Abstract
Bacterial biofilms are large aggregates of cells embedded in an extracellular matrix of self-produced polymers. In macrocolony biofilms of Escherichia coli, this matrix is generated in the upper biofilm layer only and shows a surprisingly complex supracellular architecture. Stratified matrix production follows the vertical nutrient gradient and requires the stationary phase σS (RpoS) subunit of RNA polymerase and the second messenger c-di-GMP. By visualizing global gene expression patterns with a newly designed fingerprint set of Gfp reporter fusions, our study reveals the spatial order of differential sigma factor activities, stringent control of ribosomal gene expression and c-di-GMP signalling in vertically cryosectioned macrocolony biofilms. Long-range physiological stratification shows a duplication of the growth-to-stationary phase pattern that integrates nutrient and oxygen gradients. In addition, distinct short-range heterogeneity occurs within specific biofilm strata and correlates with visually different zones of the refined matrix architecture. These results introduce a new conceptual framework for the control of biofilm formation and demonstrate that the intriguing extracellular matrix architecture, which determines the emergent physiological and biomechanical properties of biofilms, results from the spatial interplay of global gene regulation and microenvironmental conditions. Overall, mature bacterial macrocolony biofilms thus resemble the highly organized tissues of multicellular organisms.
Highlights
A biofilm is defined as an aggregate of microbial cells that are embedded in a self-produced matrix of extracellular polymeric substances (EPS) and adhere to each other or to a surface [1,2,3,4]
In addition to this practical relevance, the ‘biofilm lifestyle’ became an attractive topic for molecular microbiologists when it was proposed that bacterial cells in a biofilm are in a specific physiological state that was conceived as the result of a developmental genetic programme [7,8], which is usually realized in several distinct steps involving checkpoints and commitments as exemplified in bacterial sporulation [9]
Heterogeneity of sS/c-di-GMP-driven matrix production within macrocolony biofilms appears on a long-range scale as a nutrient gradient-driven stratification as well as on a shortrange scale in the highly structured intermediate biofilm layer with its directly adjacent matrix-surrounded and matrix-free cells
Summary
A biofilm is defined as an aggregate of microbial cells that are embedded in a self-produced matrix of extracellular polymeric substances (EPS) and adhere to each other or to a surface [1,2,3,4]. A hallmark of biofilms is their pronounced tolerance against antibiotics and disinfectants, which causes severe medical and technical problems [5,6] In addition to this practical relevance, the ‘biofilm lifestyle’ became an attractive topic for molecular microbiologists when it was proposed that bacterial cells in a biofilm are in a specific physiological state that was conceived as the result of a developmental genetic programme [7,8], which is usually realized in several distinct steps involving checkpoints and commitments as exemplified in bacterial sporulation [9]. Recent studies with Escherichia coli macrocolony biofilms that grow for extended times on agar plates have shown that the extracellular matrix is produced in distinct biofilm zones, and seems arranged in a complex supracellular architecture.
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