SMU.746-SMU.747, a putative membrane permease complex, is involved in aciduricity, acidogenesis, and biofilm formation in Streptococcus mutans

Abstract

Dental caries induced by Streptococcus mutans is one of the most prevalent chronic infectious diseases worldwide. The pathogenicity of S. mutans relies on the bacterium's ability to colonize tooth surfaces and survive a strongly acidic environment. We performed an ISS1 transposon mutagenesis to screen for acid-sensitive mutants of S. mutans and identified an SMU.746-SMU.747 gene cluster that is needed for aciduricity. SMU.746 and SMU.747 appear to be organized in an operon and encode a putative membrane-associated permease. SMU.746- and SMU.747-deficient mutants showed a reduced ability to grow in acidified medium. However, the short-term or long-term acid survival capacity and F1F0 ATPase activity remained unaffected in the mutants. Furthermore, deletion of both genes did not change cell membrane permeability and the oxidative and heat stress responses. Growth was severely affected even with slight acidification of the defined medium (pH 6.5). The ability of the mutant strain to acidify the defined medium during growth in the presence of glucose and sucrose was significantly reduced, although the glycolysis rate was only slightly affected. Surprisingly, deletion of the SMU.746-SMU.747 genes triggered increased biofilm formation in low-pH medium. The observed effects were more striking in a chemically defined medium. We speculate that the SMU.746-SMU.747 complex is responsible for amino acid transport, and we discuss its possible role in colonization and survival in the oral environment.

FIG 1

(A) Confirmation of acid-sensitive phenotype. ISS1 transposon mutants that displayed an initial acid-sensitive phenotype were further verified by spotting of 5.0 μl from a 10-fold dilution series, with a starting optical density (A₆₀₀) of 2.0 made in 0.85% NaCl, onto THY agar plates at pH 5.5 or 7.0. Experiments were repeated at least three times, and the relevant areas of the representative plates are shown. UA159 is the wild-type strain, while 3S3F, 7A5G, and 61B11C are independent mutants. (B) Genetic organization of the SMU.745-SMU.747 region of S. mutans UA159. Locations of ISS1 insertion are shown with inverted triangles. The lollipop indicates putative transcription termination. Plasmids used for promoter analysis are indicated. (C) Plasmids used for construction of the deletion mutants and complementation are shown. Arrows indicate gene orientation.

FIG 2

Synteny among the genomic regions of S. mutans (GenBank accession no. AE014133), Streptococcus agalactiae (AL735626), Streptococcus pneumoniae (AE007317), Streptococcus pyogenes (AE004092), and Streptococcus thermophilus (CP000023) containing SMU.745-SMU.747 homologs, indicated by dark gray. Syntenic regions are shown with gray boxes.

FIG 3

(A) Effect of low pH on growth on THY plates. (B) Growth kinetics of the S. mutans mutants and the parental UA159 strains in liquid medium. (C) IBSL32 with the pIBL36 or pIB184Km plasmid in liquid medium. For growth on pH 5.0 and 7.0 THY agar plates, diluted overnight cultures (10 μl) were spot dried and plates were inoculated for 48 h at 37°C in 5% CO₂. Growth kinetics was measured as described in Materials and Methods. Strains were grown in THY medium at pH 5.0, 5.5, and 7.0 (marked as black dotted, gray, and black lines, respectively). Samples are as follows: S. mutans UA159 (diamonds), IBSL16 (squares), IBSL28 (triangles), IBSL32 (circles), IBSL32/pIBL36 (pentagons), and IBSL32/pIB184Km (asterisks).

FIG 4

Biofilm formation of S. mutans UA159 and the SMU.746-SMU.747 mutant in THY (A) or CDM plus 1% sucrose (B) medium. Amounts of biofilm formation (A₅₇₀/A₅₉₅) are shown as black, gray, and white bars for the wild-type, IBSL32, and IBSL32/pIBL36 strains, respectively; and horizontal lines mark planktonic growth. The data are averages with standard deviations of the results from at least four independent experiments. (C) Microscopy photographs of 2-day-old biofilm formed by S. mutans UA159 (left panel) and the SMU.746-SMU.747 mutant strain (right panel) in THY pH 5.5 medium (magnification, ×10 [upper panel] or ×100 [lower panel]); red lines represent 100 and 10 μm for the upper and the lower panels, respectively.

FIG 5

Effect of acid stress on cell survival and acidogenesis of S. mutans strains in different media. (A) Acid killing of S. mutants UA159 (diamonds) and IBSL32 mutant (circles) strains. Aliquots were plated on THY agar upon suspension in 0.1 M glycine (pH 2.8, gray lines; pH 7.0, black lines) and each 12.5 min thereafter. (B) A long-term survival assay was carried out as described in Materials and Methods. Aliquots of overnight cultures were plated daily on THY agar. The data are the averages with standard deviations of the results from two independent experiments. Black lines represent THY plus 1% glucose; gray lines are CDM plus 1%glucose. WT, IBSL32, and IBSL32pIBL36 strains are marked as diamonds, circles, and triangles, respectively. (C) Growth and acid production in THY and TY plus 1% glucose measured in 18-h cultures. (D) Growth and acid production in CDM with 1% glucose or sucrose in 2-day-old cultures. The data represent average values of 5 independent experiments.

FIG 6

Drop of external pH in S. mutans cell suspensions after induction with 1% glucose. Cells were resuspended in 0.05 M phosphate-citrate buffer, pH 7.0 (A) or pH 5.0 (B). Samples are as follows: S. mutans UA159 (WT), IBSL32, and complemented IBSL32. Controls represent average pH values for all 3 strains without induction. The data are averages with standard deviations of results from three independent experiments.