Enterotoxin B production by Staphylococcus aureus under controlled fatty acid nutrition induced by cerulenin.

Cells of Staphylococcus aureus, strain S-6, can grow in the presence of 100 microgram of cerulenin/ml if the basal medium is supplemented with certain saturated or unsaturated fatty acids. The production of enterotoxin B (SEB) is markedly influenced by both the ratio of saturated to unsaturated fatty acid and by the melting point of the unsaturated fatty acid supplement. The results presented suggest that a certain degree of membrane fluidity promotes maximum SEB production and that greater or lesser degrees of membrane fluidity prohibit substantial SEB formation but fail to affect final growth density.

Extreme sensitivity of staphylococcal enterotoxin B and C production to inhibition by cerulenin.

Production of staphylococcal enterotoxins B and C was completely inhibited by concentrations of cerulenin (4 mug/ml and 2 mug/ml, respectively) that did not affect either growth rate or final growth density. Type A toxin formation was not similarly inhibited.

Effects of exogenous fatty acids on growth and enterotoxin B formation by Staphylococcus aureus 14458 and its membrane mutant.

Growth and enterotoxin B (SEB) formation by Staphylococcus aureus 14458 and its membrane mutant can be depressed or stimulated by addition of graded amounts of saturated or unsaturated fatty acid mixtures. Under some conditions depression of SEB formation is separable from growth inhibition. Individual fatty acids most active in altering growth and (or) SEB production have been identified. Small amounts of unsaturated fatty acid mixture not only antagonize some effects of saturated fatty acid mixtures, but also appear to potentiate inhibition of growth and SEB formation by the saturated fatty acid mixture. 2-Admantanone, a compound which increases cell membrane fluidity, strongly suppresses SEB formation while stimulating growth. The results are discussed in relation to possible effects of exogenous fatty acids on the composition of membrane phospholipids and attendant changes in SEB production.

Cerulenin-inhibited cells of Staphylococcus aureus resume growth when supplemented with either a saturated or an unsaturated fatty acid.

In the presence of an inhibitory concentration of cerulenin, cells of Staphylococcus aureus can resume growth when supplemented with either a saturated or an unsaturated fatty acid. A requirement for both types of acids for growth could not be demonstrated.

Enterotoxin B formation by fermentation mutants of Staphylococcus aureus.

An appreciable fraction of carbohydrate-negative (car) mutants of Staphylococcus aureus strains ATCC 14458, 778, and S-6 exhibit increased enterotoxin B (SEB) production. In addition, some lac and mtl mutants of these strains also display enhanced SEB formation. All such mutants appear to be point mutations. Mutagen-induced reversions of high SEB producing car, mtl, or lac mutants yield varying amounts of SEB and some clones seem to be restored to the characteristics of the parent type. A few sequentially isolated lac, mtl double mutants of strain 778 elaborate much more or much less SEB than either the lac or the mtl single mutants.

Derivation of high enterotoxin B-producing mutants of Staphylococcus aureus from the parent strains.

Certain pH-sensitive (membrane) mutants of Staphylococcus aureus, strains 14458 and 778, produce significantly more type-B enterotoxin (SEB) than the parent type. Some carbohydrate mutants (car) from these parent strains also are superior to the parent in SEB formation. By isolating car mutants from high-SEB-producing membrane mutants, it is possible to derive a double mutant producing from six to 50 times as much SEB as the parent type. Inversion of the sequence by isolating pH-sensitive mutants from car mutants does not yield clones with strikingly higher SEB production than the parent strain. The successful isolation sequence (pH-sensitive mutant first and car mutants derived from it) is relatively simple and virtually assures detection of a truly high-SEB-producing clone. The total number of clones whose direct assay for SEB formation is necessary for detection of a high-producing mutant is on the order of 50 to 60.

Membrane mutations and production of enterotoxin B and alpha hemolysin in Staphylococcus aureus.

Staphylococcus aureus strain S-6, which produces enterotoxin type B (SEB), and strain 10-275, a high toxin-producing mutant derived from S-6, display pronounced differences in dye sensitivity, osmotic stability, and bacitracin sensitivity. Such characteristics are consistent with the concept that strain 10-275 is a membrane mutant of strain S-6. Some membrane mutants of S. aureus strain 14458 exhibit about two- to three-fold increases in SEB production whereas other membrane mutants show about twofold increases in alpha-hemolysin production. It is suggested that specific and independent membrane mutations control the secretory processes resulting in the extracellular elaboration of these exoproteins.

Chromosome mapping in Staphylococcus aureus.

The genome of Staphylococcus aureus was mapped by enumerating mutants induced by nitrosoguanidine during synchronous chromosomal replication following release from phenethanol inhibition. Both chromosomal replication time and cell division time were 120 min for this strain of S. aureus. Duplication of genes occurred within a 10-min period of the 120 min required for chromosomal replication. A high-resolution method was devised to determine the gene order of four genes that duplicated in the same 10-min interval of replication of the chromosome. A genomic map locating the positions of 10 genes was derived.

Inducible lysis clostridium tetani.

Lysis was induced in seven strains of Clostridium tetani by exposure to mitomycin C. The search for a suitable indicator strain to detect bacteriophage in lysates has, so far, been unsuccessful. Inhibition studies on macromolecular synthesis during induction have shown that deoxyribonucleic acid, ribonucleic acid, and protein syntheses are all involved in the lysis induced by mitomycin C. In experiments comparing toxin and protein content in induced and uninduced cells of C. tetani, the toxin-protein ratio proved to be the same in both systems up to the point of lysis. Several possible hypotheses deduced from these results are discussed.