Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall

Science

Volumn 285, Issue 5428, 1999, Pages 760-763

  Mazmanian, Sarkis K ^a   Liu, Gwen ^a   Ton That, Hung ^a   Schneewind, Olaf ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BACTERIAL ENZYME; MEMBRANE PROTEIN;

ARTICLE; CELL WALL; ENZYME ANALYSIS; GENE MUTATION; GENE OVEREXPRESSION; MEMBRANE BINDING; NONHUMAN; NUCLEOTIDE SEQUENCE; PRIORITY JOURNAL; STAPHYLOCOCCUS AUREUS;

AMINO ACID SEQUENCE; AMINOACYLTRANSFERASES; BACTERIAL PROTEINS; CELL WALL; CYSTEINE ENDOPEPTIDASES; GENES, BACTERIAL; GRAM-POSITIVE BACTERIA; MEMBRANE PROTEINS; MOLECULAR SEQUENCE DATA; MUTAGENESIS; PEPTIDOGLYCAN; PROTEIN SORTING SIGNALS; STAPHYLOCOCCUS AUREUS;

BACTERIA (MICROORGANISMS); POSIBACTERIA; STAPHYLOCOCCUS AUREUS;

EID: 0033618622     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.285.5428.760     Document Type: Article

References (30)

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15. 35S]-Promix (Amersham) for 5 min, and surface protein processing was quenched by addition of 75 μl of 100% trichloroacetic acid (TCA). TCA precipitates were collected by centrifugation, washed in acetone, and dried. Samples were suspended in 1 ml of 0.5 M tris-HCl (pH 7.5) and incubated for 1 hour at 37°C after addition of 50 μl of lysostaphin (2 mg/ml). Proteins were again precipitated with TCA and washed with acetone; after immunoprecipitation with α-enterotoxin B, they were analyzed by 14% SDS-polyacrylamide gel electrophoresis and Phosphorimager.
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24. 4 (predicted mass 6285, observed 6286).
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26. 490-524 by Eco RI-Bam HI digestion and inserted into the pT181 derivative pWilS. S. aureus SM317 (pGL4) was grown on TSB tet agar. A plasmid library of S. aureus OS2 chromosomal DNA was obtained by partial digestion with Sau 3A1. DNA fragments of 3 to 5 kb were purified and cloned into Bam HI-digested pC194-mcs, which contains the multiple cloning site of pUC19 inserted into the Hind III site of pC194. SM317 (pGL4) was transformed with the pC194-mcs plasmid library and transformants were selected on TSB tet-cm agar.
27. The DNA insertions of pGL1631 and 1834 were mapped and sequenced by synthesizing oligonucleotide primers. The primers for the amplification of srtA from the chromosomal DNA of S. aureus strains OS2 (pGL1897) and SM317 (pGL1898) were 5′-AAG-GATCCAAAAGGAGCGGTATACATTGC-3′ and 5′-AAGGATCCTACCTTTTCCTCTAGCTGAAG-3′.
28. In another report we show that purified SrtA protein catalyzes the in vitro transpeptidation of substrates bearing an LPXTG motif (H. Ton-That, G. Liu, S. K. Mazmanian, K. F. Faull, O. Schneewind, in preparation).
29. Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
30. We thank W. W. Navarre for help in determining the surface protein anchor structure of strain SM317, and D. Missiakas, P. Model, M. Russel, and members of our laboratory for critical reading of this manuscript. S.K.M. was supported by the Predoctoral Training Program in Genetic Mechanisms at UCLA (T32GM07104). H.T.-T. was supported by the Microbial Pathogenesis Training Grant at UCLA (AI 07323). Work in O.S.'s laboratory is supported by grant AI33987 from the National Institute of Allergy and Infectious Diseases.