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     

Ultrastructure of the Exosporium and Underlying Inclusions in Spores of Bacillus megaterium Strains

Spores of selected strains of Bacillus megaterium were prepared by various methods and examined with the electron microscope. An exosporium like that of B. cereus, with a nap and basal layer, was found in spores of a B. megaterium strain that reportedly contains a capsule-like exosporium. The exosporium occasionally appeared to be doubled or have an apical opening. Pili-like filaments were discerned on the surface. Beneath the exosporium were found large deposits of planar inclusions, which in cross section appeared laminated and in surface views consisted of a patchwork of striated packets with a periodicity of approximately 5 nm. The inclusions were usually attached to the exosporium, but in ultrastructure they differed from both the exosporium and coat. In two other strains of B. megaterium, one or two coats occurred but a typical exosporium was not present.     

Effects of heat-, CaCl2- and ethanol-treatments on activation of Bacillus spores

The effects of heat, CaCl2, and ethanol on activation of Bacillus spores were determined by monitoring the absorbance decrease during germination in inosine. Bacillus cereus T, B. subtilis A and B. megaterium QM B1551 spores were activated by heat- and CaCl2-treatments. Ethanol activated B. megaterium and B. subtilis spores yet did not activate B. cereus spores. CaCl2- and ethanol-activations were less effective than heat-activation as judged by optimal germination rates and germination extents. The presence of CaCl2 during heat-treatment inhibited heat-activation of all three Bacillus spores without affecting viability or dipicolinic acid content of the spores. The electrophoretic patterns of coat plus outer membrane proteins extracted from Bacillus spores treated with CaCl2 and heat in the presence of CaCl2 were similar to each other and were distinctively different from the patterns of proteins from unactivated spores or the spores treated with heat and/or ethanol.     

Glucose metabolism via the Embden-Meyerhof pathway is not involved in ATP production during spore germination of bacillus megaterium QM B1551. A study with a mutant lacking hexokinase

In order to investigate contributions by glucose metabolism via the Embden-Meyerhof pathway and that via the direct oxidation route to gluconate to initial ATP production during spore germination, respiratory activity and RNA synthesis were compared between the mutant lacking hexokinase and the parent spores of Bacillus megaterium QM B1551. We found that time courses of those metabolic events were almost identical between those spores, thus clearly indicating that NADH formed by a spore-specific enzyme glucose dehydrogenase (EC 1.1.1.47) is solely responsible for aerobic production of ATP at this stage.     

-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.     

Effects of alpha- and beta-D-glucose on germination of spores of Bacillus megaterium QM B1551.

The effects of D-glucose anomers on the germination of dormant spores of Bacillus megaterium QM B1551 were studied, alpha-D-Glucose (1 mM) slightly initiated the germination of the dormant spores during 10 min incubation at 37 degrees C, while about 60% of the dormant spores became germinated with beta-D-glucose (1 mM) in the same conditions. From the above observations and the finding that only a trace amount of alpha- or beta-D-glucose may bind with the dormant spores, it is speculated that the beta-D-glucose-stereospecific receptor site for the germination exists on the surface of the dormant spores of the bacillus.     

The ExsA protein of Bacillus cereus is required for assembly of coat and exosporium onto the spore surface

The outermost layer of spores of the Bacillus cereus family is a loose structure known as the exosporium. Spores of a library of Tn917-LTV1 transposon insertion mutants of B. cereus ATCC 10876 were partitioned into hexadecane; a less hydrophobic mutant that was isolated contained an insertion in the exsA promoter region. ExsA is the equivalent of SafA (YrbA) of Bacillus subtilis, which is also implicated in spore coat assembly; the gene organizations around both are identical, and both proteins contain a very conserved N-terminal cortex-binding domain of ca. 50 residues, although the rest of the sequence is much less conserved. In particular, unlike SafA, the ExsA protein contains multiple tandem oligopeptide repeats and is therefore likely to have an extended structure. The exsA gene is expressed in the mother cell during sporulation. Spores of an exsA mutant are extremely permeable to lysozyme and are blocked in late stages of germination, which require coat-associated functions. Two mutants expressing differently truncated versions of ExsA were constructed, and they showed the same gross defects in the attachment of exosporium and spore coat layers. The protein profile of the residual exosporium harvested from spores of the three mutants--two expressing truncated proteins and the mutant with the original transposon insertion in the promoter region--showed some differences from the wild type and from each other, but the major exosporium glycoproteins were retained. The exsA gene is extremely important for the normal assembly and anchoring of both the spore coat and exosporium layers in spores of B. cereus.     

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.     

