Germination of Single Bacterial Spores

Changes in refractility and optical density occurring in individual spores of Bacillus cereus T and B. megaterium QM B1551 during germination were investigated by use of a Zeiss microscope photometer. The curves revealed that the germination process in single spores had two distinct phases; an initial rapid phase was followed by a second slower phase. Under the experimental condition employed, the first phase of germination of B. cereus spores lasted for approximately 75 +/- 15 sec, whereas the second phase lasted for 3 to 4.5 min. In B. megaterium spores, the first phase was observed to last for approximately 2 min and the second phase for more than 7 min. The duration of the second phase was dependent on conditions employed for germination. The kinetics of the first phase were strikingly similar under all conditions of physiological germination. Time-lapse phase-contrast microscopy of germinating spores also revealed the biphasic nature of germination. It was postulated that the first phase represents changes induced by an initial partial hydration of the spore and release into the medium of dipicolinic acid, whereas the second phase reflects degradation of the cortex and hydration of the core.     

Water vapor, aqueous ethyl alcohol, and heat activation of Bacillus megaterium spore germination

Dormant spores of Bacillus megaterium were activated for germination on glucose by heating them in aqueous suspension (but not if heated dry), by treating them with aqueous ethyl alcohol at 30 C, or by exposing them to water vapor at room temperature. The degree of water vapor activation depended upon the relative humidity, the time, and the temperature of exposure. Activation increased the extent and rate of glucose-induced germination and decreased the average microlag. Extended water vapor treatment also activated spores for germination induced by KI and by l-alanine. Spores activated by any of the three treatments were deactivated by treatment at 66 C, either for 18 hr in 100% ethyl alcohol or for 40 hr over P(2)O(5). Deactivated spores were reactivated by heat, by 5 m ethyl alcohol, or by water vapor. It is postulated that heating and ethyl alcohol may change the structure of liquid water, so that it is more like water vapor and can more readily penetrate to and hydrate a critical (enzymatic?) spore site, leading to activation.     

Germination of Unactivated Spores of Bacillus cereus T

Heat-activated spores of Bacillus cereus T germinate rapidly in the presence of l-alanine alone or inosine alone. In contrast, unactivated spores can not germinate in the presence of either germinant alone but rapidly in the presence of both germinants. The highest level of cooperative action of l-alanine and inosine on the germination was observed when they were present in a ratio 1 :1. Preincubations of unactivated spores with l-alanine or inosine had opposite effects on the subsequent germination in the presence of both germinants: preincubation with l-alanine stimulated the initiation of subsequent germination, while preincubation with inosine inhibited it. These results suggest that germination of unactivated spores initiated by l-alanine and inosine includes two steps, the first initiated by l-alanine and the second prompted by inosine. The effect of preincubation of unactivated spores with l-alanine was not diminished by washings. The pH dependence of the preincubation of unactivated spores was not so marked as that of the subsequent germination in the presence of inosine.     

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.     

Response of Bacillus spores to combinations of germinative compounds

Foerster, Harold F. (University of Texas, Austin), and 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.     

Influence of cations on lysozyme-induced germination of coatless spores of Clostridium perfringens 8-6

Bacterial endospore germination is powerfully influenced by inorganic salts, cations having especially important effects. Spores of Clostridium perfringens 8-6 are unusual in lacking a spore coat; these spores germinate only in the presence of lysozyme, which readily digests the exposed cortex. Lysozyme-induced germination showed the same response to ionic strength and valence of cations as does lysozyme hydrolysis of peptidoglycan, and close parallels are evident in the influence of inorganic cations on germination of normal spores. La3+ and transition element cations inhibited lysozyme-induced germination at low concentration, again demonstrating parallels with their action on lysozyme digestion of peptidoglycan and on the germination of normal spores. The poly-cations poly(L-lysine) and Ruthenium Red inhibited at extremely low concentrations. Mn2+ and Co2+, at appropriately low concentrations, stimulated lysozyme germination of 8-6 spores and also lysis of Micrococcus lysodeikticus.     

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.     

