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     

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     

Repression of sporulation in Bacillus subtilis by L-malate.

L-Malate repressed sporulation in the wild-type strain of Bacillus subtilis. When 75 mM L-malate was added to the growth medium at the time of inoculation, the appearance of heat-resistant spores was delayed 6 to 8 h. The synthesis of extracellular serine protease, alkaline phosphatase, glucose dehydrogenase, and dipicolinic acid was similarly delayed. Sporulation was not repressed when malate was added to the culture at t4 or later. A mutant was selected for ability to sporulate in the presence of malate. This strain could also sporulate in the presence of glucose. The malate-resistant mutant grew poorly with malate as sole carbon source, although it possessed an intact citric acid cycle, and it showed increased levels of malic enzyme. This indicates a defect in the metabolism of malate in the mutant. A mutant lacking malate dehydrogenase activity was also able to sporulate in the presence of malate. A model for the regulation of sporulation by malate is presented and discussed. Citric acid cycle intermediates other than malate did not affect sporulation. In contrast to previous results, sporulation of certain citric acid cycle mutants could be greatly increased or completely restored by the addition of intermediates after the enzymatic block. The results indicate that the failure of citric acid cycle mutants to sporulate can be adequately explained by lack of energy and lack of glutamate.     

Isolation and characterization of Bacillus megaterium mutants containing decreased levels of spore protease.

A proteolytic activity present in spores of Bacillus megaterium has previously been implicated in the initiation of hydrolysis of the A, B, and C proteins which are degraded during spore germination. Four mutants of B. megaterium containing 20 to 30% of the normal level of spore proteolytic activity have been isolated. Partial purification of the protease from wild-type spores by a reviewed procedure resulted in the resolution of spore protease activity on the A, B, and C proteins into two peaks--a major one (protease II) and a minor one (protease I). The protease mutants tested lacked active protease II. All of the mutants exhibited a decreased rate of degradation of the A, B, and C proteins during spore germination at 30 degrees C, but degradation of the proteins did occur. Degradation of the A, B, and C proteins during germination of the mutant spores was decreased neither by blockade of ATP production nor by germination at 44 degrees C. Initiation of spore germination was normal in all four mutants, and all four mutants went through outgrowth, grew, and sporulated normally in rich medium. Similarly, outgrowth of spores of two of the four mutants was normal in minimal medium at 30 degrees C. In the two mutants studied, the kinetics of loss of spore heat resistance and spore UV light resistance during germination were identical to those of wild-type spores. This indicates that the A, B, and C proteins alone are not sufficient to account for the heat or UV light resistance of the dormant spore.     

Coexistence of parentalBacillus brevis and its gramicidin S-negative mutant during spore germination stages

Bacillus brevis strain Nagano and its gramicidin S-negative mutant, BI-7, were compared in separate as well as in mixed cultures with respect to germination of their spores in several media. Mixed-culture experiments were facilitated by the observation that colonies of wild and mutant cultures are distinctly different in appearance on nutrient agar. We found that there was complete coexistence in both strains throughout the outgrowth phase of germination, during which gramicidin S-induced suicide normally occurs in the wild-type prior to vegetative growth. Coexistence was also observed in media supporting germination but not growth, i.e., alanine-salts and alanine-water. The same was found when spores of the two strains were incubated in a soil suspension. We found that both strains become sensitive to starvation in a salts mixture only after development into vegetative cells, the mutant strain being more sensitive than the parent in this regard, but again coexistence was observed in mixed culture.     

Mechanisms of induction of germination of Bacillus subtilis spores by high pressure

Spores of Bacillus subtilis lacking all germinant receptors germinate >500-fold slower than wild-type spores in nutrients and were not induced to germinate by a pressure of 100 MPa. However, a pressure of 550 MPa induced germination of spores lacking all germinant receptors as well as of receptorless spores lacking either of the two lytic enzymes essential for cortex hydrolysis during germination. Complete germination of spores either lacking both cortex-lytic enzymes or with a cortex not attacked by these enzymes was not induced by a pressure of 550 MPa, but treatment of these mutant spores with this pressure caused the release of dipicolinic acid. These data suggest the following conclusions: (i) a pressure of 100 MPa induces spore germination by activating the germinant receptors; and (ii) a pressure of 550 MPa opens channels for release of dipicolinic acid from the spore core, which leads to the later steps in spore germination.     

