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ABSTRACT Bacteria form multicellular aggregates called biofilms. A crucial component of these aggregates is a protective matrix that holds the community together. Biofilm matrix composition varies depending upon bacterial species but typically includes exopolysaccharides (EPS), proteins, and extracellular DNA.Pseudomonas aeruginosais a model organism for the study of biofilms, and in non-mucoid biofilms, it uses the structurally distinct EPS Psl and Pel, the EPS-binding protein CdrA, and eDNA as key matrix components. An interesting phenomenon that we and others have observed is that the periphery of a biofilm aggregate can be EPS-rich and contain very few cells. In this study, we investigated two possible models of assembly and dynamics of this EPS-rich peripheral region: (i) newly synthesized EPS is inserted and incorporated into the existing EPS-rich region at the periphery during biofilm aggregate growth or (ii) EPS is continuously turned over and newly synthesized EPS is deposited at the outermost edge of the aggregate. Our results support the latter model. Specifically, we observed that new EPS is continually deposited at the aggregate periphery, which is necessary for continued aggregate growth but not aggregate stability. We made similar observations in another paradigm biofilm-forming species,Vibrio cholerae. This pattern of deposition raises the question of how EPS is retained. Specifically, forP. aeruginosabiofilms, the matrix adhesin CdrA is thought to retain EPS. However, current thinking is that cell-associated CdrA is responsible for this retention, and it is not clear how CdrA might function in the relatively cell-free aggregate periphery. We observed that CdrA is enzymatically degraded during aggregate growth without negatively impacting biofilm stability and that cell-free CdrA can partially maintain aggregation and Psl retention. Overall, this study shows that the matrix ofP. aeruginosabiofilms undergoes both continuous synthesis of matrix material and matrix turnover to accommodate biofilm aggregate growth and that cell-free matrix can at least partially maintain biofilm aggregation and EPS localization. Furthermore, our similar observations forV. choleraebiofilms suggest that our findings may represent basic principles of aggregate assembly in bacteria. IMPORTANCEHere, we show that, to accommodate growing cellular biomass, newly produced Psl is deposited over existing Psl at the periphery of biofilm aggregates. We demonstrated thatV. choleraeemploys a similar mechanism with its biofilm matrix EPS, VPS. In addition, we found that the protease LasB is present in the biofilm matrix, resulting in degradation of CdrA to lower molecular weight cell-free forms. We then show that the released forms of CdrA are retained in the matrix and remain functional. Together, our findings support that theP. aeruginosabiofilm matrix is dynamic during the course of aggregate growth and that other species may employ similar mechanisms to remodel their matrix. 

Abstract Biofilms are ubiquitous surface-associated bacterial communities embedded in an extracellular matrix. It is commonly assumed that biofilm cells are glued together by the matrix; however, how the specific biochemistry of matrix components affects the cell-matrix interactions and how these interactions vary during biofilm growth remain unclear. Here, we investigate cell-matrix interactions inVibrio cholerae, the causative agent of cholera. We combine genetics, microscopy, simulations, and biochemical analyses to show thatV. choleraecells are not attracted to the main matrix component (Vibriopolysaccharide, VPS), but can be attached to each other and to the VPS network through surface-associated VPS and crosslinks formed by the protein Bap1. Downregulation of VPS production and surface trimming by the polysaccharide lyase RbmB cause surface remodeling as biofilms age, shifting the nature of cell-matrix interactions from attractive to repulsive and facilitating cell dispersal as aggregated groups. Our results shed light on the dynamics of diverse cell-matrix interactions as drivers of biofilm development. 

Many pathogenic bacteria form biofilms as a protective measure against environmental and host hazards. The underlying structure of the biofilm matrix consists of secreted macromolecules, often including exopolysaccharides. To escape the biofilm, bacteria may produce a number of matrix-degrading enzymes, including glycosidic enzymes that digest exopolysaccharide scaffolds. The human pathogenVibrio choleraeassembles and secretes an exopolysaccharide called VPS (Vibriopolysaccharide) which is essential in most cases for the formation of biofilms and consists of a repeating tetrasaccharide unit. Previous studies have indicated that a secreted glycosidase called RbmB is involved inV.choleraebiofilm dispersal, although the mechanism by which this occurs is not understood. To approach the question of RbmB function, we recombinantly expressed and purified RbmB and tested its activity against purified VPS. Using a fluorescence-based biochemical assay, we show that RbmB specifically cleaves VPSin vitrounder physiological conditions. Analysis of the cleavage process using mass spectrometry, solid-state NMR, and solution NMR indicates that RbmB cleaves VPS at a specific site (at the α-1,4 linkage between D-galactose and a modified L-gulose) into a mixture of tetramers and octamers. We demonstrate that the product of the cleavage contains a double bond in the modified guluronic acid ring, strongly suggesting that RbmB is cleaving using a glycoside lyase mechanism. Finally, we show that recombinant RbmB fromV.choleraeand the related aquatic speciesVibrio coralliilyticusare both able to disrupt livingV.choleraebiofilms. Our results support the role of RbmB as a polysaccharide lyase involved in biofilm dispersal, as well as an additional glycolytic enzyme to add to the toolbox of potential therapeutic antibacterial enzymes.