The Influence of Biofilms in the Biology of Plasmids

Formation of a bacterial biofilm. Bacterial biofilm development involves three stages. (1) Initial attachment of single group or small groups of bacteria to a surface, often aided by attachment structures such as pili. (2) Growth of these attached cells as well as attachment of additional cells increases the biomass of the biofilm. Concurrently, the bacteria produce an extracellular matrix made of up various components including DNA, protein, and polysaccharides that help the biofilm retain its structure and keep the biofilm cells attached to the surface and to each other. (3) During and after the formation of a large biofilm, individual cells or even large pieces of the biofilm may break off. These detached cells may go on to live a planktonic lifestyle or seed new biofilms. Dark lines indicate the components of the matrix used to attach cells to each other and the surface such as eDNA ( 1 ), while orange extracellular material indicates other matrix components used to retain biofilm structure and surface attachment.

The interplay between conjugation and biofilm development. (Top) A model proposed by Ghigo ( 15 ), based on his analysis of conjugation between E. coli strains. Planktonic populations of donor cells (green), carrying plasmids such as R1, whose conjugation functions are normally repressed, contain a few spontaneously depressed individuals. When these depressed cells encounter a biofilm containing recipient cells (white), they can attach via their sex pili (red) and transfer the plasmid. In newly generated transconjugants, there is a transient period where repression of conjugation is not operative. This can be followed by “epidemic spread” of the plasmid through the biofilm population, and the associated production of sex pili also can increase the biofilm biomass directly. (Bottom) In E. faecalis, expression of conjugation is regulated by peptide-mating pheromones produced by recipient cells. In the scenario depicted on the left, the pheromone produced by recipient cells (white) in a biofilm turns on expression of conjugation in planktonic donor cells (blue) in close proximity, and the resulting synthesis of pheromone-induced surface adhesins (thick, gray layer) promotes both an increase in biofilm resulting from increased attachment of planktonic cells, and also leads to plasmid transfer within the biofilm. In the right, the development of a mixed biofilm as a result of attachment of both donors and recipients to the same surface may allow for signaling and conjugation between sessile donor and recipient cells in close proximity ( 34 ).

Three mechanistic models that could account for increased ratios of plasmids/chromosome in a fraction of E. faecalis biofilm cells. Figures depict actively growing “planktonic-like” diplococcal cells prior to cell division. Each cell half contains two copies of the chromosome (represented by thick lines), where a new round of replication has initiated on one chromosome (indicated by the “bubble”). Each cell half also contains three copies of a plasmid, with the dashed lines indicating rolling circle replication initiated by either a single-stranded replication initiator protein or a conjugative relaxase. (It should be noted that some plasmids showing increased copy number in biofilms actually use a “theta” mode of replication similar to the chromosome [ 35 ]). Each diagram contains one diplococcal cell (left) with a plasmid copy number similar to that of planktonic cells and a second cell with an altered copy number, representing a subpopulation of the biofilm community. The three diagrams illustrate three possible mechanisms by which the plasmid/chromosome ratio could be increased in these subpopulations. (A) Secretion of eDNA into the biofilm matrix by a fraction of cells reduces the ratio of chromosome/plasmid. (B) Biofilm growth reduces chromosomal copy number in subpopulations by inhibiting initiation of chromosome replication. (C) In a subpopulation of biofilm cells, changes in cellular physiology could disrupt normal mechanisms limiting conjugative nicking or vegetative plasmid replication initiation, leading to increases in copy number. Additionally, for some well-studied plasmids, such as ColE1, blockage of chromosome replication initiation (B) can lead to “runaway” plasmid replication ( 36 ). Based on published results ( 1 , 34 ), all of these models are postulated to operate in the early stages of biofilm development, when the adherent cells are still growing, and there is no significant death or lysis.