Sporulation temperature affects initiation of germination and inactivation by high hydrostatic pressure of Bacillus cereus

The influence of sporulation temperature (20, 30 and 37 °C) on the heat resistance and initiation of germination and inactivation by high pressure on Bacillus cereus ATCC 14579 spores was investigated. Spores sporulated at 37 °C were the most heat-resistant. However, spores sporulated at 20 °C were more resistant to the initiation of germination and inactivation by high pressure. Spores were more sensitive to pressure at higher treatment temperatures. At 25 °C, there was an optimum pressure (250 MPa) for the initiation of germination for the three suspensions; at higher temperatures an increase of pressure up to 690 MPa caused progressively more germination. Resistance to the germinability and inactivation by high pressure of the spore population was distributed heterogeneously. Semilogarithmic curves of the ungerminated and survival fraction of B. cereus spores were concave. The resistant fraction of the spore population was lower at higher treatment temperatures. At 60 °C after 30 s of treatment at 690 MPa almost 5 log cycles of the population of B. cereus sporulated at 20 °C was germinated, and more than 7 log cycles of the population of B. cereus sporulated at 30 and 37 °C. The same treatment inactivated 4, 6 and 7 log cycles of the population of B. cereus sporulated at 20, 30 and 37 °C, respectively.     

Initiation of bacterial spore germination

To investigate the problem of initiation in bacterial spore germination, we isolated, from extracts of dormant spores of Bacillus cereus strain T and B. licheniformis, a protein that initiated spore germination when added to a suspension of heat-activated spores. The optimal conditions for initiatory activity of this protein (the initiator) were 30 C in 0.01 to 0.04 m NaCl and 0.01 m tris(hydroxymethyl)aminomethane (pH 8.5). The initiator was inhibited by phosphate but required two co-factors, l-alanine (1/7 of K(m) for l-alanine-inhibited germination) and nicotinamide adenine dinucleotide (1.25 x 10(-4)m). In the crude extract, the initiator activity was increased 3.5-fold by heating the extract at 65 C for 10 min, but the partially purified initiator preparation was completely heat-sensitive (65 C for 5 min). Heat stability could be conferred on the purified initiator by adding 10(-3)m dipicolinic acid. A fractionation of this protein that excluded l-alanine dehydrogenase and adenosine deaminase from the initiator activity was developed. The molecular weight of the initiator was estimated as 7 x 10(4). The kinetics of germination in the presence of initiator were examined at various concentrations of l-alanine and nicotinamide adenine dinucleotide.     

Initiation of spore germination in Bacillus subtilis: relationship to inhibition of L-alanine metabolism

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 K(i) 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.     

RNA synthesis during spore germination in Bacillus subtilis.

Summary We have analyzed the RNA synthesized during spore germination in Bacillus subtilis. Early in germination there is little incorporation of [³H]uridine into RNA. A large increase in incorporation into RNA was found at 45–60 min into germination which was in part due to increases in the specific activity of the UTP pool. When corrected for specific activity changes, the instantaneous rate of RNA synthesis showed a seven to tenfold increase between 30 and 45 min of germination. Polyacrylamide gel electrophoresis studies showed that the RNA synthesized during germination appeared very similar to the RNA made during vegetative growth. DNA-RNA hybridization studies indicated that mRNA and rRNA were synthesized throughout germination. Their relative proportions remained constant and were very similar to the composition of RNA synthesized during vegetative growth.In partial fulfillment of the requirements for the doctoral degree by A.S. in the Department of Microbiology at the New York University School of Medicine     

L-Proline site for triggering Bacillus megaterium spore germination.

Germination of $\text{Bacillus megaterium}$ QM B1551 spores can be triggered by L-proline chloromethyl ketone at ～ 10 fold lower concentrations than L-proline. [³H] L-proline chloromethyl ketone bound to several protein fractions, one of which was decreased in a mutant (JV137) that cannot be triggered by L-proline. Treatment of spores with [³H] acetic anhydride specifically inhibited L-proline triggered germination, and also covalently modified the same protein fraction which appears to be bound to the spore membrane. These results indicate that it is possible to identify a protein fraction in spores that may play a key role in triggering spore germination.     

