Mechanism of Excretion of a Bacterial Proteinase: Demonstration of Two Proteolytic Enzymes Produced by a Sarcina Strain (Coccus P)

Abstract

A Sarcina strain (Coccus P) produces two proteolytic enzymes. One is found only extracellularly, is far more prevalent, and is actively excreted during exponential growth. It is the enzyme responsible for the known strong proteolytic activity of the cultures of this strain. A second protease is, however, produced which remains associated with the intact cells but is released by the protoplasts. The two enzymes appear unrelated in their derivation. Calcium ions play an essential role in preventing autodigestion of the excreted enzyme.

Mechanism of Excretion of a Bacterial Proteinase: Factors Controlling Accumulation of the Extracellular Proteinase of a Sarcina Strain (Coccus P)

“…B. to Harvard University in May 1969, in partial fulfillment of the requirements for that degree. 2 excreted proteinase of Coccus P, it was shown (17) that this irreversible inactivation is due to autodigestion occurring in the absence of Ca2 . An antiwetting agent, Ficoll, delays this autodigestion, suggesting that the function of Ca2+ is to stabilize an already active form of the enzyme molecule rather than to act as a constituent of the prosthetic group required for activity.…”

“…MATERIALS AND METHODS Bacterial strains. The bacterial strains used were wild type of Coccus P, the proteinase C-less CPIO mutant already described (17), and a lysine-requiring mutant which was obtained from the wild type by nitrosoguanidine mutagenesis (17) and selected by the penicillin technique (7).…”

“…The liquid medium used was minimal medium, MMCP, described by Gorini and Lord (9). For low-calcium conditions (10-6 M), no external Ca2+ was added; for high-calcium conditions, the Ca2+ concentration was raised to 10-3 M as described before (17).…”

“…The effect of Ficoll (Pharmacia) was tested at 5% concentration in the low-Ca medium, since it was already established (17) that 10% Ficoll did not affect bacterial growth and did not raise the Ca2+ concentration over the 10-6 M low-Ca level. To the high-Ca cultures, 5% Ficoll was generally added to reduce the loss of enzymatic activity resulting from prolonged shaking.…”

“…Growth conditions: flask cultures. The inoculation procedure was as previously described (17), and experiments were started when cultures reached a cell density of 2 x 108 cells/ml. Samples were withdrawn, and the culture growth was stopped as described for each exper-iment.…”