The atlA operon of Streptococcus mutans: role in autolysin maturation and cell surface biogenesis

Abstract

The Smu0630 protein (AtlA) was recently shown to be involved in cell separation, biofilm formation, and autolysis. Here, transcriptional studies revealed that atlA is part of a multigene operon under the control of at least three promoters. The morphology and biofilm-forming capacity of a nonpolar altA mutant could be restored to that of the wild-type strain by adding purified AtlA protein to the medium. A series of truncated derivatives of AtlA revealed that full activity required the C terminus and repeat regions. AtlA was cell associated and readily extractable from with sodium dodecyl sulfate. Of particular interest, the surface protein profile of AtlA-deficient strains was dramatically altered compared to the wild-type strain, as was the nature of the association of the multifunctional adhesin P1 with the cell wall. In addition, AtlA-deficient strains failed to develop competence as effectively as the parental strain. Mutation of thmA, which can be cotranscribed with atlA and encodes a putative pore-forming protein, resulted in a phenotype very similar to that of the AtlA-deficient strain. ThmA was also shown to be required for efficient processing of AtlA to its mature form, and treatment of the thmA mutant strain with full-length AtlA protein did not restore normal cell separation and biofilm formation. The effects of mutating other genes in the operon on cell division, biofilm formation, or AtlA biogenesis were not as profound. This study reveals that AtlA is a surface-associated protein that plays a critical role in the network connecting cell surface biogenesis, biofilm formation, genetic competence, and autolysis.

FIG. 1.

Transcriptional analysis of the atlA region of wild-type and the atlA polar mutant (630P) of S. mutans UA159. (A) Schematic diagram of the atlA locus and RT-PCR in the junctions J1 (between atlA and smu0631), J2 (between smu0631 and pepT), and J3 (between pepT and thmA). Following reverse transcription with reverse primer smu0631-rt-RV (for J1), pepT-antisense (for J2), and thmA-antisense (for J3), PCR amplification was performed with primer sets of smu0630-rt-FW/smu0631-rt-RV, smu0631-rt-FW/pepT-antisense, and pepT-sense/thmA-antisense. The PCR products were run on Tris-acetate-EDTA gels. Lane 1, RT-PCR product; lane 2, negative control lacking RT; lane 3, positive control of PCR from chromosomal DNA of UA159. The higher M_r band in lane 1 of the 630P strain appears to arise from amplification of a product generated by low levels of read-through from the polar kanamycin cassette. We have measured read-through from the polar cassette by real-time PCR at about 1% of the atlA-smu0631 cotranscript (data not shown). (B) Real-time PCR. For measuring pepT and thmA mRNAs, RNA from UA159 was used for RT with a thmA-antisense primer. (C) Promoter activity of atlA, pepT, and thmA genes as measured by CAT assays. The promoter fusion of each gene with the cat gene was inserted in a single copy into the chromosome of the wild-type strain. Data presented are means ± standard deviations (error bars) of three independent experiments. *, P < 0.005 (Student's t test).

FIG. 2.

Growth phenotypes of S. mutans strains. (A) Growth in BHI broth was monitored in a Bioscreen C system. Data points are averages of triplicate samples. (B) Biofilm formation of S. mutans UA159 (wild type) and its derivatives (SAB66, SAB67, and SAB71) in BM supplemented with glucose or sucrose. See text for more details. Data are representative of at least two separate experiments. The error bars represent standard deviations. *, P < 0.005 (Student's t test).

FIG. 3.

Restoration activity of histidine-tagged derivatives of AtlA (630). Schematic illustration of the different AtlA constructs (A). Amino acid coordinates are shown in parentheses. Restoration of 630NP to wild-type behavior is shown for culture confluence (B) and biofilm formation (C) after treatment of a strain lacking AtlA (630NP) with various concentrations of the 630D1 and 630D5 proteins. The 630NP strain was grown in BHI medium for the observation of planktonic growth and in BM supplemented with glucose at a final concentration of 20 mM for 24 h to monitor biofilm formation. Biofilms were assayed in polystyrene microtiter plates by staining with crystal violet and were quantified by adding an ethanol-acetone mixture and reading the optical density at 575 nm. Data are representative of at least two separate experiments performed in triplicate.

FIG. 4.

SDS-PAGE analysis of different cell extracts from wild-type (WT) and 630NP (NP) strains of S. mutans: bead-beaten SDS-boiling extracts (A), 4% SDS extracts (B), bead-beaten Tris extracts (C), and surface-associated fractions extracted with 0.2% (wt/vol) Zwittergent in PBS (D). Following SDS-PAGE, proteins were either stained with Coomassie blue (top) or transferred onto a nitrocellulose membrane and subjected to Western blotting using an anti-630D1 polyclonal antiserum at the dilution of 1:350 (bottom). Lane M, size marker.

FIG. 5.

Effect of ThmA on the conversion of AtlA from a 107-kDa to a 79-kDa peptide. (A) Protein profiles in Coomassie-stained SDS-PAGE gel of UA159 (wild type; lane 1) and its derivatives, SAB66 (ΔpepT, polar mutant; lane 2), SAB67 (ΔpepT, nonpolar mutant; lane 3), and SAB71 (ΔthmA mutant; lane 4). (B) Effect of ThmA on the processing of exogenous 630D1. Two strains, 630NP (lane 1 and 2) and SAB78 (lanes 3 and 4), were grown in BHI broth; samples either were left untreated (lanes 1 and 3), or purified 630D1 was added to a final concentration of 20 ng/ml culture for 1 h (lanes 2 and 4). Following SDS-PAGE, proteins were stained with Coomassie blue (top) or transferred onto a nitrocellulose membrane and subjected to Western blotting using an anti-630D1 polyclonal antiserum (dilution 1:350) (bottom). Lane M, size marker. WT, wild type.

FIG. 6.

Western blot analysis of different cell extracts from wild-type (WT) and 630NP (NP) strains of S. mutans: bead-beaten SDS-boiling extracts (A), surface-associated fractions extracted with 0.2% (wt/vol) Zwittergent in PBS (B), 4% SDS extracts (C), and supernatant fraction, concentrated approximately 60-fold by ammonium sulfate (D). Following SDS-PAGE, proteins were transferred onto a nitrocellulose membrane and subjected to Western blotting using an anti-P1 monoclonal antiserum at the dilution of 1:700. Lane M, size marker.