Autoinducer 2 affects biofilm formation by Bacillus cereus

Abstract

Cell-free supernatants from growing Bacillus cereus strain ATCC 10987 induced luminescence in a Photorhabdus luminescens DeltaluxS mutant, indicating the production of functional autoinducer 2 (AI-2). The exogenous addition of in vitro synthesized AI-2 had an inhibitory effect on biofilm formation by B. cereus and promoted release of the cells from a preformed biofilm.

FIG. 1.

Biofilm formation by B. cereus ATCC 10987. (A) Photograph of a B. cereus biofilm stained with crystal violet. Panels: 1, LB medium inoculated with strain ATCC 10987; 2, LB medium alone. (B) OD₅₉₅ of solubilized crystal violet from microtiter plate assay (filled circles) and CFU/ml of attached cells (open circles) in biofilms over time. After various times of incubation, biofilm density was measured as described in the text. The data represent the means of three independent experiments. The error bars represent standard deviations.

FIG. 2.

Growth-dependent AI-2 production by B. cereus ATCC 10987. B. cereus ATCC 10987 was grown in LB medium (open circles). At various time points, the amount of AI-2 in CFS (filled squares) was measured by using the P. luminescens bioassay. Addition of LB medium alone to P. luminescens wild-type strain TT01 (100% RLU) and to the ΔluxS mutant P12102 (0% RLU) was used to define the reference levels of luminescence. The data indicated by squares represent the means (± standard deviations) of three independent preparations.

FIG. 3.

Effect of AI-2 on biofilm formation by B. cereus ATCC 10987. (A) Different concentrations of in vitro synthetized AI-2 were added to microtiter wells inoculated with strain ATCC 10987 in LB medium. After 24 h of incubation, the biofilm density was measured. The data represent the means (± standard deviations) of triplicate experiments. (B) Time course of biofilm formation in the presence (gray bars) or absence (white bars) of 1 μM AI-2. Experiments were run in triplicate.

FIG. 4.

Effect of AI-2 on mature biofilms. After 24 h of incubation in microtiter plates, free cells were removed and replaced by fresh medium alone or fresh medium containing 3, 4.5, or 6 μM AI-2. Incubation was continued for 24 h, and biofilm density was measured. The means of three independent experiments are indicated.

FIG. 5.

Genetic organization and sequence analysis of the lsr region in B. cereus ATCC 10987 and E. coli K-12. Putative transcription start sites are indicated by broken arrows. For each gene product, the similarity between the B. cereus and E. coli proteins is indicated as a percentage of identity. LsrB, periplasmic AI-2 binding protein; LsrC and LsrD, channel proteins; LsrA, ATPase; LsrF, a protein similar to aldolases; LsrG and Bce3018, proteins with no significant similarity with known proteins; LsrR, repressor of the lsr operon; LsrK, AI-2 kinase. The genes encoding the ABC transporters are represented by striped boxes, the LsrR-like regulators are represented by gray boxes, and the processing enzymes are represented by checkered boxes (lsrF-like) or a diagonally striped box (lsrG).