title: The effect of phenethyl alcohol, and other antimetabolites, on growth and extracellular protein production in Staphylococcus aureus
Creators: name: Wheller, Denis
Publisher: name: University of Glasgow
Year: 1984

description: Staphylococcus aureus can excrete at least 31 different proteins which are either wholly extracellular or cell-associated to some degree. Conditions required for production of the two classes are likely to be different since the former are produced late in the growth cycle whereas the latter are probably constitutive. Two proteins were chosen to represent each class and the effect of various antimetabolites on their production was investigated. Particular attention was paid to the effect of phenethyl alcohol since it was hoped to clarify its mode of action. Acid phosphatase was chosen as the example of a cell-associated protein. Since its degree of association was uncertain this was investigated in two ways. A culture which had been grown, overnight was separated by centrifugation and the phosphatase activity in the supernatant and in the cells was determined. Activity associated with the cells was fractionated by washing to remove loosely bound enzyme, protoplasting to determine activity in the wall and periplasm, and lysis to determine activity in the membranes and cytoplasm. It was found that 40% of the total activity was extracellular, 35% was associated with the outer layers of the cell and the remainder was in the protoplast. Electron microscopy revealed considerable activity in the cytoplasm, membranes and walls much of which could be easily washed out. The phosphatase activity appeared to have three pH optima at 6.1, 6.8 and 7.8. These results confirmed that the acid phosphatase was largely cell-associated. Alpha-haemolysin was the other protein chosen, since it was widely accepted to be a wholly extracellular enzyme. Production of the two proteins under different conditions was assessed. In rich media, such as the glucose-casamino acids-yeast extract diffusate medium most commonly used in these experiments, alpha-haemolysin activity was first detected at a population density of 0.68+/- 0.11 measured by extinction at E600nm. Evidence of growth linking; indeed production of the haemolysin increased most markedly while the culture was slowing down at the end of log phase. In poorer media with lower nitrogen content or with alternative carbon sources production of alpha-haemolysin began much later and yield was greatly reduced. Some other extracellular proteins were' assessed in a few experiments and seemed to parallel the appearance of the alpha-haemolysin. Acid phosphatase activity was always detected and its production appeared to be proportional to the population density, although it was not directly growth-linked. The effect of phenethyl alcohol on growth and extracellular protein production was examined by varying concentration of inhibitor and by varying either the time of addition or the cell number. Concentrations of phenethyl alcohol above about 25mM were totally inhibitory to growth due to damage to membrane. Analysis of the results indicated that the effect on growth was competitive up to 25mM and non-competitive above this concentration. Production of alpha-haemolysin was delayed until an E600nm +/- 0.2 and the yield was reduced by 80%, when treated with phenethyl alcohol at 15mM. Above this concentration it was not detected. Acid phosphatase production was inhibited in line with growth inhibition. Inhibition of both growth and extracellular protein production was independent of growth rate and population density, although if inhibitor was added when the culture was nearing stationary phase and maximum protein level this was not always easy to detect. Certain amino acids had been suggested to be involved in alpha-haemolysin production. Phenethyl alcohol did not directly mimic the effect of phenylalanine analogues but addition of excess histidine to complex media did partially reverse the inhibition of alpha-haemolysin production. A comparison of the effect of phenethyl alcohol analogues indicated that the inhibitory effects increased with decreased polarity of the side chain, suggesting that membrane penetration might be involved. (Abstract shortened by ProQuest.).