Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Dec;177(24):7202–7209. doi: 10.1128/jb.177.24.7202-7209.1995

Superlytic hemolysin mutants of Serratia marcescens.

M Hilger 1, V Braun 1
PMCID: PMC177601  PMID: 8522529

Abstract

Hemolysis by Serratia marcescens is caused by two proteins, ShlA and ShlB. ShlA is the hemolysin proper, and ShlB transports ShlA through the outer membrane, whereby ShlA is converted into a hemolysin. Superhemolytic ShlA derivatives that displayed 7- to 20-fold higher activities than wild-type ShlA were isolated. ShlA80 carried the single amino acid replacement of G to D at position 326 (G326D), ShlA87 carried S386N, and ShlA80III carried G326D and N236D. Superhemolysis was attributed to the greater stability of the mutant ShlA derivatives because they aggregated less than the wild-type hemolysin, which lost activity within 3 min at 20 degrees C. In contrast to the highly hemolytic wild-type ShlA at 0 degrees C, the hyperlytic hemolysins were nonhemolytic at 0 degrees C, suggesting that the hyperlytic derivatives differed from wild-type ShlA in adsorption to and insertion into the erythrocyte membrane. However, the size of the pores formed at 20 degrees C by superhemolytic hemolysins could not be distinguished from that of wild-type ShlA. In addition to the N-terminal sequence up to residue 238, previously identified to be important for activation and secretion, sites 326 and 386 contribute to hemolysin activity since they are contained in regions that participate in hemolysin inactivation through aggregation.

