Live Cell Imaging of Germination and Outgrowth of Individual Bacillus subtilis Spores; the Effect of Heat Stress Quantitatively Analyzed with SporeTracker

Spore-forming bacteria are a special problem for the food industry as some of them are able to survive preservation processes. Bacillus spp. spores can remain in a dormant, stress resistant state for a long period of time. Vegetative cells are formed by germination of spores followed by a more extended outgrowth phase. Spore germination and outgrowth progression are often very heterogeneous and therefore, predictions of microbial stability of food products are exceedingly difficult. Mechanistic details of the cause of this heterogeneity are necessary. In order to examine spore heterogeneity we made a novel closed air-containing chamber for live imaging. This chamber was used to analyze Bacillus subtilis spore germination, outgrowth, as well as subsequent vegetative growth. Typically, we examined around 90 starting spores/cells for ≥4 hours per experiment. Image analysis with the purposely built program “SporeTracker” allows for automated data processing from germination to outgrowth and vegetative doubling. In order to check the efficiency of the chamber, growth and division of B. subtilis vegetative cells were monitored. The observed generation times of vegetative cells were comparable to those obtained in well-aerated shake flask cultures. The influence of a heat stress of 85°C for 10 min on germination, outgrowth, and subsequent vegetative growth was investigated in detail. Compared to control samples fewer spores germinated (41.1% less) and fewer grew out (48.4% less) after the treatment. The heat treatment had a significant influence on the average time to the start of germination (increased) and the distribution and average of the duration of germination itself (increased). However, the distribution and the mean outgrowth time and the generation time of vegetative cells, emerging from untreated and thermally injured spores, were similar.     

Nonoccluded virus-like particles in the mollusc Agriolimax reticulatus (Stylommatophora: Limacinae)

A novel slide-culture technique was used to study germination and outgrowth of Bacillus popilliae spores without disturbing the microenvironment. Infective spores formed in larvae required 24 hr to begin outgrowth, whereas noninfective spores from colonies initiated outgrowth in 12 hr. Dissolution of the paraspore coincided with outgrowth and not with germination of the attached spore. Germination and outgrowth were asynchronous events and required several days in all cell populations, except for free spores.     

Growth from Spores of Clostridium perfringens in the Presence of Sodium Nitrite

The method by which sodium nitrite may act to prevent germination or outgrowth, or both, of heat-injured spores in canned cured meats was investigated by using Clostridium perfringens spores. Four possible mechanisms were tested: (i) prevention of germination of the heat-injured spores, (ii) prior combination with a component in a complex medium to prevent germination of heat-injured spores, (iii) inhibition of outgrowth of heat-injured spores, and (iv) induction of germination (which would render the spore susceptible to thermal inactivation). Only the third mechanism was effective with the entire spore population when levels of sodium nitrite commercially acceptable in canned cured meats were used. Concentrations of 0.02 and 0.01% prevented outgrowth of heat-sensitive and heat-resistant spores, respectively. Nitrite-induced germination occurred with higher sodium nitrite concentrations.     

Germination of Heat- and Alkali-Altered Spores of Clostridium perfringens Type A by Lysozyme and an Initiation Protein

The normal system functioning in the utilization of metabolizable germinants by both heat-sensitive and heat-resistant spores of Clostridium perfringens was inactivated by heat or by treatment of the spores with alkali to remove a soluble coat protein layer. Altered spores were incapable of germination (less than 1%) and outgrowth (less than 0.0005%) in complex media without the addition of either lysozyme or an initiation protein produced by C. perfringens. The addition of either of these agents permitted, in the case of alkali-treated spores, both 90 to 95% germination and outgrowth, as measured by colony formation. In the case of heat-damaged spores, only 50% germination and 2% outgrowth resulted from addition of the initiation protein, whereas lysozyme permitted 85% germination and 8% outgrowth. Alteration of the spores by heat or alkali apparently inactivated the normal lytic system responsible for cortical degradation during germination. Kinetics of production of the initiation protein and conditions affecting both its activity and that of lysozyme on altered spores are described.     

