Functionally Distinct Exopolysaccharides Dynamic Remodeling of Microbial Biofilms by 2014

Biofilms are densely populated communities of microbial cells protected and held together by a matrix of extracellular polymeric substances. The structure and rheological properties of the matrix at the microscale influence the retention and transport of molecules and cells in the biofilm, thereby dictating population and community behavior. Despite its importance, quantitative descriptions of the matrix microstructure and microrheology are limited. Here, particle-tracking microrheology in combination with genetic approaches was used to spatially and temporally study the rheological contributions of the major exopolysaccharides Pel and Psl in Pseudomonas aeruginosa biofilms. Psl increased the elasticity and effective cross-linking within the matrix, which strengthened its scaffold and appeared to facilitate the formation of microcolonies. Conversely, Pel reduced effective cross-linking within the matrix. Without Psl, the matrix becomes more viscous, which facilitates biofilm spreading. The wild-type biofilm decreased in effective cross-linking over time, which would be advantageous for the spreading and colonization of new surfaces. This suggests that there are regulatory mechanisms to control production of the exopolysaccharides that serve to remodel the matrix of developing biofilms. The exopolysaccharides were also found to have profound effects on the spatial organization and integration of P. aeruginosa in a mixed-species biofilm model of P. aeruginosa-Staphylococcus aureus. Pel was required for close association of the two species in mixed-species microcolonies. In contrast, Psl was important for P. aeruginosa to form single-species biofilms on top of S. aureus biofilms. Our results demonstrate that Pel and Psl have distinct physical properties and functional roles during biofilm formation. IMPORTANCE Most bacteria grow as biofilms in the environment or in association with eukaryotic hosts. Removal of biofilms that form on surfaces is a challenge in clinical and industrial settings. One of the defining features of a biofilm is its extracellular matrix. The matrix has a heterogeneous structure and is formed from a secretion of various biopolymers, including proteins, extracellular DNA, and polysaccharides. It is generally known to interact with biofilm cells, thus affecting cell physiology and cell-cell communication. Despite the fact that the matrix may comprise up to 90% of the biofilm dry weight, how the matrix properties affect biofilm structure, maturation, and interspecies interactions remain largely unexplored. This study reveals that bacteria can use specific extracellular polymers to modulate the physical properties of their microenvironment. This in turn impacts biofilm structure, differentiation, and interspecies interactions. Received 2 July 2014 Accepted 7 July 2014 Published 5 August 2014 Citation Chew SC, Kundukad B, Seviour T, van der Maarel JRC, Yang L, Rice SA, Doyle P, Kjelleberg S. 2014. Dynamic remodeling of microbial biofilms by functionally distinct exopolysaccharides. mBio 5(4):e01536-14. doi:10.1128/mBio.01536-14. Editor Rita Colwell, University of Maryland Copyright © 2014 Chew et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Staffan Kjelleberg, LASKJELLEBERG@ntu.edu. sg. This article is a direct contribution from a Fellow of the American Academy of Microbiology. Bacteria form surface-attached biofilm communities in nature and are significant in the context of environmental sustainability and health care (1, 2). Integral to the biofilm is a matrix of extracellular polymeric substances (EPS). The EPS form a heterogeneous viscoelastic material that holds the microbial cells together and account for 50 to 90% of the total organic matter in the biofilm (3). Division of labor in metabolism, gene transfer, and quorum sensing are facilitated in the biofilm because the matrix is responsible for maintaining the spatial organization of cells in the biofilm (4, 5). The EPS are largely composed of polysaccharides, proteins, nucleic acids, humic acids, and lipids and determine the architecture of the biofilm (6). For example, biofilms assume many, often highly differentiated forms during development, including mushroom-like microcolonies and filamentous streamers, as well as a homogenous, undifferentiated layer of cells. The physical properties of the matrix determine the shape and mechanical stability of biofilms (7, 8) and affect processes such as mass transfer and detachment kinetics of the various differentiated forms. For example, EPS shearing can form sieve-like networks that result in rapid accumulation of cells and other dispersed biomass that results in the formation of biofilm streamers (9). Sloughing and RESEARCH ARTICLE July/August 2014 Volume 5 Issue 4 e01536-14 ® mbio.asm. org 1 m b.asm . rg on A uust 1, 2014 P ubished by m b.asm . rg D ow nladed fom