Properties of Hg- and Cd-spores of Bacillus megaterium QM B1551

Hg- and Cd-spores of Bacillus megaterium QM B1551 were produced in Schaeffer's medium containing mercuric chloride and cadmium chloride respectively. Metals were added to the medium at 9 hr of incubation (Stage V) to give a final concentration of 50 microM. It was found by electron microscopic and biochemical studies that the coats of both Hg- and Cd-spores were thinner than those of control spores. Of the total Hg and Cd in the spores, 77% of the Hg and 63% of the Cd were detected in the spore coats. Hg- and Cd-spores were less resistant to heat and more sensitive to germinants than control spores. Other properties of Hg- and Cd-spores were similar to those of control spores. These results suggest that the spore coat has some relationship to the heat resistance and germinability of spores.     

Isolation and properties of membranes from Bacillus megaterium spores.

Membranes from dormant and heat-activated spores of Bacillus megaterium QM B1551 were isolated and purified by gentle lysis procedures followed by differential and sucrose density gradient centrifugations. The purified membranes were enriched for inner membranes and were characterized by their density and content of proteins, phospholipids, enzymes, cytochromes, and carotenoids. These purified spore membranes could be used to investigate their role in the triggering of germination.     

Hydrophobicity of Bacillus and Clostridium spores.

The hydrophobicities of spores and vegetative cells of several species of the genera Bacillus and Clostridium were measured by using the bacterial adherence to hexadecane assay and hydrophobic interaction chromatography. Although spore hydrophobicity varied among species and strains, the spores of each organism were more hydrophobic than the vegetative cells. The relative hydrophobicities determined by the two methods generally agreed. Sporulation media and conditions appeared to have little effect on spore hydrophobicity. However, exposure of spore suspensions to heat treatment caused a considerable increase in spore hydrophobicity. The hydrophobic nature of Bacillus and Clostridium spores suggests that hydrophobic interactions may play a role in the adhesion of these spores to surfaces.     

Hydrophobicity of Bacillus and Clostridium spores

The hydrophobicities of spores and vegetative cells of several species of the genera Bacillus and Clostridium were measured by using the bacterial adherence to hexadecane assay and hydrophobic interaction chromatography. Although spore hydrophobicity varied among species and strains, the spores of each organism were more hydrophobic than the vegetative cells. The relative hydrophobicities determined by the two methods generally agreed. Sporulation media and conditions appeared to have little effect on spore hydrophobicity. However, exposure of spore suspensions to heat treatment caused a considerable increase in spore hydrophobicity. The hydrophobic nature of Bacillus and Clostridium spores suggests that hydrophobic interactions may play a role in the adhesion of these spores to surfaces.     

Utility of sodium hypochlorite for ultrastructure study of bacterial spore integuments

Rode, L. J. (The University of Texas, Austin), and M. Glenn Williams. Utility of sodium hypochlorite for ultrastructure study of bacterial spore integuments. J. Bacteriol. 92:1772-1778. 1966.-Spores of Bacillus megaterium are partially dissolved by sodium hypochlorite. Spore integuments become visible during the dissolution, and ultrastructural features may be detected. Three distinct integument types are described for B. megaterium QM B1551 with the use of this technique. Since a variety of microbial cells are affected by sodium hypochlorite, its use may be applicable to ultrastructure study of cells other than bacterial spores.     

Glucose-triggered germination of Bacillus megaterium spores.

Triggering of germination in Bacillus megaterium QM B1551 spores with D-glucose was studied. First, the interaction of glucose with spores for less than 1 min resulted in triggering almost 90% of the spores after the glucose was removed by dilution. Therefore only a brief time is needed for glucose to trigger germination, and then the continuous presence of glucose is not necessary. Detectable uptake of glucose began 2 to 3 min after absorbance loss started, and a non-metabolizable glucose analog, methyl-alpha-D-glucopyranoside, triggered germination in the absence of detectable uptake. Several inhibitors that reduced or eliminated glucose uptake did not block triggering of germination. Therefore, glucose uptake may be a relatively late event and not a prerequisite for triggering of germination.     