Germination and outgrowth of Bacillus popilliae in hemolymph slide mounts

Cabbage looper hemolymph induced rapid germination and outgrowth of spores of Bacillus popilliae. Spores germinated within sporangia but outgrowth occurred from free released spores as well as from spores retained in sporangia. With 37°C, an alkaline pH, and tyrosinase, outgrowth resulted in 1 hr. Of six strains of milky disease bacteria tested, hemolymph mediated germination and outgrowth of only those which are infective perorally for European chafer larvae, indicating a potential use as a screening tool to assess virulence for the chafer.     

The influence of gramicidin S on hydrophobicity of germinating Bacillus brevis spores

Gramicidin S is known to prolong the outgrowth stage of spore germination in the producing culture. Bacillus brevis strain Nagano and its gramicidin S-negative mutant, BI-7, were compared with respect to cell-surface hydrophobicity and germination of their spores. Parental spores were hydrophobic as determined by adhesion to hexadecane, whereas mutant spores showed no affinity to hexadecane. Addition of gramicidin S to mutant spores resulted in a high cell surface hydrophobicity and a delay in germination outgrowth. The hydrophobicity of parental spores was retained throughout most of the germination period. Hydrophobicity was lost as outgrowing spores entered into the stage of vegetative growth. The data indicate that gramicidin S is responsible for the hydrophobicity of B. brevis spores. It is suggested that in making spores hydrophobic, the antibiotic plays a role in concentrating the spores at interfaces where there is a higher probability of finding nutrients for germination and growth.Abbreviation GS Gramicidin S     

The effect of visible radiations on the germination and outgrowth of Bacillus spores

The effect of visible radiations on the germination and outgrowth of spores of Bacillus subtilis MD2 and Bacillus subtilis var. niger was determined by direct observation of populations irradiated on the surface of nutrient agar. Little effect on germination (phase darkening) was found but white light prevented outgrowth of some and retarded it for all spores. Different wavebands in the visible spectrum differed in their effect on outgrowth, the greatest retardation being found for the shorter wavelengths, 410–570 nm. Outgrowth in dark controls was always greater both in number of spores outgrown and rate of outgrowth. The results are consistent with others, suggesting an effect of singlet oxygen generated from endogenous photosensitizers by visible radiation.     

A Method for Extracting RNA from Dormant and Germinating Bacillus subtilis Strain 168 Endospores

RNA was extracted from dormant and germinating Bacillus subtilis 168 spores (intact spores and chemically decoated spores) by using rapid rupture followed by acid–phenol extraction. Spore germination progress was monitored by assaying colony forming ability before and after heat shock and by reading the optical density at 600 nm. The purity, yield, and composition of the extracted RNA were determined spectrophotometrically from the ratio of absorption at 260 nm to that at 280 nm; in a 2100 BioAnalyzer, giving the RNA yield/10⁸ spores or cells and the distribution pattern of rRNA components. The method reported here for the extraction of RNA from dormant spores, as well as during different phases of germination and outgrowth, has proven to be fast, efficient and simple to handle. RNA of a high purity was obtained from dormant spores and during all phases of germination and growth. There was a significant increase in RNA yield during the transition from dormant spores to germination and subsequent outgrowth. Chemically decoated spores were retarded in germination and outgrowth compared with intact spores, and less RNA was extracted; however, the differences were not significant. This method for RNA isolation of dormant, germinating, and outgrowing bacterial endospores is a valuable prerequisite for gene expression studies, especially in studies on the responses of spores to hostile environmental conditions.     

The Effect of Sporulation Temperature on the Resistance of Bacillus subtilis to a Chemical Inhibitor

The ability of spores of Bacillus subtilis to germinate at 50° in sublethal concentrations of chlorocresol is related to sporulation temperature as is the resistance of the subsequent outgrowth at 50° to this substance. The degree of germination, age of spores and amount of outgrowth produced are of minor importance in determining resistance of the outgrowth.     

Effect of phenolic compounds and oleuropein on the germination of Bacillus cereus T spores

The phenolic compounds extracted from olives with ethyl acetate inhibited germination and outgrowth of Bacillus cereus T spores. Purified oleuropein, a well-characterized component of olive extract, inhibited these processes also. The addition of oleuropein and olive extracts 3 or 5 min after germination began, immediately decreased the rate of change of phase bright to phase dark spores and delayed significantly outgrowth.     

