing FapC signals as a template, a biofilm forming functional amyloid from Pseudomonas . We also attempted to identify biofilm polysaccharide species by using 1 H/ 13 C chemical shifts obtained from the 2D spectrum. This study marks the first demonstration of high-resolution 2D ssNMR spectroscopy for characterizing native bacterial biofilms and expands the scope of ssNMR in studying biofilms. Our experimental pipeline can be readily applied to other in vitro biofilm model systems and natural biofilms and holds the promise of making a substantial impact on biofilm research, fostering new ideas and breakthroughs to aid in the development of strategic approaches to combat infections caused by biofilm-forming bacteria.

Spatial Structure Formation by RsmE-Regulated Extracellular Secretions in Pseudomonas fluorescens Pf0-1.

Cells in microbial communities on surfaces live and divide in close proximity, which greatly enhances the potential for social interactions. Spatiogenetic structures are manifested through competitive and cooperative interactions among the same and different genotypes within a shared space, and extracellular secretions appear to function dynamically at the forefront. A previous experimental evolution study utilizing Pseudomonas fluorescens Pf0-1 colonies demonstrated that diverse mutations in the rsmE gene were repeatedly and exclusively selected through the formation of a dominant spatial structure. RsmE's primary molecular function is translation repression, and its homologs regulate various social and virulence phenotypes. Pseudomonas spp. possess multiple paralogs of Rsm proteins, and RsmA, RsmE, and RsmI are the most prevalent. Here, we demonstrate that the production of a mucoid polymer and a biosurfactant are exclusively regulated through RsmE, contradicting the generalized notion of functional redundancy among the Rsm paralogs. Furthermore, we identified the biosurfactant as the cyclic lipopeptide gacamide A. Competition and microscopy analyses showed that the mucoid polymer is solely responsible for creating a space of low cellular density, which is shared exclusively by the same genotype. Gacamide A and other RsmE-regulated products appear to establish a physical boundary that prevents the encroachment of the competing genotype into the newly created space. Although cyclic lipopeptides and other biosurfactants are best known for their antimicrobial properties and reducing surface tension to promote the spreading of cells on various surfaces, they also appear to help define spatial structure formation within a dense community. IMPORTANCE In densely populated colonies of the bacterium Pseudomonas fluorescens Pf0-1, diverse mutations in the rsmE gene are naturally selected by solving the problem of overcrowding. Here, we show that RsmE-regulated secretions function together to create and protect space of low cell density. A biosurfactant generally promotes the spreading of bacterial cells on abiotic surfaces; however, it appears to function atypically within a crowded population by physically defining genotypic boundaries. Another significant finding is that these secretions are not regulated by RsmE's paralogs that share high sequence similarity. The experimental pipeline described in this study is highly tractable and should facilitate future studies to explore additional RsmE-regulated products and address why RsmE is functionally unique from its paralogs.