Abstract
The survival of bacteria in nature is greatly enhanced by their ability to grow within surface-associated communities called biofilms. Commonly, biofilms generate proliferations of bacterial cells, called microcolonies, which are highly recalcitrant, 3-dimensional foci of bacterial growth. Microcolony growth is initiated by only a subpopulation of bacteria within biofilms, but processes responsible for this differentiation remain poorly understood. Under conditions of crowding and intense competition between bacteria within biofilms, microevolutionary processes such as mutation selection may be important for growth; however their influence on microcolony-based biofilm growth and architecture have not previously been explored. To study mutation in-situ within biofilms, we transformed Pseudomonas aeruginosa cells with a green fluorescent protein gene containing a +1 frameshift mutation. Transformed P. aeruginosa cells were non-fluorescent until a mutation causing reversion to the wildtype sequence occurs. Fluorescence-inducing mutations were observed in microcolony structures, but not in other biofilm cells, or in planktonic cultures of P. aeruginosa cells. Thus microcolonies may represent important foci for mutation and evolution within biofilms. We calculated that microcolony-specific increases in mutation frequency were at least 100-fold compared with planktonically grown cultures. We also observed that mutator phenotypes can enhance microcolony-based growth of P. aeruginosa cells. For P. aeruginosa strains defective in DNA fidelity and error repair, we found that microcolony initiation and growth was enhanced with increased mutation frequency of the organism. We suggest that microcolony-based growth can involve mutation and subsequent selection of mutants better adapted to grow on surfaces within crowded-cell environments. This model for biofilm growth is analogous to mutation selection that occurs during neoplastic progression and tumor development, and may help to explain why structural and genetic heterogeneity are characteristic features of bacterial biofilm populations.
Highlights
Replicating cells under conditions of cellular crowding, for example within eukaryotic malignancies, are constrained by intensely competitive and nutrient limited growth conditions
Despite many recent advances in understanding genetic determinants involved in biofilm formation, the growth and development of discrete 3dimensional microcolony structures within bacterial biofilm communities remains poorly understood
We show that microevolutionary processes are involved in the structural development of bacterial biofilms
Summary
Replicating cells under conditions of cellular crowding, for example within eukaryotic malignancies, are constrained by intensely competitive and nutrient limited growth conditions. In these circumstances microevolutionary processes, such as mutation selection, are often important for growth. Bacteria often face similar constraints for growth in crowded cell populations. They largely exist within matrix-encased and densely packed communities of cells called biofilms. Cells within microcolonies often proliferate rapidly while other biofilm bacteria are non-dividing and do not increase their biovolume [7]. Studies indicate that early-stage microcolonies of this kind are clonal structures derived from a single cell within the biofilm [8,9]
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