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. 1998 Jan;180(2):290-5.
doi: 10.1128/JB.180.2.290-295.1998.

The adhesion-associated sca operon in Streptococcus gordonii encodes an inducible high-affinity ABC transporter for Mn2+ uptake

P E Kolenbrander  1 , R N Andersen, R A Baker, H F Jenkinson
Affiliations

The adhesion-associated sca operon in Streptococcus gordonii encodes an inducible high-affinity ABC transporter for Mn2+ uptake

P E Kolenbrander et al. J Bacteriol. 1998 Jan.

Abstract

ScaA lipoprotein in Streptococcus gordonii is a member of the LraI family of homologous polypeptides found among streptococci, pneumococci, and enterococci. It is the product of the third gene within the scaCBA operon encoding the components of an ATP-binding cassette (ABC) transporter system. Inactivation of scaC (ATP-binding protein) or scaA (substrate-binding protein) genes resulted in both impaired growth of cells and > 70% inhibition of 54Mn2+ uptake in media containing < 0. 5 microM Mn2+. In wild-type and scaC mutant cells, production of ScaA was induced at low concentrations of extracellular Mn2+ (< 0. 5 microM) and by the addition of > or = 20 microM Zn2+. Sca permease-mediated uptake of 54Mn2+ was inhibited by Zn2+ but not by Ca2+, Mg2+, Fe2+, or Cu2+. Reduced uptake of 54Mn2+ by sca mutants and by wild-type cells in the presence of Zn2+ was abrogated by the uncoupler carbonylcyanide m-chlorophenylhydrazone, suggesting that Mn2+ uptake under these conditions was proton motive force dependent. The frequency of DNA-mediated transformation was reduced > 20-fold in sca mutants. The addition of 0 VSports手机版. 1 mM Mn2+ to the transformation medium restored only partly the transformability of mutant cells, implying an alternate role for Sca proteins in the transformation process. Cells of sca mutants were unaffected in other binding properties tested and were unaffected in sensitivity to oxidants. The results show that Sca permease is a high-affinity mechanism for the acquisition of Mn2+ and is essential for growth of streptococci under Mn2+-limiting conditions. .

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Figures

FIG. 1
FIG. 1
Structure of the sca locus in S. gordonii PK488 and delineation of the coding sequences and directions of transcription (arrows). Sites of insertion of ermAM to generate scaC (PK3041) and scaA (OB309) mutants of S. gordonii DL1 are indicated. Segments amplified by PCR for subsequent insertional inactivation are shown as striped bars. Base pair numbers are in accordance with sequence L11577 deposited in GenBank. Abbreviations of restriction enzymes: B, BglII; E, EcoRI; Ev, EcoRV; H, HindIII; P, PstI; S, SalI. Sites confirmed to be present in both PK488 and DL1 are shown in boldface type.
FIG. 2
FIG. 2
Immunoblot detection of ScaA expression in wild-type (DL1) or scaC mutant (PK3041) cells in defined media containing Mn2+ (0.1 to 10 μM) in the absence (A) or presence (B) of 20 μM Zn2+. Cultures were grown for 12 h at 37°C and the cells were harvested. Surface proteins were extracted, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and blotted onto nitrocellulose. Panels A and B show reactions with ScaA antibodies, and as a control Panel C shows a reaction with antibodies to HppA oligopeptide-binding lipoprotein from S. gordonii (25). Protein loadings were equalized (50 μg per lane) except for PK3041 cells grown in 0.1 μM Mn2+ with Zn2+, where <10 μg of protein was loaded because of the 80%-reduced growth yield of cells (see text and Fig. 3).
FIG. 3
FIG. 3
Growth yields of wild-type and sca mutant strains after 16 h of incubation at 37°C in defined media containing different concentrations of Mn2+ (0.1 to 10 μM) in the absence (open symbols) or presence (filled symbols) of 40 μM Zn2+ (as ZnSO4 · 7H2O). Data are expressed as percentages of the growth yield of wild-type cells in 10 μM Mn2+ without added Zn2+, where 100% was equivalent to an optical density at 600 nm of 2.69 (5.4 × 109 cells/ml). Symbols: circles, DL1 (wild type); triangles, OB309 (scaA1::ermAM); squares, PK3041 (scaC::ermAM).
FIG. 4
FIG. 4
54Mn2+ uptake by wild-type or sca mutant cells. (A) Uptake by wild-type cells (open circles) in the presence of nonradioactive Mn2+ (as MnCl2 · 4H2O); (B) uptake in 0.5 μM Mn2+ by wild-type cells (open circles) and scaA (open triangles) and scaC (open squares) mutant cells and the effect of Zn2+ on uptake by wild-type cells; (C) uptake in the presence of 0.5 μM Mn2+ by wild-type cells (control; open circles) or in the presence of 50 mM dGlc and the effect of the addition of 100 μM CCCP (solid circles) in the absence or presence of 40 μM Zn2+; (D) effect of Zn2+, dGlc, or CCCP on uptake in 0.5 μM Mn2+ by scaA (triangles) and scaC (squares) mutants. Values are plotted as percentages of initial 54Mn2+ uptake, where 100% uptake was equivalent to 3.8 pmol of Mn2+/108 cells.
FIG. 5
FIG. 5
Growth and transformability to Str of a wild-type strain (A) and scaA (B) and scaC (C) mutant strains in transformation medium (<0.1 μM Mn2+) without (0) or with Mn2+ (10 or 100 μM) or with CSP (400 ng/ml added 20 min prior to the addition of 50 ng of Str-transforming DNA/ml). Early-exponential-phase cells were incubated with DNA for 30 min, 10 μg of DNAse I/ml was then added, and the cultures were incubated for a further 90 min before the numbers of total CFU (filled bars) and Str CFU (open bars) were determined by viable-cell agar plate count. Values are percentages of Str transformants.
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