Studies on gramicidin S-mediated suicide during germination outgrowth ofBacillus brevis spores

We have confirmed the finding of Murray et al. [Lett Appl Microbiol 1: 63–65, 1985] that most of theBacillus brevis spores undergoing the gramicidin S-delayed outgrowth stage of germination are killed by gramicidin S, the antibiotic produced during sporulation. We found, however, that 1% of the population resists this suicidal event even when high concentrations of gramicidin S are added and outgrowth is further delayed. It is obviously this small fraction of the population which, at the end of the long outgrowth stage, develops into vegetative cells. Previous work indicates that this minor population is not genetically resistant to gramicidin S. We conclude that the long delay in germination outgrowth is brought about by two effects of gramicidin S: (1) killing; and (2) decreasing the rate of one or more of the cellular metabolic activities necessary for outgrowth.     

Microcycle Sporogenesis of Bacillus cereus in a Chemically Defined Medium

A chemically defined medium which allowed germination, outgrowth, and subsequent resporulation of Bacillus cereus T spores, without intervening cell division (microcycle sporogenesis), is described. No medium replacement was required. The second-stage spores were heat-stable and had similar germination characteristics and dipicolinic acid content to primary spores. Deoxyribonucleic acid (DNA) replication began soon after germination and there was a doubling in the DNA content of the cells within 2 hr.     

Permeability of dormant spores of Bacillus subtilis to gramicidin S

Abstract Gramicidin S, dissolved in ethanol, penetrated into the inside of the dormant spores of Bacillus subtilis , had a partial inhibitory effect on l-alanine-initiated germination and completely inhibited their outgrowth and vegetative growth. The activity of particulate NADH oxidase of the antibiotic-treated dormant spores was also influenced significantly. Abnormal morphological changes were observed in germinated spores from gramicidin S-treated dormant spores. An immunoelectron microscopy method with colloidal gold-IgG complex showed that the penetration site of gramicidin S inside dormant spores was mainly the core region. These facts suggest that gramicidin S induces the damage of not only the outer membrane-spore coat complex but also the inner membrane surrounding the spore protoplast, and is able to penetrate into the core region of B. subtilis dormant spores.     

Effect of different factors on the germination of spores in S and P- variants of Bacillus brevis]

The effect of temperature, duration of heating and the presence of L-alanine and L-glutamine in the medium on the spore germination was studied with the S and P- variants of Bacillus brevis which did not contain gramicidin S and with the R and P+ varants obtained on a defined medium with beta-phenyl-beta-alanine, an inhibitor of the biosynthesis of gramicidin S. The experiments were carried out according to the scheme of complete factor experiment. Germination of the spores was found upon their incubation in a defined medium with L-alanine within two hours after their preliminary heating at 80 degrees C during 45 minutes (S variant), at 60 degrees C during 45 minutes (R variant+trace amounts of gramicidin S), at 80 degrees C during 15 minutes (P+ variant/trace amounts of gramicidin S). Germination of the spores of the P- variant was best upon heating to 60 degrees C during 45 minutes. Gramicidin S is presumed to inhibit, to a certain extent, germination of the spores of its producing culture.     

Mechanisms of Induction of Germination of Bacillus subtilis Spores by High Pressure

Spores of Bacillus subtilis lacking all germinant receptors germinate >500-fold slower than wild-type spores in nutrients and were not induced to germinate by a pressure of 100 MPa. However, a pressure of 550 MPa induced germination of spores lacking all germinant receptors as well as of receptorless spores lacking either of the two lytic enzymes essential for cortex hydrolysis during germination. Complete germination of spores either lacking both cortex-lytic enzymes or with a cortex not attacked by these enzymes was not induced by a pressure of 550 MPa, but treatment of these mutant spores with this pressure caused the release of dipicolinic acid. These data suggest the following conclusions: (i) a pressure of 100 MPa induces spore germination by activating the germinant receptors; and (ii) a pressure of 550 MPa opens channels for release of dipicolinic acid from the spore core, which leads to the later steps in spore germination.     

Induction of petite mutations during germination and outgrowth of Saccharomyces cerevisiae ascospores.

The germination and outgrowth of Saccharomyces cerevisiae ascospores were studied by determining the sensitivity of the ascospores to the action of chemical mutagens. Survival of the ascospores after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment was low during the first 2 h of germination and then increased and remained constant. Survival of the ascospores after 2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl]aminopropylamino)acridine-2HC1 (ICR-170) treatment was constant from 0 to 5 h, but as the ascospores completed outgrowth at 6 h they became more sensitive to killing by ICR-170. Survival of the ascospores remained high during treatment with 2-methoxy-6-chloro-9-(3-[ethyl-2-hydroxyethyl]aminopropylamino)acridine-2HC1 (ICR-170-OH) or 2,7-diamino-10-ethyl-9-phenyl-phenanthridinium bromide. The main classes of mutations screened for were petites and auxotrophs. The induction of petites and auxotrophs by MNNG was independent of the stage of germination and outgrowth treated. Petite induction by ICR-170 was dependent upon the stage of germination and outgrowth treated. The early hours of germination (0 to 3 h) were not sensitive to petite induction. However, there was maximal petite induction at 5 h into germination and outgrowth, followed by a decline. During this same time period, ICR-170 induced less than 1% auxotrophic colonies. This finding is very unusual because ICR-170 induced 15% auxotrophic colonies in starved log-phase cultures of S. cerevisiae. The acridine ICR-170-OH induced no mutations during germination and outgrowth of the ascospores. Ethidium bromide induced petites, and the petite frequency became maximal at 5 h of germination and outgrowth, a result similar to that obtained with ICR-170.     