Ultrahigh-Temperature Activation of a Low-Temperature Bacillus subtilis Spore Germination System

Exposure of Bacillus subtilis A spores to 115 to 125 C for several seconds activated a low-temperature germination system that remained dormant after a heat treatment that activated the normal l-alanine- and glucose-stimulated germination systems. The low-temperature germination system was characterized by an optimum temperature lower than that of the l-alanine or glucose germination systems (30 C versus 45 C), germination in the absence of exogenous germination stimulants, and the capacity for heat-induced deactivation and subsequent reactivation. The rates of activation at 115 to 125 C were exponential and were not influenced by a previous heat treatment that activated the l-alanine- or glucose-stimulated germination systems. Although activation of the low-temperature germination system was accompanied by suppression of l-alanine-stimulated germination, it did not appear to be a modification of the l-alanine germination system.     

Quantification of viable endospores from a Greenland ice core

Endospores (i.e., bacterial spores) embedded in polar ices present an opportunity to investigate the most durable form of life in an ideal medium for maintaining long-term viability. However, little is known about the endospore distribution and viability in polar ices. We have determined germinable endospore concentrations of bacterial spores capable of germination in a Greenland ice core (GISP2 94 m, ID# G2-271) using two complementary endospore viability assays (EVA), recently developed in our laboratory. These assays are based on bulk spectroscopic analysis (i.e., spectroEVA), and direct microscopic enumeration (i.e., microEVA) of ice core concentrates. Both assays detect dipicolinic acid (DPA) release during l-alanine induced germination via terbium ion (Tb3+)-DPA luminescence. Using spectroEVA, the germinable and total bacterial spore concentrations were found to be 295+/-19 spores mL(-1) and 369+/-36 spores mL(-1), respectively, (i.e., 80% of the endospores were capable of germination). Using microEVA, the germinating endospore concentration was found to be 27+/-2 spores mL(-1). The total cell concentration, as determined by DAPI stain fluorescence microscopy, was 7.0 x 10(3)+/-6.7 x 10(2) cells mL(-1). Culturing attempts yielded 2 CFU mL(-1) (4 degrees C). We conclude that endospores capable of germination in the GISP2 ice cores are readily determined using novel endospore viability assays.     

gerR, a novel ger operon involved in L-alanine- and inosine-initiated germination of Bacillus cereus ATCC 14579

Bacillus cereus endospores germinate in response to particular nutrients. Spores are able to sense these nutrients in the environment by receptors encoded by the gerA family of operons. Analysis of the Bacillus cereus ATCC 14579 genome revealed seven gerA family homologues. Using a transposon Tn917-based insertional mutagenesis approach followed by an enrichment procedure to select for l-alanine-induced germination mutants, we isolated a mutant with a defect in the l-alanine germination pathway. The transposon disrupted the last gene of a tricistronic gerA family operon, designated gerR, with the order gerRA, gerRC, gerRB. A second mutant was created by insertion of pMUTIN4 in gerRC. Both mutants showed the same phenotype for nutrient-induced germination. Spores of the gerR mutant strains were blocked in their l-alanine-initiated germination pathway and showed a delayed inosine-induced germination response. Apparently, germination mediated by l-alanine and inosine cannot be compensated for completely by the other germinant receptors, and this points towards an essential role of the gerR-encoded receptor in the receptor complex. In food products, spores of the mutant strains showed a reduced germination response compared to spores of the parental strain. High-pressure-initiated germination was not affected by the gerR mutations, as experiments with 100 and 550 MPa showed no difference with spores of the parental strain.     

Sequence of events during germination of putrefactive anaerobe 3679 spores

The sequence of changes during germination of putrefactive anaerobe 3679h spores was studied under aerobic conditions in a solution containing l-alanine and sodium pyrophosphate. Evidence that specific changes occurred in two distinct regions of the spore is given by data on several criteria that were used to measure germination. During the initial stage of germination, the absorbancy decreased, dipicolinic acid was released, the spores lost their resistance to heat and toxic chemicals, and the spore periphery (cortex) darkened gradually under phase-contrast microscopy. The final stage of germination was characterized by changes in the central spore region (core), notably phase darkening of the spore center and stainability with mercurochrome, and by a slight additional absorbancy decrease.     

