A mRNA signal for the type III secretion of Yop proteins by Yersinia enterocolitica

Science

Volumn 278, Issue 5340, 1997, Pages 1140-

  Anderson, Deborah M ^a   Schneewind, Olaf ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ARTICLE; CELL KILLING; CYTOTOXICITY; PRIORITY JOURNAL; PROTEIN SECRETION; REPORTER GENE; SIGNAL TRANSDUCTION; YERSINIA ENTEROCOLITICA;

AMINO ACID SEQUENCE; BACTERIAL OUTER MEMBRANE PROTEINS; BACTERIAL PROTEINS; BASE SEQUENCE; CODON; FRAMESHIFT MUTATION; MEMBRANE PROTEINS; MOLECULAR SEQUENCE DATA; MUTATION; NUCLEIC ACID CONFORMATION; POINT MUTATION; PROTEIN BIOSYNTHESIS; RECOMBINANT FUSION PROTEINS; RNA, BACTERIAL; RNA, MESSENGER; YERSINIA ENTEROCOLITICA;

EID: 1842413699     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.278.5340.1140     Document Type: Article

References (30)

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11. 2-terminal sequence of purified YopE was confirmed by Edman degradation, and the molecule was subjected to electrospray ionization mass spectrometry. An average compound mass of 23,018.75 [mass-to-charge ratio (m/z) = 1212, 1280, 1355, 1440, 1645, 1772, 1919)] was observed, in agreement with a calculated compound mass of 23,016.10, given a standard error rate of 0.01 % (±2 daltons).
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17. 2-or COOH-terminal fusions, we replaced the central Nde I to Bam HI cassette with two fragments joined at a Kpn I site. The npt gene [E. Beck et al., Gene 19, 327 (1982)] was amplified by PCR with abutted Kpn I and Bam HI sites. The YopE and YopN fusions were amplified with flanking Nde I and Kpn I sites. Fusions harboring the first 10 or 15 codons of Yop mRNA were created by annealing oligonucleotides with overlapping ends for cloning between the Nde I and Kpn I sites. All constructs were verified by DNA sequencing.
18. 2-or COOH-terminal fusions, we replaced the central Nde I to Bam HI cassette with two fragments joined at a Kpn I site. The npt gene [E. Beck et al., Gene 19, 327 (1982)] was amplified by PCR with abutted Kpn I and Bam HI sites. The YopE and YopN fusions were amplified with flanking Nde I and Kpn I sites. Fusions harboring the first 10 or 15 codons of Yop mRNA were created by annealing oligonucleotides with overlapping ends for cloning between the Nde I and Kpn I sites. All constructs were verified by DNA sequencing.
19. Overnight cultures of Y. enterocolitica were diluted 1:50 into fresh M9 medium with casamino acids and grown for 2 hours at 26°C before incubation for 3 hours at 37°C. A portion (30 ml) of the induced culture (absorbance at 600 nm of 0.5) was centrifuged at 17,000g for 13 min, and 20 ml of supernatant was removed and precipitated with 5% trichloroacetic acid (TCA). The remainder of the supernatant was discarded, and the sedimented material was suspended in water (750 μl). A portion (500 μl) of this suspension was precipitated with ice-cold 10% TCA (500 μl). All TCA precipitates were washed in acetone, dissolved in sample buffer, and analyzed by immunoblotting with rabbit antiserum. Immunoreactive species were identified as a chemiluminescent signal and quantitated by laser-densitometry scanning of developed x-ray films. Immunoblotting for cytoplasmic chloramphenicol acetyltransferase served as an internal control for correct fractionation of Yersinia cultures.
20. 1-15-Npt was investigated with the membrane-impermeable reagent sulfosuccinimidobiotin (Pierce) [M. P. Lisanti et al., Proc. Natl. Acad. Sci. U.S.A. 85, 9557 (1988)]. The amount of fusion protein covalently modified with sulfosuccinimidobiotin was similar to that found to be soluble in the supernatant of centrifuged Yersinia cultures, suggesting that nonsecreted polypeptides remained within the bacterial cytosol. This result was confirmed by fractionating Yersinia cultures into medium, periplasm, cytosol, and membrane compartments [N. G. Davis, J. D. Boeke, P. Model, J. Mol. Biol. 181, 111 (1985)] followed by immunoblotting. Similar amounts of fusion proteins were found to be soluble in the culture medium and the bacterial cytosol. Upon ultracentrifugation of cell extracts, a small amount of hybrid protein sedimented together with the membranes, which may result from its association with the type III secretion machinery.
21. 1-15-Npt was investigated with the membrane-impermeable reagent sulfosuccinimidobiotin (Pierce) [M. P. Lisanti et al., Proc. Natl. Acad. Sci. U.S.A. 85, 9557 (1988)]. The amount of fusion protein covalently modified with sulfosuccinimidobiotin was similar to that found to be soluble in the supernatant of centrifuged Yersinia cultures, suggesting that nonsecreted polypeptides remained within the bacterial cytosol. This result was confirmed by fractionating Yersinia cultures into medium, periplasm, cytosol, and membrane compartments [N. G. Davis, J. D. Boeke, P. Model, J. Mol. Biol. 181, 111 (1985)] followed by immunoblotting. Similar amounts of fusion proteins were found to be soluble in the culture medium and the bacterial cytosol. Upon ultracentrifugation of cell extracts, a small amount of hybrid protein sedimented together with the membranes, which may result from its association with the type III secretion machinery.
22. Overnight cultures of Yersinia were diluted 1:20 into 20 ml of M9 minimal medium, grown for 2 hours at 26°C, and induced for 3 hours by temperature shift to 37°C. One milliliter of culture was labeled with 100 μCi of Pro-Mix for 1 min and precipitated with ice-cold TCA. The SDS-solubilized samples were immunoprecipitated with antiserum to purified YopH or Npt, separated by SDS-polyacrylamide gel electrophoresis, and quantified by Phosphorlmager.
23. 32P]cytidine 5′-triphosphate-labeled DNA sequences prepared by random primer labeling of restriction fragments corresponding to the full-length open reading frame of either npt or yopH.
24. 2-terminal amino acid sequence was determined by Edman degradation.
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26. -9 and were picked from the plates and patched onto fresh TSB agar supplemented with neomycin, chloramphenicol, and EGTA. Nitrocellulose filters were placed directly on the colonies and incubated in 1% SDS and lysozyme for 10 min. Colonies that reacted with antibodies to Npt were subsequently analyzed by immunoblotting for secretion of the Npt hybrid. Plasmid was isolated and transformed into W22703 to determine the linkage of the suppressor mutations to this DNA. Mutations were identified by DNA sequencing.
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30. We thank S. Daefler, M. Russel, R. Simons, and members of our laboratory for critically reading the manuscript, and G. R. Cornelis for the Y. enterocolitica strains. Supported by a grant from the Stein-Oppenheimer Foundation, the Department of Microbiology and Immunology (O.S.), and the Microbial Pathogenesis Training grant Al 07323 from the Public Health Service to the Department of Microbiology and Immunology at UCLA School of Medicine (D.M.A).