Essential role of small, acid-soluble spore proteins in resistance of Bacillus subtilis spores to UV light.

Bacillus subtilis strains containing deletions in the genes coding for one or two of the major small, acid-soluble spore proteins (SASP; termed SASP-alpha and SASP-beta) were constructed. These mutants sporulated normally, but the spores lacked either SASP-alpha, SASP-beta, or both proteins. The level of minor SASP did not increase in these mutants, but the level of SASP-alpha increased about twofold in the SASP-beta- mutant, and the level of SASP-beta increased about twofold in the SASP-alpha- mutant. The growth rates of the deletion strains were identical to that of the wild-type strain in rich or poor growth media, as was the initiation of spore germination. However, outgrowth of spores of the SASP-alpha(-)-beta- strain was significantly slower than that of wild-type spores in all media tested. The heat resistance of SASP-beta- spores was identical to that of wild-type spores but slightly greater than that of SASP-alpha- and SASP-alpha(-)-beta- spores. However, the SASP-alpha- and SASP-alpha(-)-beta- spores were much more heat resistant than vegetative cells. The UV light resistances of SASP-beta- and wild-type spores were also identical. However, SASP-alpha(-)-beta- spores were slightly more sensitive to UV light than were log-phase cells of the wild-type or SASP-alpha(-)-beta- strain (the latter have identical UV light resistances); SASP-alpha- spores were slightly more UV light resistant than SASP-alpha(-)-beta- spores. These data strongly implicate SASP, in particular SASP-alpha, in the UV light resistance of B. subtilis spores.     

The Bacillus anthracis SleL (YaaH) protein is an N-acetylglucosaminidase involved in spore cortex depolymerization

Bacillus anthracis spores, the infectious agents of anthrax, are notoriously difficult to remove from contaminated areas because they are resistant to many eradication methods. These resistance properties are due to the spore's dehydration and dormancy and to the multiple protective layers surrounding the spore core, one of which is the cortex. In order for B. anthracis spores to germinate and resume growth, the cortex peptidoglycan must be depolymerized. This study reports on analyses of sleL (yaaH), which encodes a cortex-lytic enzyme. The inactivation of sleL does not affect vegetative growth, spore viability, or the initial stages of germination, including dipicolinic acid release. However, mutant spores exhibit a slight delay in the loss of optical density compared to that of wild-type spores. Mutants also retain more diaminopimelic acid and N-acetylmuramic acid during germination than wild-type spores, suggesting that the cortex peptidoglycan is not being hydrolyzed as rapidly. This finding is supported by high-pressure liquid chromatography analysis of the peptidoglycan structure used to confirm that SleL acts as an N-acetylglucosaminidase. When sleL is inactivated, the cortex peptidoglycan is not depolymerized into small muropeptides but instead is retained within the spore as large fragments. In the absence of the sleL-encoded N-acetylglucosaminidase, other cortex-lytic enzymes break down the cortex peptidoglycan sufficiently to allow rapid germination and outgrowth.     

A polysaccharide deacetylase gene (pdaA) is required for germination and for production of muramic delta-lactam residues in the spore cortex of Bacillus subtilis

The predicted amino acid sequence of Bacillus subtilis yfjS (renamed pdaA) exhibits high similarity to those of several polysaccharide deacetylases. Beta-galactosidase fusion experiments and results of Northern hybridization with sporulation sigma mutants indicated that the pdaA gene is transcribed by E(sigma)(G) RNA polymerase. pdaA-deficient spores were bright by phase-contrast microscopy, and the spores were induced to germination on the addition of L-alanine. Germination-associated spore darkening, a slow and partial decrease in absorbance, and slightly lower dipicolinic acid release compared with that by the wild-type strain were observed. In particular, the release of hexosamine-containing materials was lacking in the pdaA mutant. Muropeptide analysis indicated that the pdaA-deficient spores completely lacked muramic delta-lactam. A pdaA-gfp fusion protein constructed in strain 168 and pdaA-deficient strains indicated that the protein is localized in B. subtilis spores. The biosynthetic pathway of muramic delta-lactam is discussed.     

