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Spores of the standard transformable Marburg strain of Bacillus subtilis can be initiated to germinate by l-alanine alone. We isolated mutants which required for this process, in addition to l-alanine, the combination of d-glucose + d-fructose + K(+) or NH(4) (+) ions. In place of fructose, autoclaved or caramelized glucose could be used. Even the standard type strain required the addition of these three agents when d-alanine was present or when the temperature was raised. These findings show that l-alanine normally performs two functions during initiation, one of which is absent in the mutants or is blocked by d-alanine or elevated temperature. One of our mutants was not absolutely dependent on the addition of external l-alanine, because it could be initiated at a reduced rate by the sole addition of glucose + K(+) or NH(4) (+). When K(+) or NH(4) (+) was replaced by Na(+), the initiation rate was greatly reduced. The divalent metal ions Mg(++), Mn(++), and Ca(++) could not satisfy the cation requirement.

Cells of Bacillus subtilis sporulate when they are transferred, at any time of growth in nutrient sporulation medium, to a potassium-phosphate buffer containing slowly utilizable carbon sources such as l-aspartate, citrate, l-glutamate, or lactate. Transfer to buffer containing more rapidly utilizable carbon sources such as malate or glucose leads to sporulation only when the cells either had reached the end of growth or when the transfer medium also contains glycine. Acetate, which as a sole carbon source does not allow growth, also does not alone permit sporulation; however, the presence of both acetate (0.05 m) and glycine or l-serine (0.01 m) in the buffer medium allows sporulation if the cells are transferred to this medium after they have grown in the nutrient sporulation medium beyond the end of the exponential growth phase (T(0)). The development, required before transfer, does not seem to involve the end of a round of deoxyribonucleic acid duplication, as experiments with tryptophan-starved cells have indicated. Glycine or serine cannot be replaced by any of the known metabolites, which are partially derived from them. Amino acid analysis of nutrient sporulation medium showed that glycine (but not serine) is present at a concentration of 0.3 mm at the beginning of the developmental period, thus allowing, in combination with an acetyl-coenzyme A (CoA) precursor, sporulation but not growth. Acetyl-CoA is required not only for adenosine-triphosphate synthesis but also for some other reactions.

Sporulation mutants that were unable to incorporate uracil during the developmental period recovered this capacity with the addition of ribose and in most cases with the addition of glutamate. Of the mutants that responded to both ribose and glumate, all but three also responded to citrate, and all but five responded to acetate. One of the exceptional strains was deficient in aconitase and another one in aconitase and isocitrate dehydrogenase; both required glutamate for growth. For the mutants which did not respond to glutamate, the products made from (14)C-glutamate were determined by thin-layer chromatography. Significant differences were found which enabled the identification of mutant blocks. The deficiency of the corresponding enzyme activity was verified. Several mutants were deficient in alpha-ketoglutarate dehydrogenase, and one lacked succinic dehydrogenase. These mutants could still grow on glucose as sole carbon source, but not on glutamate. The intact Krebs cycle is therefore not required for vegetative growth of aerobic Bacillis subtilis, but it is indispensable for sporulation.

l-Alanine initiates the germination of spores of Bacillus subtilis by entering two metabolic pathways. The products of one pathway, which is inhibited by d-alanine or by elevated temperature, can also be derived from a combination of fructose, glucose, and K(+). The present study demonstrated that the products of the other pathway can be derived from l-asparagine or l-glutamine or, to a lesser extent, from several other amino acids. Hence, the combination of l-asparagine (or l-glutamine), fructose, glucose, and K(+) can initiate spore germination in the absence of l-alanine. Spores preincubated in a combination of asparagine and fructose do not lose refractility, optical density, or heat resistance, and do not take up methylene blue stain. The spores do, however, undergo some reaction which prepares them for a more rapid response to the later addition of glucose and K(+). This preincubation reaction has an optimal temperature of about 44 C.

Fatty acids of different chain lengths were added to cultures of Bacillus subtilis growing in nutrient sporulation medium, and the effects of these fatty acids on growth, oxygen uptake, adenosine triphosphate (ATP) concentration, and membrane protein composition were examined. All fatty acids inhibited growth, the effect being reduced in the presence of glycolytic compounds and reversed by transfer to medium without fatty acids. The inhibition of growth was correlated with a reduction in both the rate of oxygen consumption and the concentration of ATP per cell. The concentration required to obtain a certain degree of inhibition increased with decreasing molecular weight of the fatty acid. However, the reduced nicotinamide adenine dinucleotide oxidation system of cell envelope preparations (i.e., the electron transport system) was not inhibited. Submaximal growth inhibition was accompanied by the relative increase of a membrane protein band revealed by urea-acetic acid gel electrophoresis. This increase was blocked by actinomycin or chloramphenicol. All of the above changes could also be produced by 2,4-dinitrophenol. The inhibition results are best explained by assuming that the fatty acids reversibly react with the cell membrane or proteins in it; they could either alter the membrane structure or uncouple the electron transport chain from two types of proteins, those used for ATP regeneration and others needed for the transport of certain compounds into the cells.