Effects of temperature and heat activation on germination of individual spores of Bacillus subtilis

Phase intensity changes of individual germinating spores of Bacillus subtilis were determined by phase-contrast light microscopy and image analysis. Two germination phases were investigated. The length of the time period before a change in phase brightness was evident and the duration of the phase intensity change until a constant greylevel was maintained. The incubation temperature (37 and 20 °C) and heat activation (10 min at 65 °C) had a distinct effect on both phases. At 37 °C, spores of B. subtilis 604 started to show a decrease in brightness in l -alanine buffer after 3–39 min and needed 10–39 min to complete the phase change. At 20 °C, lag times of 10–100 min were observed and the spores needed 30–100 min to reach a constant greylevel. Heat activation and subsequently exposure to l -alanine buffer at 20 °C reduced the lag phase to 6–90 min and the phase change was finished after 30–60 min. Our results indicate enzymatic involvement before and during the phase intensity change of germinating spores.     

-aminobutyric acid as a required germinant for mutant spores of Bacillus megaterium

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. gamma-Aminobutyric acid, obtained from yeast extract by column chromatography, was shown to be required for germination by the mutant spores. gamma-Aminobutyric acid and l-alanine at final concentrations of 1 mm each, in solutions of KI (40 mm), equaled the potency of yeast extract (1 mg/ml) in the germination of the mutant spores. One of several other amino acids could be substituted, though less effectively, for l-alanine. alpha-Aminobutyric acid, beta-aminobutyric acid, beta-alanine, and 5-aminovaleric acid were ineffective substitutes for gamma-aminobutyric acid in mutant spore germination.     

Inhibition by potassium ion of the pregerminative response to L-alanine of unactivated spores of Bacillus cereus T.

The effect of potassium ion on L-alanine-inosine-induced germination of unactivated spores of Bacillus cereus T was studied. Unactivated spores germinated in 0.1 M sodium phosphate buffer (NaPB), but not 0.1 M potassium phosphate buffer (KPB), at pH 8.0 and at 30 C. Inhibition of germination was also observed on incubation of unactivated spores in NaPB containing potassium chloride. Previously it was demonstrated that germination of unactivated spores involves at least two steps, one induced by L-alanine, and the other by inosine. Potassium ion seems to inhibit the response of the spores to inosine, because: (1) Spores that had been preincubated with L-alanine in NaPB or KPB, germinated in NaPB but not KPB in the presence of inosine. (2) During germination in NaPB, incorporation of L-[14C]alanine showed bimodal kinetics with a rapid first phase and a second continuous phase, but in KPB the second phase of incorporation did not occur. The events occurring before germination of unactivated spores are discussed with reference to the initiation of germination.     

Role of Calcium in Biphasic Germination of Bacillus cereus T Spores Sensitized to Lysozyme

Biphasic germination induced by inosine in the presence of Ca²⁺ was examined using Bacillus cereus T spores treated with sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) at pH 10. The first phase of the germination was stimulated by Ca²⁺ in the concentration-dependent manner, showing the optimal concentration at 0.5-1.0 mM. The second phase appeared to be insensitive to the cation. The optimal temperatures for the first and the second phase were 25 C and 40 C, respectively; the optimal pHs for the two phases were 7-9 and around 7.5, respectively. Heat resistance and dipicolinic acid of the SDS-DTT-treated spores were lost mostly during the first phase. A Ca²⁺-specific chelator, glycoletherdiamine-N,N,N',N'-tetraacetic acid (GEDTA), inhibited the first phase evoked by Ca²⁺, while it had no inhibitory effect on the second phase. In contrast, the divalent cations examined, except Mg²⁺ and Sr²⁺, affected not only the first phase but also the second phase. The order of inhibitory effect on the first phase was Hg²⁺ > Zn²⁺ > Ba²⁺, Co²⁺, Cu²⁺ > Mn²⁺; on the second phase, it was Hg²⁺ > Cu²⁺ > Zn²⁺ > Co²⁺ > Mn²⁺ > Ba²⁺.     