Full Text

The Full Text of this article is available as a PDF (616.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bernheimer A. W. Assay of hemolytic toxins. Methods Enzymol. 1988;165:213–217. doi: 10.1016/s0076-6879(88)65033-6. [DOI] [PubMed] [Google Scholar]
  2. Braun V., Günther H., Neuss B., Tautz C. Hemolytic activity of Serratia marcescens. Arch Microbiol. 1985 May;141(4):371–376. doi: 10.1007/BF00428852. [DOI] [PubMed] [Google Scholar]
  3. Braun V., Hobbie S., Ondraczek R. Serratia marcescens forms a new type of cytolysin. FEMS Microbiol Lett. 1992 Dec 15;100(1-3):299–305. doi: 10.1111/j.1574-6968.1992.tb14056.x. [DOI] [PubMed] [Google Scholar]
  4. Braun V., Neuss B., Ruan Y., Schiebel E., Schöffler H., Jander G. Identification of the Serratia marcescens hemolysin determinant by cloning into Escherichia coli. J Bacteriol. 1987 May;169(5):2113–2120. doi: 10.1128/jb.169.5.2113-2120.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cramer W. A., Cohen F. S., Merrill A. R., Song H. Y. Structure and dynamics of the colicin E1 channel. Mol Microbiol. 1990 Apr;4(4):519–526. doi: 10.1111/j.1365-2958.1990.tb00619.x. [DOI] [PubMed] [Google Scholar]
  7. Cramer W. A., Heymann J. B., Schendel S. L., Deriy B. N., Cohen F. S., Elkins P. A., Stauffacher C. V. Structure-function of the channel-forming colicins. Annu Rev Biophys Biomol Struct. 1995;24:611–641. doi: 10.1146/annurev.bb.24.060195.003143. [DOI] [PubMed] [Google Scholar]
  8. Flyg C., Kenne K., Boman H. G. Insect pathogenic properties of Serratia marcescens: phage-resistant mutants with a decreased resistance to Cecropia immunity and a decreased virulence to Drosophila. J Gen Microbiol. 1980 Sep;120(1):173–181. doi: 10.1099/00221287-120-1-173. [DOI] [PubMed] [Google Scholar]
  9. Fowler V., Branton D. Lateral mobility of human erythrocyte integral membrane proteins. Nature. 1977 Jul 7;268(5615):23–26. doi: 10.1038/268023a0. [DOI] [PubMed] [Google Scholar]
  10. König W., Faltin Y., Scheffer J., Schöffler H., Braun V. Role of cell-bound hemolysin as a pathogenicity factor for Serratia infections. Infect Immun. 1987 Nov;55(11):2554–2561. doi: 10.1128/iai.55.11.2554-2561.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Leininger E., Roberts M., Kenimer J. G., Charles I. G., Fairweather N., Novotny P., Brennan M. J. Pertactin, an Arg-Gly-Asp-containing Bordetella pertussis surface protein that promotes adherence of mammalian cells. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):345–349. doi: 10.1073/pnas.88.2.345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lugtenberg B., Meijers J., Peters R., van der Hoek P., van Alphen L. Electrophoretic resolution of the "major outer membrane protein" of Escherichia coli K12 into four bands. FEBS Lett. 1975 Oct 15;58(1):254–258. doi: 10.1016/0014-5793(75)80272-9. [DOI] [PubMed] [Google Scholar]
  13. Létoffé S., Delepelaire P., Wandersman C. Cloning and expression in Escherichia coli of the Serratia marcescens metalloprotease gene: secretion of the protease from E. coli in the presence of the Erwinia chrysanthemi protease secretion functions. J Bacteriol. 1991 Apr;173(7):2160–2166. doi: 10.1128/jb.173.7.2160-2166.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Miyazaki H., Yanagida N., Horinouchi S., Beppu T. Characterization of the precursor of Serratia marcescens serine protease and COOH-terminal processing of the precursor during its excretion through the outer membrane of Escherichia coli. J Bacteriol. 1989 Dec;171(12):6566–6572. doi: 10.1128/jb.171.12.6566-6572.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ondraczek R., Hobbie S., Braun V. In vitro activation of the Serratia marcescens hemolysin through modification and complementation. J Bacteriol. 1992 Aug;174(15):5086–5094. doi: 10.1128/jb.174.15.5086-5094.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Pagani L., Luzzaro F., Ronza P., Rossi A., Micheletti P., Porta F., Romero E. Outbreak of extended-spectrum beta-lactamase producing Serratia marcescens in an intensive care unit. FEMS Immunol Med Microbiol. 1994 Nov;10(1):39–46. doi: 10.1111/j.1574-695X.1994.tb00009.x. [DOI] [PubMed] [Google Scholar]
  17. Parker M. W., Buckley J. T., Postma J. P., Tucker A. D., Leonard K., Pattus F., Tsernoglou D. Structure of the Aeromonas toxin proaerolysin in its water-soluble and membrane-channel states. Nature. 1994 Jan 20;367(6460):292–295. doi: 10.1038/367292a0. [DOI] [PubMed] [Google Scholar]
  18. Parker M. W., Postma J. P., Pattus F., Tucker A. D., Tsernoglou D. Refined structure of the pore-forming domain of colicin A at 2.4 A resolution. J Mol Biol. 1992 Apr 5;224(3):639–657. doi: 10.1016/0022-2836(92)90550-4. [DOI] [PubMed] [Google Scholar]
  19. Poole K., Schiebel E., Braun V. Molecular characterization of the hemolysin determinant of Serratia marcescens. J Bacteriol. 1988 Jul;170(7):3177–3188. doi: 10.1128/jb.170.7.3177-3188.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Relman D. A., Domenighini M., Tuomanen E., Rappuoli R., Falkow S. Filamentous hemagglutinin of Bordetella pertussis: nucleotide sequence and crucial role in adherence. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2637–2641. doi: 10.1073/pnas.86.8.2637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ruan Y., Braun V. Hemolysin as a marker for Serratia. Arch Microbiol. 1990;154(3):221–225. doi: 10.1007/BF00248958. [DOI] [PubMed] [Google Scholar]
  22. Scheffer J., König W., Braun V., Goebel W. Comparison of four hemolysin-producing organisms (Escherichia coli, Serratia marcescens, Aeromonas hydrophila, and Listeria monocytogenes) for release of inflammatory mediators from various cells. J Clin Microbiol. 1988 Mar;26(3):544–551. doi: 10.1128/jcm.26.3.544-551.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Schiebel E., Braun V. Integration of the Serratia marcescens haemolysin into human erythrocyte membranes. Mol Microbiol. 1989 Mar;3(3):445–453. doi: 10.1111/j.1365-2958.1989.tb00190.x. [DOI] [PubMed] [Google Scholar]
  24. Schiebel E., Schwarz H., Braun V. Subcellular location and unique secretion of the hemolysin of Serratia marcescens. J Biol Chem. 1989 Sep 25;264(27):16311–16320. [PubMed] [Google Scholar]
  25. Schmitz G., Braun V. Cell-bound and secreted proteases of Serratia marcescens. J Bacteriol. 1985 Mar;161(3):1002–1009. doi: 10.1128/jb.161.3.1002-1009.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schönherr R., Hilger M., Broer S., Benz R., Braun V. Interaction of Serratia marcescens hemolysin (ShlA) with artificial and erythrocyte membranes. Demonstration of the formation of aqueous multistate channels. Eur J Biochem. 1994 Jul 15;223(2):655–663. doi: 10.1111/j.1432-1033.1994.tb19038.x. [DOI] [PubMed] [Google Scholar]
  27. Schönherr R., Tsolis R., Focareta T., Braun V. Amino acid replacements in the Serratia marcescens haemolysin ShIA define sites involved in activation and secretion. Mol Microbiol. 1993 Sep;9(6):1229–1237. doi: 10.1111/j.1365-2958.1993.tb01252.x. [DOI] [PubMed] [Google Scholar]
  28. Slatin S. L., Qiu X. Q., Jakes K. S., Finkelstein A. Identification of a translocated protein segment in a voltage-dependent channel. Nature. 1994 Sep 8;371(6493):158–161. doi: 10.1038/371158a0. [DOI] [PubMed] [Google Scholar]
  29. St Geme J. W., 3rd, Falkow S., Barenkamp S. J. High-molecular-weight proteins of nontypable Haemophilus influenzae mediate attachment to human epithelial cells. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2875–2879. doi: 10.1073/pnas.90.7.2875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  31. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  32. Swihart K. G., Welch R. A. The HpmA hemolysin is more common than HlyA among Proteus isolates. Infect Immun. 1990 Jun;58(6):1853–1860. doi: 10.1128/iai.58.6.1853-1860.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zhou C., Yang Y., Jong A. Y. Mini-prep in ten minutes. Biotechniques. 1990 Feb;8(2):172–173. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES