Transport and processing of staphylococcal α-lysin and σ-lysin

Lee, Ker Yin (1984) Transport and processing of staphylococcal α-lysin and σ-lysin. PhD thesis, University of Glasgow.

Abstract

The main object of the investigation described in this thesis was to determine whether staphylococcal α-lysin and σ-lysin are synthesized as larger precursor molecules from which signal sequences are processed during transmembrane translocation. σ -lysin was first detected at the mid-logarithmic phase of growth of S. aureus NCTC 10345, and production, which appeared to obey exponential kinetics, continued well into the stationary phase of growth. It appeared as a very diffuse band just behind the dye front when supernate proteins were separated by SDS-PAGE. Rifampicin, at a concentration of 200μg ml-1, rapidly blocked the incorporation of [3H] uridine into the RNA of S. aureus. The same concentration of rifampicin required considerably longer time to inhibit cell growth at both the mid-logarithmic and late logarithmic-early stationary phases of growth; growth of the bacteria stopped after 1 and 2 h respectively in each phase. Addition of rifampicin to the late logarithmic-early stationary phase culture had less effect on growth than the mid-logarithmic phase culture. The stability of mRNA for the staphylococcal σ -lysin was determined by measuring the residual lysin synthesis after inhibition of DNA-dependent RNA polymerase activity with rifampicin. At the late logarithmic-early stationary phase of growth the 6-lysin mRNA was very stable with a half-life of about 20 min. Total cellular RNA was extracted from cells from late logarithmic- early stationary cultures phase of S. aureus NCTC 10345 and translated with an E. coli S-30 extract (cell-free system). The rate of amino acid incorporation was linear for 15 min and the staphylococcal mRNA stimulated the translation to a level approximately 20-fold above the background. -1 Protein synthesis was almost abolished by chloramphenicol (50 μg ml-1), was dependent on an exogenous energy source (ATP, GTP, PEP) and was barely activated by the protease inhibitor, PMSF (1mg ml-1) or the RNase inhibitor, heparin (1mg ml-1). σ -lysin was identified amongst the translation products by immunoprecipitation and immunoabsorption. The σ - lysin synthesized in vitro was of a size similar to mature 6-lysin and seemed not to require a signal sequence for secretion from the cell. Phenethyl alcohol (PEA), at the maximum concentration which did not inhibit growth (0.3% v/v) inhibited the production of α-lysin and exoproteases but not that of σ -lysin in S. aureus Wood 46. The inhibition of α-lysin was reversible and transient accumulation of cell- associated α-lysin occurred in the presence of PEA. A precursor of α-lysin, of molecular weight approximately 3,000 daltons (peptide elongation of about 20-30 amino acids) larger than extracellular α-lysin, was immunologically detected in the SDS extracts of membranes and whole cells of PEA-treated S. aureus Wood 46 cultures. Also, a degraded form of α-lysin, of molecular weight approximately 27,000 daltons was only detected in membranes prepared from cells lysed by lysostaphin but not in membranes from cells lysed with an X-press or SDS extracts of whole cells. It was concluded from these results that α-lysin is synthesized with a 3 kdal N-terminal signal sequence which is removed during transmembrane translocation whereas σ-lysin seems not to require a signal sequence for secretion from the cell.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Birkbeck, Dr. T. H.
• Date of Award: 1984
• Depositing User: Mrs Monika Milewska-Fiertek
• Unique ID: glathesis:1984-38946
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 27 Nov 2018 14:54
• Last Modified: 26 Oct 2022 08:12
• Thesis DOI: 10.5525/gla.thesis.38946
• URI: https://theses.gla.ac.uk/id/eprint/38946