Properties of Bacillus megaterium and Bacillus subtilis mutants which lack the protease that degrades small, acid-soluble proteins during spore germination.

During germination of spores of Bacillus species the degradation of the spore's pool of small, acid-soluble proteins (SASP) is initiated by a protease termed GPR, the product of the gpr gene. Bacillus megaterium and B. subtilis mutants with an inactivated gpr gene grew, sporulated, and triggered spore germination as did gpr+ strains. However, SASP degradation was very slow during germination of gpr mutant spores, and in rich media the time taken for spores to return to vegetative growth (defined as outgrowth) was much longer in gpr than in gpr+ spores. Not surprisingly, gpr spores had much lower rates of RNA and protein synthesis during outgrowth than did gpr+ spores, although both types of spores had similar levels of ATP. The rapid decrease in the number of negative supertwists in plasmid DNA seen during germination of gpr+ spores was also much slower in gpr spores. Additionally, UV irradiation of gpr B. subtilis spores early in germination generated significant amounts of spore photoproduct and only small amounts of thymine dimers (TT); in contrast UV irradiation of germinated gpr+ spores generated almost no spore photoproduct and three to four times more TT. Consequently, germinated gpr spores were more UV resistant than germinated gpr+ spores. Strikingly, the slow outgrowth phenotype of B. subtilis gpr spores was suppressed by the absence of major alpha/beta-type SASP. These data suggest that (i) alpha/beta-type SASP remain bound to much, although not all, of the chromosome in germinated gpr spores; (ii) the alpha/beta-type SASP bound to the chromosome in gpr spores alter this DNA's topology and UV photochemistry; and (iii) the presence of alpha/beta-type SASP on the chromosome is detrimental to normal spore outgrowth.     

Identification of Bacillus anthracis proteins associated with germination and early outgrowth by proteomic profiling of anthrax spores

The use of anthrax spores as a bioweapon has spurred efforts aimed at identifying key proteins expressed in Bacillus anthracis. Because spore germination and outgrowth occur prior to and are required for disease manifestations, blocking germination and early outgrowth with novel vaccines or inhibitors targeting critical B. anthracis germination and outgrowth-associated factors is a promising strategy in mitigating bioterror. By screening 587 paired protein spots that were isolated from dormant and germinating anthrax spores, respectively, we identified 10 spore proteins with statistically significant germination-associated increases and decreases. It is likely that proteins whose levels change during germination may play key roles in the germination and outgrowth processes, and they should be listed as priority targets for development of prophylactic and therapeutic agents against anthrax. The 31 new proteins identified in this study also complement an emerging proteomic database of B. anthracis.     

Role of the Nfo and ExoA apurinic/apyrimidinic endonucleases in repair of DNA damage during outgrowth of Bacillus subtilis spores

Germination and outgrowth are critical steps for returning Bacillus subtilis spores to life. However, oxidative stress due to full hydration of the spore core during germination and activation of metabolism in spore outgrowth may generate oxidative DNA damage that in many species is processed by apurinic/apyrimidinic (AP) endonucleases. B. subtilis spores possess two AP endonucleases, Nfo and ExoA; the outgrowth of spores lacking both of these enzymes was slowed, and the spores had an elevated mutation frequency, suggesting that these enzymes repair DNA lesions induced by oxidative stress during spore germination and outgrowth. Addition of H₂O₂ also slowed the outgrowth of nfo exoA spores and increased the mutation frequency, and nfo and exoA mutations slowed the outgrowth of spores deficient in either RecA, nucleotide excision repair (NER), or the DNA-protective α/β-type small acid-soluble spore proteins (SASP). These results suggest that α/β-type SASP protect DNA of germinating spores against damage that can be repaired by Nfo and ExoA, which is generated either spontaneously or promoted by addition of H₂O₂. The contribution of RecA and Nfo/ExoA was similar to but greater than that of NER in repair of DNA damage generated during spore germination and outgrowth. However, nfo and exoA mutations increased the spontaneous mutation frequencies of outgrown spores lacking uvrA or recA to about the same extent, suggesting that DNA lesions generated during spore germination and outgrowth are processed by Nfo/ExoA in combination with NER and/or RecA. These results suggest that Nfo/ExoA, RecA, the NER system, and α/β-type SASP all contribute to the repair of and/or protection against oxidative damage of DNA in germinating and outgrowing spores.     