Photocontrol of the Germination of Onoclea Spores: II. Analysis of Germination Processes by Means of Anaerobiosis

The oxygen requirements during the three phases of photoinduced germination of Onoclea sensibilis L. spores were analyzed by temporarily applying nitrogen atmosphere. The dark preinduction phase, during which the spores imbibe water and establish sensitivity to irradiation, involves an oxidative process which can be reversibly inhibited and stimulated by nitrogen and air, respectively. The induction phase of germination is characterized by a pure photochemical reaction, independent of temperature and oxygen. The postinduction phase, when the photoproduct triggers dark processes eventually leading to the protrusion of the rhizoidal or protonematal cells, involves an oxidative process which occurs within the first 10 hours of this phase. This oxidative process differs in kinetic characteristics from that in the preinduction phase. The oxidative process is inhibited by nitrogen treatment, but following nitrogen inhibition the ability of the spores to germinate can be reinstated by a long period of air intervening between the nitrogen treatment and a second irradiation. This suggests that enzymes or reactants which are needed in the postinduction process decay under anaerobic conditions and are resynthesized when the spores are transferred to air. Spores take up acetocarmine stain towards the latter part of the postinduction phase. Stain uptake is apparently succeeded very closely by cell division, and some time later by protrusion of the germling cells.     

Effects of cyanide, salicylhydroxamic acid, and temperature on respiration and germination of spores of the fern Sphaeropteris cooperi

During the first 96 h of culture, germinating spores of the fern Sphaeropteris cooperi (F. v. Muell.) Tryon showed a gradual rise in respiratory activity to a maximum of about 6.5 μl 0₂ h⁻¹ mg⁻¹ dry wt. This was followed by a transitory decline in rate, concluded by a second respiratory rise preceding the emergence of the rhizoid after 192 h of culture. Oxygen uptake during the first 120 h of germination was insensitive to 1 m M potassium cyanide (KCN) but was inhibited by 1 m M salicylhydroxamic acid (SHAM); however, beyond this time cyanide showed increasing inhibitory effectiveness whereas SHAM became less effective. Regardless of time of application, KCN had no effect on germination. Maximum inhibition of germination by SHAM was achieved if applied up to 120 h after culture initiation, after which spores became insensitive to SHAM. Heat treatment (50°C for 90 min) during the cyanide-resistant phase of respiration (0 h–120 h) induced cyanide-sensitive respiration and completely inhibited spore germination. Elevated temperatures had little effect if applied during the cyanide-sensitive phase (beyond 120 h). Temperature inhibited spores regained their ability to germinate if maintained in culture until the cyanide-resistant pathway was restored and then subjected to a second photoinductive light treatment. These results suggest the presence and possible involvement of the cyanide-resistant, alternative respiratory pathway during germination of Sphaeropteris cooperi 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.     

High calcium content in Streptomyces spores and its release as an early event during spore germination.

The metal ion content of spores of five Streptomyces species was studied. A general feature of this study was the finding of a very high calcium content (1.1 to 2.1% of the dry weight). Accumulation of calcium occurred preferentially during the sporulation process. Spore calcium was located in the integument fraction, and more than 95% of the calcium was removed from intact spores by ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid. Several divalent cations (Mg2+, Mn2+, Zn2+, and Fe2+) which induced darkening of spores and loss of heat resistance also caused the release of calcium from spores. In addition, darkening of spores was blocked by metabolic inhibitors, whereas calcium excretion was not affected. Two different categories of events in the initiation of germination may be differentiated; first, calcium release from spores which is not energy dependent and is a consequence of triggering of germination by some divalent cations, and second, some other events including loss of heat resistance, loss of spore refractility, and a decrease in absorbance, with at least one energy-dependent step.     

Identification of a Germination System Involved in the Heat Injury of Bacillus Subtilis Spores

Bacillus subtilis A spores were injured by exposure to heat treatments of 110 to 132 C. Injury was demonstrated by the inability to form colonies on fortified nutrient agar (FNA) unless the medium was supplemented with CaCl(2) and Na(2) dipicolinate (CNA). A preliminary heat treatment fully heat-activated the spores, was not lethal, and did not prevent injury by subsequent secondary heat treatment. Exposure of heat-activated spores to 122 C reduced germination in FNA. The primary germination agents in FNA were identified, and a defined germination medium of glucose, NaCl, l-alanine, and sodium phosphate (GNAP) was developed. Germination of heat-activated spores in GNAP was equivalent to germination in FNA. Injury measured by colony formation on FNA and CNA was correlated to injury measured by reduced germination in both FNA and GNAP. Inactivation of the FNA and GNAP germination systems by secondary treatment exhibited similar kinetics. Therefore, injury expressed as the inability to form colonies on FNA involved alteration of the GNAP germination system.