N-methyl anthranilate) on the L-alanine-induced initiation of spore germination was examined in Bacillus subtilis 168. Methyl anthranilate irreversibly inhibited.
JOURNAL OF BACTERIOLOGY, Sept. 1974, p. 805-810 Copyright 0 1974 American Society for Microbiology
Vol. 119, No. 3
Printed in USA.
Initiation of Spore Germination in Bacillus subtilis: Relationship to Inhibition of L-Alanine Metabolism CHANDAN PRASAD1
Laboratory of Molecular Biology, National Institute of Neurological Diseases and Stroke, Bethesda, Maryland 20014 Received for publication 28 May 1974
The inhibitory effects of anthranilic acid esters (methyl anthranilate and N-methyl anthranilate) on the L-alanine-induced initiation of spore germination was examined in Bacillus subtilis 168. Methyl anthranilate irreversibly inhibited alanine initiation by a competitive mechanism. In its presence, the inhibition could be reversed only by the combined addition of D-glucose, D-fructose, and K+. Both L-alanine dehydrogenase and L-glutamate-pyruvate transaminase, enzymes which catalyze the first reaction in L-alanine metabolism, were competitively inhibited by methyl anthranilate. The K1 values for germination initiation (0.053 mM) and of L-glutamate-pyruvate transaminase (0.068 mM) were similar, whereas that for L-alanine dehydrogenase (0.4 mM) was six to seven times higher. Since a mutant lacking L-alanine dehydrogenase activity germinated normally in L-alanine alone, it is speculated that the major pathway of L-alanine metabolism during initiation may be via transmination reaction. It has been reported previously that methyl anthranilate (MA) inhibits the initiation of spore germination of aerobic bacilli (15). MA inhibited the initiation of germination in nutrient broth or by L-alanine under conditions in which it did not affect ougrowth, cell division, or sporulation. This communication shows that the analogues of MA also inhibit germination, and the enzymes inhibited by these drugs are characterized.
initial reading taken as 100%. In some cases the value A/A' (AO = A.2. at zero time) was also plotted. The percent inhibition of germination was computed as follows: % Inhibition of germination =
_ Ac,
AA:) x 100
where A,0 and A,0 = AO values for samples with and without (control) inhibitor, respectively, and AA, and AA, = (AO - A) values for samples with and without inhibitor. Unless indicated otherwise, the germination was measured at 37 C, and all germination mixtures received enough concentrated spore suspension to get AO = 0.3-0.5. The calculation of Km for germination was made by using maximal rate of decrease in A62,. Preparation of spore extract. Spore extracts for enzyme assays were prepared as described earlier
MATERIALS AND METHODS Bacteria. All strains were derived from the transformable Bacillus subtilis Marburg strain 168 of Spizizen. Standard strain 60015 (which requires indole and methionine) from this laboratory was used in most of the experiments. An L-alanine dehydrogenase-minus mutant (60229) was also used in some (14). Enzyme assays. L-Leucine and L-alanine dehydrostudies (3). genases were assayed as described earlier (14). Sporulation, harvesting, heat activation, and L-Glutamate-pyruvate transaminase was assayed initiation procedures. The procedures for sporula- by measuring the formation of ["4C]glutamate from tion, harvesting, heat activation, and initiation were [I4C a-ketoglutarate. The reaction mixture (100 ulidescribed previously (22). The initiating agents were ters) (micromoles): Tris-hydrochloride used at the following millimolar concentrations: KCl, buffer contained (pH 7.4), 10.0; L-alanine, 5.0; [14C la-ketogluta400; L-alanine, 5; L-asparagine, 2.5; D-glucose, 5; rate (3.2 gCi/Mmol), 0.15; phosphate, 10-4; D-fructose, 5; and tris(hydroxymethyl)aminomethane and crude extract (100 topyridoxal gg of protein). The (Tris)-hydrochloride buffer (pH 7.4), 100. In some reaction was run at 37 C in a 200 glass tube (10 by 75 mm) experiments nutrient broth (Difco) was also used. and stopped by immersing the tube in boiling water The rate of germination initiation was measured by 2 min. The tube was cooled and then spun at 3,000 the decrease of absorbance at 625 nm (A.625). The data for x g for 10 min at room temperature. A 5- to 10-jsliter are presented as percent reduction in A with the volume of supernatant was spotted on an MN 300 I Present address: Laboratory of Biochemical Genetics, cellulose plate, dried, and chromatographed in the following solvent system: isopropanol-88% formic National Heart and Lung Institute, Bethesda, Md. 20014. 805
J. BACTERIOL.
PRASAD
806
tested at concentrations of 1 or 2.5 mM, except N-phenylanthranilic acid which was tested at 10.0 mM. Of 10 compounds examined, only MA, ethyl anthranilate, and N-methyl MA inhibited the initiation by 90% or more at a concentration of 1.0 mM or less. On the basis of these results, MA was selected for study of its mode of action. Variation of inhibition with MA concentration. The degree of inhibition of germination initiation in nutrient broth containing 100 ,ug of chloramphenicol per ml increased linearly with increasing concentrations of MA (Fig. 1). The temperature of incubation and pH of nutrient broth did not show any significant RESULTS effect on the degree of inhibition. Time course of MA inhibition. The inhibiInhibition of initiation in nutrient broth by analogues of anthranilic acid. Previous work tion of initiation of spores by MA (1 mM) is (15) had indicated that the addition of MA to a shown in Fig. 2. If MA was added any time after suspension of B. subtilis, B. cereus, B. megater- the initiation of germination, it took about 10 to ium, or B. coagulans spores in G medium (11) 12 min before further reduction of the A.25 inhibits the initiation of germination. In the ceased. An explanation for such a lag may be present work, experiments were undertaken to that it takes about 10 to 12 min for MA to delineate conditions and characteristics of this penetrate the spores and inhibit the reaction(s) inhibition. To determine the relationship be- leading to initiation. Nature of inhibition of alanine-induced tween the structure and inhibitory potential of different analogues of anthranilic acid, heat- germination and its reversal by glucose, frucshocked spores and inhibitor were incubated in tose, and K+. The germination of 60015 spores nutrient broth at 37 C for 30 min, and the A.2, can be initiated by L-aianine alone or by a was measured. The percent inhibition of germi- combination of D-fructose, D-glucose, L-asparanation was calculated. Table 1 shows the effect gine, and K+ (21). Previous experiments indiof different anthranilic acid analogues on the cated that the metabolism of these compounds initiation of germination. The compounds were in spores produces at least three common inter-
acid-water-8-quinolinol (40:2:10:0.026, vol/vol/vol/ wt). After the plates were dried, the spot corresponding to glutamic acid was cut, and the radioactivity was counted in 10 ml of scintillation fluid (4 pints of toluene [ca. 1.9 liters], 2 pints of Triton-X 100 [ca. 0.55 liter] and 150 ml of Liquifluor). All other enzymes were assayed as described by Bergmeyer (1). Enzyme specific activity is expressed as nanomoles of substrate transformed, or product formed, per minute per milligram of protein. Protein was measured by the method of Lowry et al. (9) using bovine serum albumin, fraction V (Armour Pharmaceutical Co., Kankakee, Ill.), as standard.
TABLE 1. EFFECT OF BENZOIC ACID ESTERS AND THEIR ANALOGUES ON THE INITIATION OF GERMINATION OF BACILLUSSUBTILIS SPORES 0
I
C-OR1 R2
R^