Germination and outgrowth of Schizosaccharomyces pombe spores isolated by a simple batch centrifugation technique

Spores of the fission yeast Schizosaccharomyces pombe have been separated from vegetative cells by a simple and rapid centrifugation (800 g for 20 min) through a 35% Hypaque solution to a purity greater than 95%. Approximately 35% of the spores were recovered. Regrowth in EMM2 plus glucose showed that over 97% of the spores germinated within the first 2 h and outgrowth continued between 5 and 10 h. Sucrose induced germination in greater than 95% of the spores with a 1 h delay and outgrowth in 50% of the spores with a 3 h delay. There was little protein synthesis during germination but the protein content increased linearly coincident with outgrowth. The RNA content increased slightly during germination, but increased linearly 1 h before the onset of outgrowth and protein synthesis. After 8 h of regrowth, coincident with the onset of DNA synthesis, the rate of RNA synthesis was accelerated. The DNA content had increased 1.7-fold after 10 h of regrowth from a haploid level of 1.36 x 10(-8) microgram spore-1.     

Effects of inactivation or overexpression of the sspF gene on properties of Bacillus subtilis spores.

Inactivation of the Bacillus subtilis sspF gene had no effect on sporulation, spore resistance, or germination in a wild-type strain or one lacking DNA protective alpha/beta-type small, acid-soluble proteins (SASP). Overexpression of SspF in wild-type spores or in spores lacking major alpha/beta-type SASP (alpha- beta- spores) had no effect on sporulation but slowed spore outgrowth and restored a small amount of UV and heat resistance to alpha- beta- spores. In vitro analyses showed that SspF is a DNA binding protein and is cleaved by the SASP-specific protease (GPR) at a site similar to that cleaved in alpha/beta-type SASP. SspF was also degraded during spore germination and outgrowth, and this degradation was initiated by GPR.     

Effect of Sodium Nitrite, Sodium Chloride, and Sodium Nitrate on Germination and Outgrowth of Anaerobic Spores

The effects of meat-curing agents on germination and outgrowth of putrefactive anaerobe 3679h (PA 3679h) spores were studied in microcultures. Nitrite concentrations up to 0.06% at pH 6.0 or between 0.8 and 1% at pH 7.0 allowed emergence and elongation of vegetative cells but blocked cell division. The newly emerged cells then lysed. With more than 0.06% nitrite at pH 6.0 or more than 0.8 to 1% at pH 7.0, the spores lost refractility and swelled, but vegetative cells did not emerge. Even as much as 4% nitrite failed to prevent germination (complete loss of refractility) and swelling of the spores. Sodium chloride concentrations above 6% prevented complete germination (i.e., the spores retained a refractile core). In the presence of 3 to 6% sodium chloride, most of the spores germinated and produced vegetative cells, but cell division was often blocked. Sodium nitrate had no apparent effect on germination and outgrowth at concentrations up to 2%.     

Interaction of Apurinic/Apyrimidinic Endonucleases Nfo and ExoA with the DNA Integrity Scanning Protein DisA in the Processing of Oxidative DNA Damage during Bacillus subtilis Spore Outgrowth