Bacillus megaterium spore germination is influenced by inoculum size

AIMS: The effect of spore density on the germination (time-to-germination, percent germination) of Bacillus megaterium spores on tryptic soy agar was determined using direct microscopic observation. METHODS AND RESULTS: Inoculum size varied from approximately 10(3) to 10(8) cfu ml(-1) in a medium where pH=7 and the sodium chloride concentration was 0.5% w/v. Inoculum size was measured by global inoculum size (the concentration of spores on a microscope slide) and local inoculum size (the number of spores observed in a given microscope field of observation). Both global and local inoculum sizes had a significant effect on time-to-germination (TTG), but only the global inoculum size influenced the percentage germination of the observed spores. CONCLUSIONS: These results show that higher concentrations of Bacillus megaterium spores encourage more rapid germination and more spores to germinate, indicating that low spore populations do not behave similarly to high spore populations. SIGNIFICANCE AND IMPACT OF THE STUDY: A likely explanation for the inoculum size-dependency of germination would be chemical signalling or quorum sensing between Bacillus spores.     

Screening for Bacillus subtilis mutants deficient in pressure induced spore germination: identification of ykvU as a novel germination gene

Exposure to high pressure induces germination in spores of Bacillus subtilis. To investigate the mechanisms of this process and to compare the pressure and nutrient induced germination pathways, a random transposon knock-out library of B. subtilis was constructed and screened for clones with a compromised pressure induced germination at 100 MPa. Two mutants were isolated and their transposon insertion was mapped to gerAC and ykvU respectively. While GerAC is required for production of the l-alanine receptor which has been implicated in pressure-induced germination before, YkvU is shown here to be a novel germination determinant in B. subtilis, affecting germination by high (100 MPa) and very high (600 MPa) pressure, by nutrients and by dodecylamine, but not by Ca(2+)-dipicolinic acid.     

Sequence of events during Bacillus megaterim spore germination

Levinson, Hillel S. (U.S. Army Natick Laboratories, Natick, Mass.), and Mildred T. Hyatt. Sequence of events during Bacillus megaterium spore germination. J. Bacteriol. 91:1811-1818. 1966.-An integrated investigation of the sequence of events during the germination of Bacillus megaterium spores produced on three different media-Liver "B" (LB), synthetic, and Arret and Kirshbaum (A-K)-is reported. Heat-activated spores were germinated in a mixture of glucose and l-alanine. For studies of dipicolinic acid (DPA) release and increase in stainability and phase-darkening, germination levels were stabilized by the addition of 2 mm HgCl(2). Heat resistance was measured by conventional plating techniques and by a new microscopic method. The sequence (50% completion time) of LB spore germination events was: loss of resistance to heat and to toxic chemicals (3.0 min); DPA loss (4.7 min); stainability and Klett-measured loss of turbidity (5.5 min); phase-darkening (7.0 min); and Beckman DU-measured loss of turbidity (7.2 min). The time difference between 50% completion of stainability and complete phase darkening was 1.5 min, in excellent agreement with the microgermination time of 1.49 min as determined by observation of spores darkening under phase optics. Alteration of the sporulation medium modified the 50% completion times of these germination events, and, in some cases, their sequence. In the A-K spores, the rates of loss of heat resistance and DPA were substantially higher than those of the other germination events, whereas in spores produced in the LB and synthetic media all germination events followed an approximately parallel time course. This is discussed from the point of view of spore population heterogeneity and germination mechanisms.     

Nuclear and cell division in Bacillus subtilis: cell development from spore germination.

The changes in the morphology of the nucleoids and the mesosomes in Bacillus subtilis cells during synchronous outgrowth after spore germination were followed in large-scale three-dimensional cell reconstructions. Shortly after outgrowth of the cell begins in Spizizen medium with glucose, the mesosome becomes an elongated structure in close contact with a rounded nucleoid. When nuclear replication reaches full activity, the mesosome develops into a single, complicated versatile system, with tubules that traverse the cytoplasm and have elaborations in and near the nucleoplasm. Later the system may retract to form large rounded mesosomes; the tubules and strings of vesicles within these mesosomes probably have been collected from the cytoplasm. Shortly after the first cell division, both sister cells have two nucleoids, but with longer generation times induced by growth in media containing acetate instead of glucose; these sister cells have only one nucleoid each. In acetate-grown cells rounded nucleoids that have no contact with a mesosome may represent nucleoids in a temporary stage of rest. On the other hand, the nucleoids of cells growing in glucose-containing medium are always penetrated by mesosomal material, superficially or deeply. Since the mesosome appears capable of traversing the nuclear fibrils, and even reaching the last strands connecting the dividing nucleoids, it is suggested that this organelle may play a vital role in the Bacillus division cycle.