The BclB glycoprotein of Bacillus anthracis is involved in exosporium integrity

Anthrax is a highly fatal disease caused by the gram-positive, endospore-forming, rod-shaped bacterium Bacillus anthracis. Spores, rather than vegetative bacterial cells, are the source of anthrax infections. Spores of B. anthracis are enclosed by a prominent loose-fitting structure called the exosporium. The exosporium is composed of a basal layer and an external hair-like nap. Filaments of the hair-like nap are made up largely of a single collagen-like glycoprotein called BclA. A second glycoprotein, BclB, has been identified in the exosporium layer. The specific location of this glycoprotein within the exosporium layer and its role in the biology of the spore are unknown. We created a mutant strain of B. anthracis DeltaSterne that carries a deletion of the bclB gene. The mutant was found to possess structural defects in the exosporium layer of the spore (visualized by electron microscopy, immunofluorescence, and flow cytometry) resulting in an exosporium that is more fragile than that of a wild-type spore and is easily lost. Immunofluorescence studies also indicated that the mutant strain produced spores with increased levels of the BclA glycoprotein accessible to the antibodies on the surface. The resistance properties of the mutant spores were unchanged from those of the wild-type spores. A bclB mutation did not affect spore germination or kinetics of spore survival within macrophages. BclB plays a key role in the formation and maintenance of the exosporium structure in B. anthracis.     

Predominance of gluconate formation from glucose during germination of Bacillus megaterium QM B1551 spores.

Metabolic pathways of glucose during germination of Bacillus megaterium QM B1551 spores were studied by using specifically labeled glucose and gluconate. The Embden-Meyerhof pathway, the pentose cycle, and the direct oxidation route of glucose to gluconate (the gluconate pathway) were all operative at this stage; among those, gluconate accumulation was most predominant, especially in the early stage. Potassium fluoride, an enolase inhibitor, abolished the catabolism by the Embden-Meyerhof pathway totally without affecting gluconate accumulation. Under these conditions glucose was exclusively oxidized to gluconate. Gluconate thus accumulated could be metabolized further via phosphorylation by gluconate kinase. Remarkable gluconate accumulation was also demonstrated in several other spores requiring alanine as an effective germinant. NADH formed by the direct glucose oxidation may serve as a initial ATP source to phosphorylate glucose in germinating spores.     

Red pigment in Bacillus megaterium spores

Bacillus megaterium QM B1551 spores contained a unique red pigment in their membranes that was not found in other species. This red pigment, presumably a carotenoid, was synthesized about the time of dipicolinic acid synthesis during sporulation and was associated with the forespores. A yellow pigment was synthesized during sporulation in rich medium and was found in the mother cell compartment. Although the yellow pigment was also associated with spores, it could be removed by two different extraction procedures without impairing germination; it was absent when sporulation occurred in a minimal medium. Although the yellow pigment of the mother cell appeared to be dispensable, the red pigment may serve a more critical function, such as membrane stabilization.     

Red pigment in Bacillus megaterium spores.

Bacillus megaterium QM B1551 spores contained a unique red pigment in their membranes that was not found in other species. This red pigment, presumably a carotenoid, was synthesized about the time of dipicolinic acid synthesis during sporulation and was associated with the forespores. A yellow pigment was synthesized during sporulation in rich medium and was found in the mother cell compartment. Although the yellow pigment was also associated with spores, it could be removed by two different extraction procedures without impairing germination; it was absent when sporulation occurred in a minimal medium. Although the yellow pigment of the mother cell appeared to be dispensable, the red pigment may serve a more critical function, such as membrane stabilization.     

Biochemistry of L-proline-triggered germination of Bacillus megaterium spores.

The mechanism by which L-proline triggers germination in Bacillus megaterium QM B1551 spores was investigated. First, brief exposure of spores to L-proline, followed by dilution, was sufficient to trigger germination. Once germination was triggered, the spores continued initiation of germination and did not require high concentrations of L-proline. Triggering of germination was pH and temperature dependent. Second, enzymes for L-proline catabolism were absent in spores, and several non-metabolizable analogs of L-proline were effective trigger compounds. Third, triggering of germination occurred in the presence of inhibitors of proton motive force production, oxygen uptake, and metabolism. Fourth, uptake of L-proline occurred after the triggering of germination. These results argue that neither uptake nor metabolism of L-proline was necessary to trigger germination. Instead, L-proline probably causes a biophysical alteration in the spores that triggers the biochemical changes in germination.     

Germination and peptidoglycan solubilization in Bacillus megaterium spores.

During initiation of Bacillus megaterium QM B1551 spore germination, trichloroacetic acid-soluble, nondialyzable peptidoglycan fragments with an average molecular weight of 20,000 were excreted. This solubilization of peptidoglycan was measured in vitro as the amount of trichloroacetic acid-soluble hexosamine released from a suspension of broken spores. HgC12, a potent inhibitor of initiation, had no effect on the in vitro solubilization of peptidoglycan. In vivo, HgC12 had no effect on peptidoglycan release from spores that had lost heat resistance, but HgC12 did block complete absorbance loss. These results suggest that mercury inhibits some reactions that normally occur before loss in heat resistance but not the subsequent peptidoglycan release, and mercury inhibits other reactions involved with complete absorbance loss.