Inhibition of Bacillus cereus spore outgrowth by covalent modification of a sulfhydryl group by nitrosothiol and iodoacetate

Abstract

Nitrosothiols with the general structure RSN-O were studied as a model system of bacteriostatic action toward outgrowing bacterial spores. With a Taft plot analysis, the influence of the structure of the R group on the inhibitory effectiveness of a series of nitrosothiols showed that effectiveness as an inhibitor of Bacillus cereus T outgrowth correlated with the electron withdrawal of R, but that size, shape, charge, hydrophobicity, and transportability had little influence. This was interpreted to mean that nitrosothiols do not traverse the membrane to act. The Taft plot together with competition data between nitrosothiol and iodoacetate indicated that the mode of nitrosothiol action is the covalent modification of a sulflhydryl group, probably to form RSN(OH)-SX, where -SX is derived from a sensitive spore sulflhydryl group. Cooperativity effects indicated that outgrowth inhibition is accompanied by a conformational change occurring upon sulflhydryl group modification, which is communicated among at least three to five subunits. Uptake of label during spore germination indicated that most of the sulfhydryl groups which can be modified are associated with the inhibitory event. These data suggest that this sulfhydryl group may be sufficiently unique that inhibitors designed to interfere specifically with it could have value as bacteriostatic agents.

Modification of membrane sulfhydryl groups in bacteriostatic action of nitrite

“…Figure 2 shows double-reciprocal plots of inhibition data obtained at different pH values. These plots are comparable to those obtained for nitrosothiols in that they are linear and the lines intersect at a point in the upper left quadrant (8,11). Moreover, interference with iodoacetate incorporation at each pH occurred as if nitrous acid were the interfering component.…”

“…This is consistent with nitrous acid as the active component (as was proposed many years ago [14]), with nitrite itself acting as a reservoir to replenish nitrous acid as it is consumed by various reactions. The cooperativity index, morphological stage at which inhibition occurs, and rapid reversibility were comparable to those observed with the S-nitrosothiols (5,(8)(9)(10)(11).…”

“…We previously explored the bacteriostatic mechanism of S-nitrosothiols (RSN=O), which are structural analogs of nitrous acid (HON=O), by examining their effects on the germination and outgrowth of spores of Bacillus cereus T (5, [8][9][10][11]. Studying the effects on these bacterial spores is appropriate, since a primary value of nitrite as a food preservative is its ability to protect against the toxic effects caused by growth of bacterial sporeformers (1).…”

“…Studying the effects on these bacterial spores is appropriate, since a primary value of nitrite as a food preservative is its ability to protect against the toxic effects caused by growth of bacterial sporeformers (1). Results obtained with these analogs indicated that the bacteriostatic mechanism involves covalent attachment of the analog to membrane sulfhydryl groups of germinated spores (5,(8)(9)(10)(11)). If S-nitrosothiols are a suitable model for the study of nitrite action, we should obtain similar results with nitrite.…”

Lactoferrin and transferrin fragments react with nitrite to form an inhibitor of Bacillus cereus spore outgrowth

“…Both the nitrosothiol inhibitors (12,14) and the heat-derived inhibitor (8) (Fig. 1) display similar reversibility of action and highly cooperative concentration-activity effects.…”