, ANm J. W. FOSTER. Response of Bacillus spores to combinations of germinative compounds. J. Bacteriol. 91:1168-1177. 1966.-Spores of 21 strains of Bacillus megaterium and 25 other strains representing 13 species of Bacillus were produced under standardized conditions. The germination of a washed spore suspension of each strain was measured as a response to various combinations of 30 different germinative compounds. The strains were first typed with respect to their response to "primary" germination compounds, i.e., glucose, L-alanine, inosine, and L-alanine-inosine mixture, and also Na+ and K+. The second stage was the determination of the response to various organic and inorganic anions and cations, each strain being supplied with the "primary" compounds best for it. Marked differences in germination patterns were observed among species and strains of the same species. No relation to established taxonomic lines was evident. A nonspecific requirement for ions was found for all strains, but not all ions were effective. A striking degree of interchangeability of germinative chemicals was found. "Fractional germination" was very common. A mixture of L-alanine and inosine and various ions was the best germinative solution for most strains. Some anomalous germination patterns were encountered. Those studied included a strain whose cells lysed spontaneously upon germination and other strains for which L-leucine had striking germinative powers.
As found previously with other Bacillus species, spores of B. stearothermophilus and "Thermoactinomyces thalpophilus" contained significant levels'of small, acid-soluble spore proteins (SASP) which were rapidly degraded during spore gerniination and which reacted with antibodies raised against B. megaterium SASP. Genes codin for a B. stearothermophilus ad a "T. thalpophilus" SASP as well as for two B. cereus SASP wire cloned, their nucleotide sequences were determined, and the amino acid sequences of the SASP coded for were compared. Strikingly, all of the amino acid residues previously found to be conserved in this group of SASP both within and between two other Bacillus species (B. megaterium and B. subtilis) were also conserved in the SASP coded for by the B. cereus genes as well as those coded for by the genes from the more distantly related organisms B. stearothermophilus and "T. thalpophilus."'This finding strongly suggests that there is significant selective pressure to conserve SASP primary sequence and thus that these proteins serve some function other than shuply amino acid storage.Ten to twenty percent of the protein of spores of a number of Bacillus species is degraded in the first minutes of spore germination, thus supplying amino acids for both new prote,in synthesis and metabolism (5,22). The proteins degraded in this process are a group of small, acid-soluble spore proteins (SASP) which are synthesized only during sporulation under transcriptional control (22). In B. megaterium three proteins (termed SASP-A, -B, and.-C) make up -85% of the protein degraded, with SASP-A and -C being very similar in primary sequence and SASP-B being more different (22). Studies of this system at the gene level have revealed that in B. megaterium, in addition to the genes encoding SASP-A and -C, there are five other genes which code for SASP which are very similar to SASP-A and -C (5, 8, 9); this multiplicity of SASP genes' has also been found in B. subtilis (2, 3). Strikingly, the amino acid sequences of the proteins coded for by these different SASP genes exhibit a high degree of homology, with -65% of all residues conserved both within and across species (3,5,9). Given this high degree of SASP sequence conservation, we felt that it might be valuable to analyze SASP genes from other sporulating bacteria, including several more distantly related than' are B. megaterium and B. subtilis. Consequently, in this communication we report the cloning and nucleotide sequence of SASP genes from B. cereus, B. stearothermophilus, and "Thermoactinomyces thalpophilus" MATERIALS AND METHODS Organisms used and isolation of DNA. The organisms used were B. cereus T, (originally obtained from H. 0. Halyorson), B. stearothermophilus NGB101 (11), and "T. thalpophilus" HA-O1 (10). Spores of B. stearothermophilus and "T. thalpophilus" were pr-epared and purified as previously described (10, 11). DNA was also isolated and purified from B. cereus and "'T. thalpophilus" as described previ-* Corresponding author. ously (5, 10). DNA from B. s...
FOERSTER, HAROLD F. (The University of Texas, Austin), AND J. W. FOSTER. Endotrophic calcium, strontium, and barium spores of Bacillus megaterium and Bacillus cereus. J. Bacteriol. 91:1333-1345. 1966.-Spores were produced by washed vegetative cells suspended in deionized water supplemented with CaCl2, SrCl2, or BaC12. Normal, refractile spores were produced in each case; a portion of
Germinated spores of Bacillus megaterium QM B1551 were irradiated with ultraviolet light, and spore-forming survivors were screened for germination requirements. Spore strains which failed to germinate in a variety of defined solutions germinative for spores of the parent strain were obtained. Mutant spores germinated readily in solutions containing yeast extract or one of numerous complex preparations. y-Aminobutyric acid, obtained from yeast extract by column chromatography, was shown to be required for germination by the mutant spores.-y-Aminobutyric acid and L-alanine at final concentrations of I mm each, in solutions of KI (40 mM), equaled the potency of yeast extract (I mg/ml) in the germination of the mutant spores. One of several other amino acids could be substituted, though less effectively, for L-alanine. a-Aminobutyric acid, ,B-aminobutyric acid, falanine, and 5-aminovaleric acid were ineffective substitutes for-y-aminobutyric acid in mutant spore germination.
Spore glutamic acid pools were examined in dormant and germinating spores using colorimetric and "4C analytical procedures. Germination of spores of Bacillus megaterium (parent strain), initiated by D-glucose, was accompanied by a rapid drop in the level of spore pool glutamate, from 12.0 Ag/mg of dry spores to 7.7 gg/mg of dry spores after 30 sec of germination. Similar decreases in extractable spore pool glutamate were observed with L-alanine-initiated germination of B. licheniformis spores. On the other hand, glutamate pools of mutant spores of B. megaterium, with a requirement of y-aminobutyric acid for spore germination, remained unchanged for 9 min of germination, at which time more than 50% of the spore population had germinated. Evidence for conversion of spore pool glutamate to y-aminobutyric acid during germination of spores of B. megaterium (parent strain) was obtained.
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