LuxS-based signaling in Streptococcus gordonii: autoinducer 2 controls carbohydrate metabolism and biofilm formation with Porphyromonas gingivalis

Abstract

Communication based on autoinducer 2 (AI-2) is widespread among gram-negative and gram-positive bacteria, and the AI-2 pathway can control the expression of genes involved in a variety of metabolic pathways and pathogenic mechanisms. In the present study, we identified luxS, a gene responsible for the synthesis of AI-2, in Streptococcus gordonii, a major component of the dental plaque biofilm. S. gordonii conditioned medium induced bioluminescence in an AI-2 reporter strain of Vibrio harveyi. An isogenic mutant of S. gordonii, generated by insertional inactivation of the luxS gene, was unaffected in growth and in its ability to form biofilms on polystyrene surfaces. In contrast, the mutant strain failed to induce bioluminescence in V. harveyi and was unable to form a mixed species biofilm with a LuxS-null strain of the periodontal pathogen Porphyromonas gingivalis. Complementation of the luxS mutation in S. gordonii restored normal biofilm formation with the luxS-deficient P. gingivalis. Differential display PCR demonstrated that the inactivation of S. gordonii luxS downregulated the expression of a number of genes, including gtfG, encoding glucosyltransferase; fruA, encoding extracellular exo-beta-D-fructosidase; and lacD encoding tagatose 1,6-diphosphate aldolase. However, S. gordonii cell surface expression of SspA and SspB proteins, previously implicated in mediating adhesion between S. gordonii and P. gingivalis, was unaffected by inactivation of luxS. The results suggest that S. gordonii produces an AI-2-like signaling molecule that regulates aspects of carbohydrate metabolism in the organism. Furthermore, LuxS-dependent intercellular communication is essential for biofilm formation between nongrowing cells of P. gingivalis and S. gordonii.

FIG. 1.

Schematic representation of the luxS locus of S. gordonii DL1 wild type (A), luxS mutant strain 1.1L (B), and complemented strain 1.1LC (C). The luxS gene was amplified from S. gordonii DL1 genomic DNA with the primers LuxF and LuxR; ermAM was ligated to a unique NdeI site (N) within the luxS gene, and the construct was transformed back onto the streptococcal chromosome (see Materials and Methods). Complemented strain 1.1LC was created by Campbell-like integration of plasmid pVALuxS carrying an intact copy of the luxS gene. The solid line indicates chromosomal DNA, and the broken line represents pVA981 DNA. (D) Expression of luxS by S. gordonii DL1 (lane 1) or mutant strains 1.1L (lane 2) or 1.1LC (lane 3). Total RNA was isolated from cells harvested at the late exponential phase of growth in TSBY medium and used in RT-PCR with luxS-specific primers LuxF3 and LuxR3.

FIG. 2.

Induction of V. harveyi BB170 luminescence by cell-free supernatants of S. gordonii DL1 wild type, the luxS mutant strain 1.1L, and the complemented strain 1.1LC. Culture supernatants were prepared from the mid-exponential-, late-exponential-, or stationary-growth phase of growth in TY-glucose medium. Sterile medium and cell-free supernatants from V. harveyi BB170 served as negative and positive controls, respectively, and data are presented as the percent light induction relative to V. harveyi BB170 positive control. (The BB170 positive control gave a ca. 5,000-fold increase compared to the negative control.)

FIG. 3.

Bacterial growth and biofilm formation of S. gordonii DL1 wild type or luxS mutant strain 1.1L. Cells from an exponential-phase culture of S. gordonii were washed once in sterile water and suspended at an OD of 600 nm (OD₆₀₀) of 0.01 in fresh prewarmed medium. Cultures were then grown in the wells of polystyrene microtiter plates at 37°C for 16 h anaerobically. Growth and biofilm formation were quantified by determining the OD₄₉₀ and OD₅₆₂ values, respectively. The results are presented as the mean ± the standard deviation for quadruplicate determinations.

FIG. 4.

Confocal microscopy of biofilm development on saliva-coated glass in flow cells with strains of P. gingivalis (green) and S. gordonii (red). P. gingivalis PLM1 adheres to S. gordonii 1.1LC (A) and accretes into biofilm microcolonies (B) similar to those formed by wild-type P. gingivalis 33277 and S. gordonii DL1 (C). P. gingivalis PLM1 and S. gordonii 1.1L coadhere (D); however accumulation of P. gingivalis PLM1 in the z dimension, to a depth greater than 10 μm, does not occur (E). The white lines in panels A and D represent the orientation in the x-y perspective, whereas B and E are the generated sagittal x-z perspectives. Bar, 10 μm.

FIG. 5.

RT-PCR analysis of mRNA expression of the indicated genes in S. gordonii DL1 and 1.1L. The results are representative of three separate preparations of total RNA. Fold induction or repression in 1.1L is indicated numerically or as “+” or “−” to show that mRNA expression could not be detected in either DL1 or 1.1L, respectively. The ratio of induction or repression in 1.1L was calculated from the relative intensity of linear range amplification products of RNA from DL1 and 1.1L normalized levels of sca mRNA (control) that was not regulated in 1.1L.

FIG. 6.

In-gel GTF activities of culture supernatant of S. gordonii strains. The position of the 174-kDa native GTF protein band is indicated. The lower-molecular-mass forms of GTF are thought to result from proteolytic degradation of the native enzyme (65). The gel shown is representative of three independent experiments.