Bacteriophage conversion of spore-negative mutants to spore-positive in Bacillus pumilus.

A pseudolysogenic phage, PMB1, was isolated from soil on the basis of its ability to increase the sporulation frequency of the oligosporogenic Bacillus pumilus strain NRS 576 (sporulation frequency, less than 1%). Several spore-negative mutants (sporulation frequency, less than 10-8) derived from strain NRS 576, which were converted to spore positive by infection with PMB1, were subsequently identified. PMB1 repeatedly grown on a given spore-negative mutant (e.g., GW2) converted GW2 cells to spore positive. Each plaque-forming unit initiated the conversion of a spore-positive clone in semisolid agar overlays. GW2 cells remained spore positive as long as they maintained PMB1. Return of PMB1-converted cells to the orginal spore-negative phenotype correlated with loss of PMB1. In liquid media, PMB1 infection increased the sporulation frequency of mutant GW2 over 106-fold. More than half of the spore-negative mutants we isolated from strain NRS 576 were converted to spore positive by PMB1 infection. PMB1-induced spores of the spore-negative mutant GW2 were somewhat more heat sensitive than uninfected or PMB1-infected spores of the spore positive parent of GW2. PMB1-induced spores of GW2 do not differ from wild-type spores in morphology by phase-contrast microscopy, dipicolinic acid content, or rate of sedimentation through Renografin gradients.     

Achievement of complete Bacillus subtilis microcycle sporulation by the addition of S-adenosylmethionine and phospholipids.

In an attempt to find factors that may be responsible for the initiation of sporulation, a system in which the germination and outgrowth phases were separate was applied to Bacillus subtilis. Outgrowth of the germinated spores to only the primary singlet cells was followed in chemically defined medium. Addition of specific metabolites induced the primary singlet cells to sporulate via microcycle sporulation. Experiments are described that led to complete sporulation by the addition of diaminopimelic acid, S-adenosyl-L-methionine and phosphatidylethanolamine.     

Germination-initiated spores of Bacillus brevis Nagano retain their resistance properties.

Initiated spores and vegetative cells of the gramicidin S-producing Bacillus brevis Nagano were compared with respect to their resistance to various forms of stress (osmotic shock-starvation, exposure to ethanol, sonic oscillation, and heat). The resistance of initiated spores to all of these stress situations was considerably greater than that of vegetative cells and approached that of dormant spores. The period during which the initiated spores remained resistant to heat was extended by addition of gramicidin S. The antibiotic may therefore be of survival value to the species in nature by slowing down the development of initiated spores in the outgrowth phase of germination, thereby extending the period during which the cells are resistant to environmental stress.     

Mating Type and Sporulation in Yeast I. Mutations Which Alter Mating-Type Control over Sporulation

In Saccharomyces cerevisiae, meiosis and spore formation as well as mating are controlled by mating-type genes. Diploids heterozygous for mating type (aα) can sporulate but cannot mate; homozygous aa and αα diploids can mate, but cannot sporulate. From an αα diploid parental strain, we have isolated mutants which have gained the ability to sporulate. Those mutants which continue to mate as αα cells have been designated CSP (control of sporulation). Upon sporulation, CSP mutants yield asci containing 4α spores. The mutant gene which allows αα cells to sporulate is unlinked to the mating-type locus and also acts to permit sporulation in aa diploid cells. Segregation data from crosses between mutant αα and wild-type aa diploids and vice versa indicate (for all but one mutant) that the mutation which allows constitutive sporulation (CSP) is dominant over the wild-type allele. Some of the CSP mutants are temperature-sensitive, sporulating at 32°, but not at 23°. In addition to CSP mutants, our mutagenesis and screening procedure led to the isolation of mutants which sporulate by virtue of a change in the mating-type locus itself, resulting in loss of ability to mate.     

Properties of spores of Bacillus subtilis blocked at an intermediate stage in spore germination

Germination of mutant spores of Bacillus subtilis unable to degrade their cortex is accompanied by excretion of dipicolinic acid and uptake of some core water. However, compared to wild-type germinated spores in which the cortex has been degraded, the germinated mutant spores accumulated less core water, exhibited greatly reduced enzyme activity in the spore core, synthesized neither ATP nor reduced pyridine or flavin nucleotides, and had significantly higher resistance to heat and UV irradiation. We propose that the germinated spores in which the cortex has not been degraded represent an intermediate stage in spore germination, which we term stage I.