Autoinducer 2 production by Streptococcus gordonii DL1 and the biofilm phenotype of a luxS mutant are influenced by nutritional conditions

Abstract

The luxS gene, present in many bacterial genera, encodes the autoinducer 2 (AI-2) synthase. AI-2 has been implicated in bacterial signaling, and this study investigated its role in biofilm formation by Streptococcus gordonii, an organism that colonizes human tooth enamel within the first few hours after professional cleaning. Northern blotting and primer extension analyses revealed that S. gordonii luxS is monocistronic. AI-2 production was dependent on nutritional conditions, and maximum AI-2 induction was detected when S. gordonii was grown in the presence of serum and carbonate. In planktonic cultures, AI-2 production rose sharply during the transition from exponential to stationary phase, and the AI-2 concentration peaked approximately 4 h into stationary phase. An S. gordonii luxS mutant that did not produce AI-2 was constructed by homologous recombination. Complementation of the mutant by insertion of an intact luxS gene into the chromosome in tandem with the disrupted gene restored AI-2 production to a level similar to that of the wild-type strain. In planktonic culture, no growth differences were observed between the mutant and wild-type strains when five different media were used. However, when grown for 4 h as biofilms in 25% human saliva under flow, the luxS mutant formed tall microcolonies that differed from those formed by the wild-type and complemented mutant strains. Biofilms of the luxS mutant exhibited finger-like projections of cells that extended into the flow cell lumen. Thus, the inability to produce AI-2 is associated with altered microcolony architecture within S. gordonii biofilms formed in saliva during a time frame consistent with initial colonization of freshly cleaned enamel surfaces.

FIG. 1.

(A) Linear map of 6.4 kb of genomic DNA flanking S. gordonii luxS. Predicted ORFs and their orientations are shown. A predicted stem-loop structure between ORF2 and luxS is indicated. (B) Schematic representations of the chromosomal regions encompassing the ermAM-disrupted gene of the S. gordonii luxS mutant and the tandem disrupted and intact gene copies of the complemented luxS mutant. The image is not drawn to scale.

FIG. 2.

Primer extension mapping of the S. gordonii luxS transcription start site. Labeled cDNA from reverse transcription reactions was run next to luxS sequencing reaction products (lanes G, A, T, and C) generated with the same primer. Reverse transcription products from reaction mixtures containing total RNA isolated from wild-type S. gordonii (lane 1), the complemented luxS mutant (lane 2), E. coli DH5α(pSF151-luxS) (lane 3), and E. coli DH5α without a plasmid (lane 4) are shown. An expanded view of the complementary nucleotide sequence surrounding the transcription start site (+1) is shown, and the putative extended −10 hexamer is highlighted with a vertical bar.

FIG. 3.

S. gordonii exhibits maximum induction of AI-2 production in THBS. Time courses of culture densities (A) and AI-2 induction levels (B) of wild-type (WT; open circles), luxS mutant (open squares), and complemented (Comp) luxS mutant (open triangles) S. gordonii strains grown in THBS are shown. Culture densities and AI-2 induction levels of the wild-type strain grown in THB lacking serum (dotted lines, no symbols) and in TY-glucose medium (filled diamonds) are also shown. All time course experiments were repeated at least six times, and the averages of three representative independent experiments with standard deviations are shown (B). KU, Klett units.

FIG. 4.

Induction of S. gordonii AI-2 production by Na₂CO₃ in a concentration-dependent manner. Time courses of culture densities (A) and AI-2 induction levels (B) of wild-type S. gordonii grown in BHIS supplemented with 0 mM (circles), 12 mM (squares), 24 mM (triangles), and 48 mM (diamonds) Na₂CO₃. All time course experiments were repeated at least six times, and the averages of three representative independent experiments with standard deviations are shown (B). KU, Klett units.

FIG. 5.

Contribution of carbonate ion to S. gordonii AI-2 production. Time courses of culture densities (A) and AI-2 induction levels (B) of wild-type S. gordonii grown in BHI medium (circles) or BHI medium supplemented with 24 mM MOPS (squares), 24 mM Na₂CO₃ (triangles), and 24 mM NaHCO₃ (diamonds). All time course experiments were repeated at least six times, and the averages of three representative independent experiments with standard deviations are shown (B). KU, Klett units.

FIG. 6.

S. gordonii luxS mutant biofilm phenotype is not apparent in BHI medium. (A) Time course (left to right) of biofilm development in medium-conditioned flow cells (10-fold-diluted BHI medium containing 2.4 mM Na₂CO₃) by luxS mutant (luxS) and wild-type (WT) S. gordonii strains. x-z reconstructions of each biofilm are shown below each x-y image. Digital zooms (magnification, 3×) of the lower left corner of each 1-h x-y image are shown as insets. (B) Digital zooms (magnification, 3×) of the center of each 1-h x-z reconstruction. Scale bars, 50 μm.

FIG. 7.

S. gordonii luxS mutant biofilm phenotype in saliva. (A) Time courses (left to right) of biofilm development in saliva-conditioned flow cells by luxS mutant (luxS), wild-type (WT), and complemented luxS mutant (Comp) S. gordonii strains. x-z reconstructions of each biofilm are shown below each x-y image. Digital zooms (magnification, 3×) of the lower left corner of each 1-h x-y image are shown as insets. (B) Digital zooms (magnification, 3×) of the center of each 1-h x-z reconstruction. Scale bars, 50 μm.