Spatiotemporal distribution of different extracellular polymeric substances and filamentation mediate Xylella fastidiosa adhesion and biofilm formation.

Richard Janissen,Marcia L A Temperini,Prasana K Sahoo,Hernandes F Carvalho,Marcelo M Nobrega,Alessandra A De Souza,Duber M Murillo,Barbara Niza,Carlos L Cesar,Monica A Cotta

doi:10.1038/srep09856

Abstract

Microorganism pathogenicity strongly relies on the generation of multicellular assemblies, called biofilms. Understanding their organization can unveil vulnerabilities leading to potential treatments; spatially and temporally-resolved comprehensive experimental characterization can provide new details of biofilm formation, and possibly new targets for disease control. Here, biofilm formation of economically important phytopathogen Xylella fastidiosa was analyzed at single-cell resolution using nanometer-resolution spectro-microscopy techniques, addressing the role of different types of extracellular polymeric substances (EPS) at each stage of the entire bacterial life cycle. Single cell adhesion is caused by unspecific electrostatic interactions through proteins at the cell polar region, where EPS accumulation is required for more firmly-attached, irreversibly adhered cells. Subsequently, bacteria form clusters, which are embedded in secreted loosely-bound EPS, and bridged by up to ten-fold elongated cells that form the biofilm framework. During biofilm maturation, soluble EPS forms a filamentous matrix that facilitates cell adhesion and provides mechanical support, while the biofilm keeps anchored by few cells. This floating architecture maximizes nutrient distribution while allowing detachment upon larger shear stresses; it thus complies with biological requirements of the bacteria life cycle. Using new approaches, our findings provide insights regarding different aspects of the adhesion process of X. fastidiosa and biofilm formation.

Highlights

{ Current address: Kavli Institute of Nanoscience, Department of Bionanoscience, Delft University of Technology, 2628CJ Delft, The Netherlands
The obtained wide-field epifluorescence microscopy (WFM) data (Fig. 1a) indicate that single bacteria are adhered vertically through the cell polar region (CPR) to the surface; cell precession movement around a vertical axis passing through the pole attached to the surface can be observed (Supplementary Movie S1)
Our Scanning Electron Microscopy (SEM) data of adhered cells (Supplementary Fig. S1) suggest that the initial, reversible cell adhesion could be mediated by fimbrial pili structures, which are mainly located at the CPR, in agreement with previous studies[27,28,29]

Summary

Introduction

Bacterial growth usually exhibits statistical similarities[3,4]; it is important to create a reference framework for biofilm growth dynamics, against which to compare morphological features of samples cultivated under different conditions, even with the same growth time To fulfill this goal, several aspects of the stages proposed in the current model[2] have yet to be observed in detail during biofilm formation, at single cell or even higher resolutions. The authors, acknowledge that their data may not be extrapolated to other systems.[11] most studies show limited snapshots of isolated stages of biofilm development for different bacteria or focus on the role of a specific actor throughout the process; notwithstanding, a solid understanding on biofilm formation should benefit from consistent observations along the whole biological cycle of a single bacterial species. When sufficiently large, biofilms occlude the xylem vessels, disturbing water and nutrient transport[21,24,25,26]

Methods

Results

Conclusion