Supramolecular Structure of the Salmonella typhimurium Type III Protein Secretion System
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C. Ginocchio, J. E. Galán, Infect. Immun. 63, 729 (1995); Rosqvist R., Hakansson S., Forsberg A., Wolf-Watz H., EMBO J. 14, 4187 (1995).
J. E. Galán, Mol. Microbiol. 20, 263 (1996); Shea J. E., Hensel M., Gleeson C., Holden D. W., Proc. Natl. Acad. Sci. U.S.A. 93, 2593 (1996); H. Ochman, F. C. Soncini, F. Solomon, E. A. Groisman,ibid., p. 7800.
C. Collazo, J. E. Galán, Mol. Microbiol. 24, 747 (1997); Y. Fu, J. E. Galán, ibid 27, 359 (1998); Hardt W.-D., Galán J. E., Proc. Natl. Acad. Sci. U.S.A. 94, 9887 (1997).
Salmonella typhimurium strains were derivatives of SJW1103 [S. Yamaguchi, H. Fujita, A. Ishihara, S. Aizawa, R. M. Macnab, J. Bacteriol.166, 187 (1986)] or SL1344 [Hoiseth S. K., Stocker B. A., Nature291, 238 (1981)]. The S. typhimurium flagellar mutants examined were ΔflhC, flgA,flgB, flgC, flgD, flgE,flgF, flgG, flhA, flhB,flhC, fliE, fliF, fliG,fliH, fliI, fliJ, fliM,fliN, Δflio-fliR, fliP,ΔfliP-fliR, fliR, and fliQ.
T. Kubori et al. unpublished results.
Pegues D. A., Hantman M. J., Behlau I., Miller S. I., ibid17, 169 (1995).
Bacteria were grown in L broth to an absorbance at 600 nm of 0.8 to 1.0 pelleted and resuspended in 1/10 of the original volume in a solution of 0.5 M sucrose and 0.15 M Trisma base. Lysozyme and EDTA were added to a final concentration of 1 mg/ml and 10 mM respectively and cells were incubated at 4°C for 1 hour. Spheroplasts were lysed by the addition of lauryldimethylamine oxide (LDAO) to a final concentration of 1% and the samples were further incubated at 4°C for 2 hours. MgSO 4 was added to a final concentration of 10 mM and samples were centrifuged at 5000 g for 20 min. The clarified sample was adjusted to pH 11 with NaOH incubated for 1 hour at 4°C and centrifuged at 60 000 g for 1 hour. The pellet was resuspended in a solution containing 0.1 M KCl-KOH (pH 11) 0.5 M sucrose and 0.1% LDAO and centrifuged at 60 000 g for 1 hour. The pellet was resuspended in TET buffer [10 mM tris-HCl (pH 8.0) 5 mM EDTA and 0.1% LDAO] and loaded onto a 30% (w/v) CsCl density gradient. Gradient fractions were centrifuged at 60 000 g for 1 hour and the pellets were washed with TET buffer.
Polypeptides separated on standard SDS-polyacrylamide gels were electroblotted onto polyvinylidene difluoride membranes and the bands of interest were applied to a Beckman LF3000 protein sequencer.
The amino acid sequences obtained from the different polypeptide species were as follows: 62 kD SEKIPVTGSG; 52 kD METSKEKTI; and 31 kD CKDKD (19).
N. A. Linderoth, M. N. Simon, M. Russel, Science 278, 1635 (1997); Koster M., et al., Mol. Microbiol. 26, 789 (1997).
The prgH and prgK genes were cloned from the wild-type strain of S. typhimurium SL1344 by polymerase change reaction, and derivative strains carrying nonpolar mutations in these genes were constructed by allele replacement (8). Salmonella typhimurium strains expressing a functional M45 epitope–tagged PrgH protein were constructed as described elsewhere [C. Collazo, J. E. Galán, Infect. Immun. 64, 3524 (1996)]. The M45 epitope tag consisted of 18 residues from the E4-6/7 protein of adenovirus (MDRSRDRLPPFETETRIL) (19) [S. Obert, R. J. O'Connor, Schmid S., Hearing P., Mol. Cell. Biol. 14, 1333 (1994)].
Single-letter abbreviations for the amino acid residues are as follows: C Cys; D Asp; E Glu; F Phe; G Gly; I Ile; K Lys; L Leu; M Met; P Pro; R Arg; S Ser; T Thr; and V Val.
We thank S. Yamaguchi for Salmonella flagellar mutant strains; M. Iwakura for amino acid sequence analysis; S. Makishima T. Kubo and N. Kobayashi for assistance in the needle preparation; and J. Bliska R. Donis and members of the Galán laboratory for critical reading of this manuscript. Supported by a Grant-in-Aid for Scientific Research from the Ministry of Education Science Sports and Culture (S.-I.A.) the American Heart Association and Public Health Service Grants from the NIH (J.E.G.).