Molecular and Functional Analysis of the Type IV Pilus Gene Cluster in Streptococcus sanguinis SK36

Abstract

Streptococcus sanguinis, dominant in the oral microbiome, is the only known streptococcal species possessing a pil gene cluster for the biosynthesis of type IV pili (Tfp). Although this cluster is commonly present in the genome of S. sanguinis, most of the strains do not express Tfp-mediated twitching motility. Thus, this study was designed to investigate the biological functions encoded by the cluster in the twitching-negative strain S. sanguinis SK36. We found that the cluster was transcribed as an operon, with three promoters located 5' to the cluster and one in the intergenic region between SSA_2307 and SSA_2305. Studies using promoter-cat fusion strains revealed that the transcription of the cluster was mainly driven by the distal 5' promoter, which is located more than 800 bases 5' to the first gene of the cluster, SSA_2318. Optimal expression of the cluster occurred at the early stationary growth phase in a CcpA-dependent manner, although a CcpA-binding consensus is absent in the promoter region. Expression of the cluster resulted in a short hairlike surface structure under transmission electron microscopy. Deletion of the putative pilin genes (SSA_2313 to SSA_2315) abolished the biosynthesis of this structure and significantly reduced the adherence of SK36 to HeLa and SCC-4 cells. Mutations in the pil genes downregulated biofilm formation by S. sanguinis SK36. Taken together, the results demonstrate that Tfp of SK36 are important for host cell adherence, but not for motility, and that expression of the pil cluster is subject to complex regulation.IMPORTANCE The proteins and assembly machinery of the type IV pili (Tfp) are conserved throughout bacteria and archaea, and yet the function of this surface structure differs from species to species and even from strain to strain. As seen in Streptococcus sanguinis SK36, the expression of the Tfp gene cluster results in a hairlike surface structure that is much shorter than the typical Tfp. This pilus is essential for the adherence of SK36 but is not involved in motility. Being a member of the highly diverse dental biofilm, perhaps S. sanguinis could more effectively utilize this structure to adhere to host cells and to interact with other microbes within the same niche.

Keywords: CcpA; Streptococcus sanguinis; adherence; biofilms; type IV pilus.

FIG 1

(A) Schematic representation and transcriptional organization of the S. sanguinis SK36 pil cluster. The gene name based on BLASTP analysis is indicated within or above the gene. The SSA_tag number is listed below the gene. The locations of PCR products shown in panel B are indicated by horizontal lines below the gene map, above the SSA_tag number. The locations of the three transcription initiation sites determined by 5′ RACE are indicated by bent arrows on solid lines. The putative promoter demonstrated by the cat fusion study is indicated by a bent arrow on a dashed line. The putative termination sites are indicated by a lollipop structure. The region that was replaced with Ωkan in strain YC9 or erm in strain YC11 is indicated by vertical arrows. (B) RT-PCR analysis demonstrating the transcripts of the pil genes. The SSA_tag number or intergenic region corresponding to the PCR products is listed above the gel photograph. Lanes 1 through 3 are PCR products generated from cDNAs of S. sanguinis SK36, YC9, and YC11, respectively. M, 1-kb DNA ladder.

FIG 2

Identification of the transcription initiation sites of the pil cluster by 5′ RACE. (A, left) Sequencing analysis of the final PCR products. The transcription initiation site is indicated by an inverted triangle. (Right) Sequence of the putative promoter 5′ to the transcription initiation site and distance of the listed sequence to the ATG codon of SSA_2318. The extended −10 element of p_pilB-1 and the putative −35 and −10 elements of p_pilB-2 and p_pilB-3 are shaded. (B) The identity of the pil-specific transcript generated by p_pilB-3 was verified by RT-PCR. (Left) Map displaying the relative locations of the three transcription initiation sites and primers used in PCR (indicated by inverted flags). (Right) PCR results. The primer pair used for each product is shown on the top of the gel photograph. C, PCR products generated from the S. sanguinis SK36 chromosome; +, RT included in the reaction mixtures; −, control reactions without RT; M, 1-kb DNA ladder.

FIG 3

CAT-specific activities of the recombinant S. sanguinis strains harboring a wild-type pilB promoter-cat translational fusion or fusions driven by the derivatives of the promoter region. The relative locations of the three promoters are shown at the top. The name of the fusion strain and its genotype are listed to the left of the graph describing CAT-specific activity. The location of spe in the fusion is indicated by an inverted triangle. The putative −10 element was replaced with 5′-CTGCAG (open boxes) and 5′-CTCGAC (filled boxes) by site-directed mutagenesis. All strains were harvested at an OD₆₀₀ of 0.8. Values are the means and standard deviations from three independent experiments. Significant differences between the fusion of the wild-type promoter and fusions of the mutated promoters were analyzed using one-way ANOVA followed by a Dunnett test. *, P < 0.01.

FIG 4

CAT-specific activities of the recombinant S. sanguinis strains harboring either a transcriptional cat (TY25) or a translational cat (TY26) fusion of p₂₃₀₅. The sequence 5′ to SSA_2305 in wild-type SK36 or cat in strains TY25 and TY26 is listed below the graph. Bases identical to SK36 are in gray. The RBS of cat is in italic type. The BamHI site was engineered for cloning purposes. Both strains were harvested at an OD₆₀₀ of 0.8. Values are the means and standard deviations from three independent experiments. N.D., not detectable.

FIG 5

Impact of CcpA on expression of the wild-type pilB promoters in response to growth phase. The CAT-specific activities of wild-type S. sanguinis SK36 (WT), its ccpA deletion derivative (ΔccpA), and the complementation strain (CΔccpA) at OD₆₀₀s of 0.3 (I) and 0.8 (II) are shown. Values are the means and standard deviations from three independent experiments. Significant differences between strains at each OD₆₀₀ value were analyzed using one-way ANOVA followed by a Tukey test. ***, P < 0.0001; **, P < 0.001; *, P < 0.01.

FIG 6

Piliation of S. sanguinis examined by TEM. Cells were immunogold labeled with anti-SSA_2315 antiserum and examined by TEM at a ×200,000 magnification, as detailed in Materials and Methods.

FIG 7

Efficiencies of adherence of S. sanguinis SK36 and its derivatives to HeLa (A) and SCC-4 (B) cells. The percent adherence was calculated as (CFU adhered/CFU of the inoculum) × 100%. The numbers are the means and standard deviations from three independent experiments. All reactions were done in triplicate. Significant differences between strains were analyzed using one-way ANOVA followed by a Tukey test. ***, P < 0.0001; **, P < 0.001; *, P < 0.01.

FIG 8

Biofilm formation of S. sanguinis SK36 and its derivatives. The final growth yield was measured at 490 nm (open bars), and the biofilm mass was quantified at 562 nm (filled bars). The values are the means and standard deviations from three independent experiments. Significant differences between wild-type SK36 and its derivatives were analyzed using one-way ANOVA followed by a Dunnett test. ***, P < 0.0001.