During the first step of biofilm formation, initial attachment is dictated by physicochemical and electrostatic interactions between the surface and the bacterial envelope. Depending upon the nature of these interactions, attachment can be transient or permanent. To achieve irreversible attachment, bacterial cells have developed a series of surface adhesins promoting specific or nonspecific adhesion under various environmental conditions. This chapter will review the recent advances in our understanding of the secretion, assembly and regulation of the bacterial adhesins during biofilm formation with a particular emphasis on the fimbrial, non-fimbrial and discrete polysaccharide adhesins in Gram-negative bacteria.

17Bacterial adhesion is affected by environmental factors, such as ionic strength, pH, 18 temperature, and shear forces, and therefore marine bacteria must have developed holdfasts 19 with different composition and structures than their freshwater counterparts to adapt to their 20 natural environment. The dimorphic a-proteobacterium Hirschia baltica is a marine budding 21 bacterium in the Caulobacterales clade. H. baltica uses a polar adhesin, the holdfast, located at 22 the cell pole opposite the reproductive stalk for surface attachment and cell-cell adhesion. The 23 holdfast adhesin has been best characterized in Caulobacter crescentus, a freshwater member 24 of the Caulobacterales, and little is known about holdfast composition and properties in marine 25 Caulobacterales. Here we use H. baltica as a model to characterize holdfast properties in 26 marine Caulobacterales. We show that freshwater and marine Caulobacterales use similar 27 genes in holdfast biogenesis and that these genes are highly conserved among the two genera. 28We also determine that H. baltica produces larger holdfast than C. crescentus and that those 29 holdfasts have a different chemical composition, as they contain N-acetylglucosamine and 30 galactose monosaccharide residues and proteins, but lack DNA. Finally, we show that H. baltica 31 holdfasts tolerate higher ionic strength than those of C. crescentus. We conclude that marine 32 Caulobacterales holdfasts have physicochemical properties that maximize binding in high ionic 33 strength environments. 34 35 IMPORTANCE 36 Most bacteria spend a large amount of their lifespan attached to surfaces, forming 37 complex multicellular communities called biofilms. Bacteria can colonize virtually any surface, 38 therefore they have adapted to bind efficiently in very different environments. In this study, we 39 compare the adhesive holdfasts produced by the freshwater bacterium C. crescentus and a 40 relative, the marine bacterium H. baltica. We show that H. baltica holdfasts have a different 41 3 morphology and chemical composition, and tolerate high ionic strength. Our results show that H. 42 baltica holdfast is an excellent model to study the effect of ionic strength on adhesion and 43 providing insights on the physicochemical properties required for adhesion in the marine 44 environment. 45 46 48 communities, known as biofilms (1). To irreversibly adhere to surfaces and form these complex 49 mutil-cellular communities, bacteria produce strong adhesins, mainly composed of proteins or 50 polysaccharides (2, 3). Bacterial adhesion is affected by different environmental conditions such 51 as pH, temperature, shear forces, and ionic strength (2,(4)(5)(6). In marine environments, bacteria 52 face 500 times higher ionic strength than in freshwater (7), therefore, marine bacteria have 53 evolved ways to overcome the effect of ionic strength and bind permanently to surfaces in high 54 salt environments such as seas and oceans. 55Caulobacterales are Alphaproteobacteria found in various habitats, from olig...

Organisms are adept in generating inorganic materials (biominerals) with structural and mechanical properties superior to those of abiotically formed minerals. Increasing efforts in interdisciplinary research are aimed at understanding how mineral-forming organisms achieve their outstanding control over the assembly and properties of minerals. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] Emergent knowledge from biomineralization research has been exploited recently for the fabrication of functional hybrid materials under mild reaction conditions in vitro, including the formation of silica and non-silica materials by proteins from sponges and diatoms. [17][18][19][20] These methods included immobilization of enzymes and other functional proteins inside a silica matrix. Such materials are attractive for sensor technology (e.g. biosensors, microarrays, microfluidic devices), as reusable biocatalysts for organic syntheses and degradation of harmful chemicals (remediation), and for drug delivery. [21a,b] While a variety of methods are available for protein immobilization by attachment to silica/silicate surfaces or physical entrapment inside silica, such approaches rely on costly reagents and bear a high risk of denaturing the protein. [22][23][24] In contrast, biomineral-forming organisms have a natural ability for immobilizing proteins, since each biomineral is composed of an inorganic matrix and tightly associated proteins.[1] Of particular interest are the silicabased cell walls of diatoms (frustules), which exhibit highly porous, nanopatterned microshapes with excellent mechanical properties. [25][26][27] Diatom silica is, therefore, desirable for many applications including use as a support matrix for biomolecules. [28][29][30][31][32] A method for the chemical attachment of DNA to diatom silica has recently been developed, [33] but attachment of functional proteins has not yet been achieved. Recent progress in analysis of the molecular mechanism of silica biogenesis in diatoms [34] has spurred us to explore a radically different approach for protein immobilization. The approach is based on genetic manipulation of the biological silicaforming machinery and enabled immobilization of the bacterial enzyme HabB in the nanoporous biosilica structures of the diatom Thalassiosira pseudonana. This is the first demonstration of an in vivo method for immobilization of an active protein in a biomineral. The method represents a paradigm for utilizing the unique capabilities of biomineralforming organisms to enable the production of nanopatterned materials with tailored functionalities.Our studies were performed with the diatom Thalassiosira pseudonana, because its biosilica-associated proteins are well characterized [35a] and a genetic transformation system for this organism has been established.[35b] T. pseudonana produces cylindrical silica structures containing a vast number of irregularly arranged, spherical nanopores with a very narrow size distribution ((18.3 AE 3.1) nm in diameter) [35c] (see the Supp...