BACTERIAL BIOFILMS

⦿ filamentous cell formation is a vital prerequisite for Xylella fastidiosa biofilm formation

The morphological plasticity of bacteria to form filamentous cells commonly represents an adaptive strategy as a consequence of stresses such as starvation and DNA damage as commonly observed in bacteria. In contrast, for diverse pathogens, such as Vibrio cholera, Caulobacter crescent, and Pseudomonas aeruginosa, filamentous cells have been observed during biofilm formation, but solid evidence that such elongated cells are necessary for either triggering or development of biofilms remains fragmentary. To identify the function of filamentous cells and the trigger of cell morphogenesis, spatially controlled cell adhesion is pivotal. Here, we exploit the highly-selective cell adhesion of the biofilm-forming phytopathogen Xylella fastidiosa to gold-patterned SiO2 substrates with well-defined geometries and dimensions. Probing both cell density and distances between cell clusters using these patterns provided evidence of quorum sensing governing filamentous cell formation. While cell morphogenesis is induced by cell cluster density, filamentous cell growth is oriented towards neighboring cell clusters and distance-dependent; large interconnected cell clusters create the early biofilm structural framework. Our findings and investigative platform could facilitate therapeutic developments targeting biofilm formation of X. fastidiosa and other pathogens.     >Download Study<

⦿ Biofilm formation of phytopathogen Xylella fastidiosa at single-cell resolution

Microorganism pathogenicity relies on the generation of multicellular biofilm assemblies. Understanding their organization and spatiotemporal formation can provide new details that can lead to new targets for disease control. Here, the step-wise biofilm formation of the phytopathogen Xylella fastidiosa was investigated using nanometer-resolution spectro-microscopy techniques, addressing further the role of different types of extracellular polymeric substances (EPS) during the bacterial life cycle. The initial stage consists of reversible adhesion of planktonic cells to the surface via electrostatic interactions through transmembrane adhesins (XadA) at the CPR (cell polar region). Secreted EPS accumulates at the CPR, covering the surrounding surface allowing both transformation to irreversible attachment and facilitated adhesion of new planktonic cells. Subsequently, bacteria form clusters, which are embedded in EPS, and bridged by up to ten-fold elongated cells (filamentation) that form the biofilm framework. Due to the facilitated cell-attachment on EPS-covered areas between interconnected cell clusters, the biofilm maturates and the EPS forms a filamentous matrix that provides mechanical support. Due to common genetic traits with other bacteria, the results may be transferrable to human biofilm-forming pathogens.     >Download Study<

⦿ Adhesin-enhanced adhesion strength of single bacteria and biofilms

Single bacteria adhesion to a surface is the first critical step in biofilm formation. Specific extracellular components facilitate adhesion by reducing the energy barrier formed between approaching surfaces, a process that depends on bacteria-host adaptation mechanisms. Further complexity to the scenario of bacterial biofilm infection arises from the ability of many species to modulate cell adhesiveness by specific transmembrane adhesion proteins (XadA), and hence host colonization, in response to changing environmental conditions. Here, we employed spatially ordered nanowire arrays to evaluate Xylella fastidiosa single-cell adhesion forces and explore their dependence on organic surface compositions. Quantitative measurements of cell adhesion forces were obtained by observing the displacement of nanowire tip positions upon interaction and adhesion of individual cells to nanowire arrays. To probe the cell holdfast in dependence of a transmembrane adhesin, XadA1-functionalized nanowire arrays directly assess the impact of XadA1 on cell-surface interaction, mimicking its presence in outer membrane vesicles (OMV), which are released in living environment. The adhesin significantly increased the cell holdfast forces up to ~ 45 nN, suggesting that adhesins are promoters of cell adhesion. From the biological point of view, adhesins provide important implications in biofilm fitness and pathogenicity.     >Download Study<

⦿ Surface physicochemical properties modulate biofilm formation efficiency

The bacterial biofilm formation depends on a number of factors, e.g. nutrients, pH, temperature, and physicochemical properties of cells and surfaces. Surface properties can be altered by adsorption of (macro)molecules from the liquid environment forming a conditioning film, which affects initial bacterial adhesion. In microbiology research, studies are focused on the question which different substrate surface properties – such as roughness, topography, viscoelasticity, hydrophobicity and charge density – play crucial roles in biofilm formation. Here, we address the role of surface properties at different length scales on adhesion of the phytopathogen Xylella fastidiosa. Different biotic and abiotic substrates were studied in respect to biofilm formation efficiency, characterized at the micro- and nanoscale. Single bacteria adhesion and biofilm growth is facilitated on hydrophilic surfaces providing higher electric surface potentials. Gene expression analyses show that different types of cellulose modulates bacterial adhesion, development rate, and architecture of mature biofilms. Probing the role of transmembrane adhesion proteins (XadA) revealed different strengths of interaction on different surfaces. Our results unequivocally support the hypothesis that different adhesion mechanisms are active along the biofilm life cycle, representing an adaptation mechanism for variations in surface composition.     >Download Study<

⦿ Modulation of Tol-Pal cell envelope complex dynamics during biofilm formation

The most important stage of biofilm-forming bacteria is the adherence of planktonic bacteria to surfaces. The adhesion stage requires the expression of transmembrane adhesins (XadA), a key element for niche- and host-specific colonization. The ubiquitous Tol/Pal system is associated with the cell membrane integrity and participates in cell division. Pal is an outer membrane-anchored lipoprotein that resides in the periplasmic space. TolB is an allosteric ß-propeller protein that acts in the bacterial periplasmic space and may interact with Pal and the cell-killing proteins categorized as group A colicins. Here, we characterized the TolB and Pal proteins of the phytopathogen Xylella fastidiosa. We confirmed the role of TolB and Pal in outer membrane integrity and biofilm formation. Small-angle X-ray scattering provided further the structure of isolated XfTolB-XfPal complexs in solution for the first time. Surprisingly, XfTolB and XfPal are localized at cell poles during bacterial cell growth.     >Download Study<

⦿ Structural changes upon light activation in transmembrane sensory rhodopsin ii

Microbial rhodopsins are a family of seven-helical transmembrane proteins containing retinal as chromophore and can be found in all three domains of life. Upon light absorption, sensory rhodopsin II (SRII) triggers in Archea two very different responses, depending on presence or absence of its cognate transducer HtrII: light activation of the NpSRII/NpHtrII complex initiates photophobic response while NpSRII alone acts as a proton pump. Using single-molecule force spectroscopy, we solved the long-standing contradictory arguments about the structural changes and interactions between SRII and its transducer. With a newly developed analysis of protein-unfolding data, we were able to reveal the localization protein-protein interactions with a resolution of six amino acids. While the majority of the results are agreement with precious models on the molecular stability of NpSRII, this study revealed several new interaction sites within the SRII and its transducer. We identified that upon light activation structural changes cause a clockwise and cell-inwards movement of the transducer, activating a signalling cascade homologous to the two-component system of eubacterial chemotaxis.     >Download Study<