Biofilm formation and sloughing in Serratia marcescens are controlled by quorum sensing and nutrient cues

Abstract

We describe here a role for quorum sensing in the detachment, or sloughing, of Serratia marcescens filamentous biofilms, and we show that nutrient conditions affect the biofilm morphotype. Under reduced carbon or nitrogen conditions, S. marcescens formed a classical biofilm consisting of microcolonies. The filamentous biofilm could be converted to a microcolony-type biofilm by switching the medium after establishment of the biofilm. Similarly, when initially grown as a microcolony biofilm, S. marcescens could be converted back to a filamentous biofilm by increasing the nutrient composition. Under high-nutrient conditions, an N-acyl homoserine lactone quorum-sensing mutant formed biofilms that were indistinguishable from the wild-type biofilms. Similarly, other quorum-sensing-dependent behaviors, such as swarming motility, could be rendered quorum sensing independent by manipulating the growth medium. Quorum sensing was also found to be involved in the sloughing of the filamentous biofilm. The biofilm formed by the bacterium consistently sloughed from the substratum after approximately 75 to 80 h of development. The quorum-sensing mutant, when supplemented with exogenous signal, formed a wild-type filamentous biofilm and sloughed at the same time as the wild type, and this was independent of surfactant production. When we removed the signal from the quorum-sensing mutant prior to the time of sloughing, the biofilm did not undergo significant detachment. Together, the data suggest that biofilm formation by S. marcescens is a dynamic process that is controlled by both nutrient cues and the quorum-sensing system.

FIG. 1.

Effect of quorum sensing and media on filamentous biofilm formation in S. marcescens. (A and B) Confocal microscopic images of 3-day-old biofilms of S. marcescens wild-type strain MG1 and the quorum-sensing mutant MG44, respectively. (C to F) MG1 (C), MG44 (D), bsmA (E), and bsmB (F) mutant 3-day-old biofilms grown in 0.1× LB medium. In each panel the top image is the x-y plane, and the arrowhead indicates the position corresponding to the x-z cross section in the lower image. Magnification, ×377. (A to D) Bar, 50 μm. (E and F) Bar, 20 μm.

FIG. 2.

Changes in medium composition lead to the formation of classic microcolony biofilms by S. marcescens: confocal microscopic images of 3-day-old wild-type biofilms of S. marcescens in MBD with 0.1% (wt/vol) sodium citrate plus 0.05% (wt/vol) CAA (A) and in MBD with 0.1% (wt/vol) l-glutamic acid plus 0.05% (wt/vol) CAA (B). In each panel the top image is the x-y plane, and the arrowhead indicates the position corresponding to the x-z cross section in the lower image. Magnification, ×400. Bar, 50 μm.

FIG. 3.

Detachment of S. marcescens biofilms: confocal microscope images of the wild type and the QS mutant grown with 500 nM BHL for the duration of the experiment prior to sloughing at 70 h (A and C) and after sloughing at 80 h (B and D). In each panel the top image is the x-y plane, and the arrowhead indicates the position corresponding to the x-z cross section in the lower image. Magnification, ×400. Bar, 50 μm.

FIG. 4.

Sloughing profiles of S. marcescens strains. Sloughing of the biofilm was measured by collecting effluent from the biofilm as described in Materials and Methods. Briefly, effluent from three flow cells per experiment was collected and vortexed to disperse clumps, and the optical density at 610 nm was determined. The sloughing profiles are the profiles for the wild type (circles), the swrI mutant MG44 grown with 500 nM BHL (triangles), and MG44 grown with 500 nM BHL for 70 h, after which the medium was switched to BHL-deficient MBD (squares). The data are data for triplicate flow cells from one experiment and are representative of three independent experiments.

FIG. 5.

Model for quorum-sensing control of surface colonization by S. marcescens.