Growth, amino acid transport, and oxygen consumption of Escherichia coli and Salmonella typhimurium are inhibited by short-chain (C(2)-C(6)) but not by medium or long-chain fatty acids (C(10)-C(18)) at concentrations at which these processes are completely inhibited in Bacillus subtilis. The resistance of gram-negative organisms is not correlated with their ability to metabolize fatty acids, since an E. coli mutant unable to transport oleic acid is still resistant. However, mutants of both E. coli and S. typhimurium in which the lipopolysaccharide layer does not contain the residues beyond the 2-keto-3-deoxyoctonate core are inhibited by medium (C(10)) but not by long-chain (C(18)) fatty acids. Furthermore, removal of a portion of the lipopolysaccharide layer by ethylenediaminetetraacetate treatment renders the organisms sensitive to medium and partially sensitive to long-chain fatty acids. The intact lipopolysaccharide layer of gram-negative organisms apparently screens the cells against medium and long-chain fatty acids and prevents their accumulation on the inner cell membrane (site of amino acid transport) at inhibitory concentrations. These results are relevant to the use of antimicrobial food additives, and they allow the characterization of gram-positive versus gram-negative bacteria and their lipopolysaccharide mutants.

Accumulation of l-alpha-glycerophosphate, in cells of Bacillus subtilis mutants lacking the nicotinamide adenine dinucleotide-independent glycerophosphate dehydrogenase activity, suppresses both growth and sporulation. After growth has stopped, the cells slowly develop one and later more asymmetric septa that are thicker than normal prespore septa and apparently contain too much cell wall material to allow further membrane development into forespores or spores. l-Malate prevents accumulation of glycerophosphate and restores sporulation of the mutant. Glucose or gluconate cannot resotre sporulation, because they still effect glycerophosphate accumulation via de novo synthesis. If that accumulation is blocked in a double mutant, which is unable to make glycerophosphate from or to metabolize it into Embden-Meyerhof compounds, then nonsuppressing amounts of glucose or gluconate can restore sporulation.

Acetate and other short chain n-fatty acids (C(1)-C(6)) inhibit strongly the uptake of l-serine or other l-amino acids but inhibit only weakly that of alpha-methylglucoside or fructose, whether measured in whole cells of Bacillus subtilis or in membrane vesicles that have been energized with reduced nicotinamide adenine dinucleotide (NADH), l-alpha-glycerol phosphate, or ascorbate plus phenazine methosulfate. The acetate inhibition is noncompetitive, as was shown for l-alpha-aminoisobutyric acid uptake by whole cells and for l-serine uptake by membrane vesicles. In membrane preparations, neither NADH oxidation nor the reduction of cytochromes by NADH are affected by fatty acids. All of these effects are similar to those of 2, 4-dinitrophenol. It is concluded that the fatty acids "uncouple" the amino acid carrier proteins from the cytochrome-linked electron transport system (to which they may be coupled via protein interaction or via a cation gradient).

Sporulation mutants of Bacillus subtilis, blocked either in the citric acid cycle or in another pathway necessary for uracil incorporation during the developmental period, were tested for their sporulation response to different carbon sources. All of the citric acid cycle mutants and all but one of the other mutants failed to respond. The one phenotypically curable mutant, 60764, responded to most metabolizable carboxylic acids, such as acetate and fatty acids, and to some other carbon sources. For an optimal response, it was necessary to add the compound at a certain concentration and time (0.08 m for acetate, when the extinction of the culture at 600 nm was 1). A liquid medium was devised in which an appreciable amount of antibiotic activity against Staphylococcus aureus was produced by our standard strain 60015. The mutant 60764 produced, even in the presence of palmitate, 20 times less antibiotic and sporulated more slowly than 60015. The antibiotic activity in both strains consisted of three major and several minor molecular species, as detected by thin-layer chromatography. When purified antibiotic was added to an exponentially growing culture of our standard strain of B. subtilis, lysis ensued, the extent of which increased with the concentration of the antibiotic added; later, resistant bacteria grew up. Three mutants unable to produce antibiotic activity were isolated and found to be deficient in sporulation. These findings show the close correlation between sporulation and antibiotic activity, but they do not prove that antibiotic activity is needed for sporulation.

Human (HeLa, Chang liver, L-132, and Intestine 407) and other mammalian (XC, SV3T3, and chick-embryo) cells in tissue culture are at least as sensitive to inhibition by lipophilic acids and nitrite as bacteria. Some of these compounds are the most frequently used antimicrobial food additives. Short-chain fatty acids (up to hexanoate) and parabens induce, at partially inhibitory concentrations, a jagged cell shape in continuous epithelial-like cell lines, such as HeLa, Chang liver, L-132, and Intestine 407. This morphological effect is not mediated or enhanced by butyryl cyclic AMP, which specifically affects fibroblasts.