Oxidative stress-induced damage, including 8-oxo-guanine and apurinic/apyrimidinic (AP) DNA lesions, were detected in dormant and outgrowing Bacillus subtilis spores lacking the AP endonucleases Nfo and ExoA. Spores of the Δnfo exoA strain exhibited slightly slowed germination and greatly slowed outgrowth that drastically slowed the spores'' return to vegetative growth. A null mutation in the disA gene, encoding a DNA integrity scanning protein (DisA), suppressed this phenotype, as spores lacking Nfo, ExoA, and DisA exhibited germination and outgrowth kinetics very similar to those of wild-type spores. Overexpression of DisA also restored the slow germination and outgrowth phenotype to nfo exoA disA spores. A disA-lacZ fusion was expressed during sporulation but not in the forespore compartment. However, disA-lacZ was expressed during spore germination/outgrowth, as was a DisA-green fluorescent protein (GFP) fusion protein. Fluorescence microscopy revealed that, as previously shown in sporulating cells, DisA-GFP formed discrete globular foci that colocalized with the nucleoid of germinating and outgrowing spores and remained located primarily in a single cell during early vegetative growth. Finally, the slow-outgrowth phenotype of nfo exoA spores was accompanied by a delay in DNA synthesis to repair AP and 8-oxo-guanine lesions, and these effects were suppressed following disA disruption. We postulate that a DisA-dependent checkpoint arrests DNA replication during B. subtilis spore outgrowth until the germinating spore''s genome is free of damage.     

Germination initiation and outgrowth of spores ofBacillus brevis strain Nagano and its gramicidin S-negative mutant

Bacillus brevis strain Nagano and its gramicidin S-negative mutant, BI-7, were compared with respect to germination of their spores produced in several media. Germination initiation occurred in the presence of nutrient broth orL-alanine but not with inosine, glucose, glycerol or fructose; the process was activated by heat. Parental and mutant spores behaved similarly in these experiments. During outgrowth, parental spores remained in this phase of germination much longer than did mutant spores, but only when the parental spores had been harvested from a sporulation medium where significant gramicidin S synthesis had occurred. When parental spores were extracted or treated with an enzyme that hydrolyzes gramicidin S, rapid outgrowth occurred. Adding exogenous gramicidin S or the extract from parental spores to mutant spores lengthened the outgrowth in a dose-dependent manner. The uptake of labeledL-alanine by parental spores was delayed compared to mutant spores in the presence or absence of chloramphenicol. These data suggest a mechanism of action for gramicidin S whereby it interferes in membrane function, such as transport or energy metabolism, in outgrowing spores.Abbreviations GS Gramicidin S - CFU colony-forming units     

Bacterial spores in food: how phenotypic variability complicates prediction of spore properties and bacterial behavior

Bacillus spores are a known cause of food spoilage and their increased resistance poses a major challenge in efficient elimination. Recent studies on bacterial cultures at the single cell level have revealed how minor differences in essential spore properties, such as core water content or germinant receptor levels, can cause the observed differences in spore germination and outgrowth behavior. Moreover, heterogeneous behavior is influenced by commonly accepted food preservation techniques, such as heating or the usage of weak organic acids. Understanding the underlying molecular mechanisms and key players involved in phenotypic heterogeneity of spores, while taking the spore's history into account, will improve predictability of the spore's behavior to various treatments and triggers.     

Mechanism of the Heat Sensitization of Bacillus subtilis Spores by Ethidium Bromide

Pretreatment with ethidium bromide (5 μg/ml) followed by a water wash had no effect on unheated Bacillus subtilis spores, but the viability of these spores after heating was much lower than that of similarly heated spores exposed to water alone. The fate of water- or ethidium bromide-treated spores, unheated or heated, was followed by allowing them to germinate and outgrow in a minimal or a complex liquid medium. Spores exposed to ethidium bromide and then heated (85°C, 10 min) exhibited a developmental block during germination and outgrowth. Many of them were blocked at the stage when the bacterium emerged from the germinated spore. When 0.35 μg of ethidium bromide per ml was added to heated spores in the germination-growth medium, the outgrowth of heated spores was inhibited to the same extent as were pretreated spores. Ethidium bromide acted in the first hour of germination of heated spores since addition after this time was ineffective in inhibiting recovery events. Repair of heat-damaged spore DNA was detected during the first 2 h of germination. The addition of ethidium bromide (final concentration, 0.35 μg/ml) inhibited DNA repair during early outgrowth. Increased sensitivity of spores to heat after pretreatment with sublethal concentrations of ethidium bromide was due to the inhibition of the repair of heat-damaged DNA.