13 C nuclear magnetic resonance studies on selectively labeled bacterial biofilms

Abstract

Bacterial biofilms of Pseudomonas aeruginosa selectively labeled by introduction of 2-(13)C-glycerol was studied by solid-state and high-resolution nuclear magnetic resonance. The (13)C nuclei were mainly integrated into mannuronate and guluronate, the two monomer units forming the bacterial alginate. The signal for the C5 position of the mannuronate, which was easily identified and well separated from other peaks, was analyzed for molecular mobility. The result indicated a high degree of motional freedom within the molecular network of the alginate. Despite the fact that the alginate was part of a solid aqueous gel phase, the reorientation mechanism of the monomer units came close to isotropic tumbling. Solid-state spectra of biofilms labeled in the described manner may serve as a valuable tool for noninvasive analyses of molecular mobility of the alginate component under various influences, thereby revealing important structural information. In addition, the effect of a monovalent electrolyte (LiCl) on the molecular mobility of alginate fragments in an aqueous solution was studied by determining the spin-lattice relaxation times, line widths and line shapes under variations of the ion concentration. The presence of ions accelerated overall motions but left rapid local motions virtually unaffected.

Magnetic resonance microscopy of biofilm structure and impact on transport in a capillary bioreactor

“…MR studies of single biofilms growing on bioreactors, a state most relevant to biomedical applications, have been limited and either did not image biofilm structure or used a negative contrast agent such as uptake of copper sulfate [10,11]. Bryers and Drummond [14] point out the hydrated gel nature of biofilms and the MR work of Mayer et al [9,20] verifies the gel structure through identification of proteins, polysaccharides, and nucleic acids in the EPS and comparison of physical response with model gel systems. The polymer gel nature of the EPS is a significant component of reduced nutrient and antimicrobial mass transport in biofilms [14].…”

“…Studies of the diffusive behavior of biofilms include studies of the diffusion of the water within the biofilm [7] and the polymer molecules themselves [8]. 13 C spectroscopy has been applied to determine the integral chemical composition of the extracellular polymeric substance and behavior of the gel as a function of environmental conditions such as electrolyte concentration [9,20]. A primary limitation of most MR studies to date, other than those of biofilms grown in porous media [3,5], is the fact that the biofilms are transferred from their natural growth state on a surface and concentrated for study in the NMR system [7][8][9]20].…”

“…13 C spectroscopy has been applied to determine the integral chemical composition of the extracellular polymeric substance and behavior of the gel as a function of environmental conditions such as electrolyte concentration [9,20]. A primary limitation of most MR studies to date, other than those of biofilms grown in porous media [3,5], is the fact that the biofilms are transferred from their natural growth state on a surface and concentrated for study in the NMR system [7][8][9]20]. MR studies of single biofilms growing on bioreactors, a state most relevant to biomedical applications, have been limited and either did not image biofilm structure or used a negative contrast agent such as uptake of copper sulfate [10,11].…”

Raman and Surface-Enhanced Raman Scattering for Biofilm Characterization

“…Nuclear magnetic resonance (NMR) is another technique used to obtain structural information on the substances in biofilm matrices [12]. Although the technique gives an opportunity to acquire information in situ from a biofilm, it is not suitable for characterizing high molecular weight molecules due to the complexity of interpreting the obtained data [13].…”

Correlated biofilm imaging, transport and metabolism measurements via combined nuclear magnetic resonance and confocal microscopy

“…Previous NMR studies of live prokaryotic cell suspensions, gelimmobilized cells and extracts have provided detailed metabolic information (Junter et al, 2002). Several NMR studies have explored extracts or concentrated samples of cells grown as biofilms (Vogt et al, 2000;Mayer et al, 2001;Hu et al, 2004).…”