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.     

Spore membrane(s) as the site of damage within heated Clostridium perfringens spores.

Clostridium perfringens spores were injured by ultrahigh-temperature treatment at 105 C for 5 min. Injury was manifested as an increased sensitivity to polymyxin and neomycin. Since many of the survivors could not germinate normally the ultrahigh-temperature-treated spores were sensitized to and germinated by lysozyme. Polymyxin reportedly acts upon the cell membrane. Neomycin may inhibit protein synthesis and has surface-active properties. Injured spores were increasingly sensitive to known surface-active agents, sodium lauryl sulfate, sodium deoxycholate, and Roccal, a quaternary ammonium compound. Injured spores sensitive to polymyxin and neomycin also were osmotically fragile and died during outgrowth in a liquid medium unless the medium was supplemented with 20% sucrose, 10% dextran, or 10% polyvinylpyrrolidone. The results suggested that a spore structure destined to become cell membrane or cell wall was the site of injury. Repair of injury during outgrowth in the presence of protein, deoxyribonucleic acid, ribonucleic acid and cell wall synthesis inhibitors was consistent with this hypothesis.     

Effect of nalidixic acid on the activation of RNA synthesis in outgrowing spores of Bacillus subtilis

Outgrowth of B. subtilis spores depends on the action of DNA gyrase (comp. Matsuda and Kameyama 1980). Application of nalidixic acid (100 micrograms/ml) to dormant spores of Bacillus subtilis prevents the outgrowth. Application of nalidixic acid (100 micrograms/ml) during the early outgrowth phase (after a 20 min germination period) does not prevent, but only delay spore outgrowth. Germination of spores is not influenced. Nalidixic acid is an effective inhibitor of RNA synthesis in outgrowing spores, whereas vegetative cells are more resistant. Spores can grow out inspite of a remarkably reduced intensity of RNA synthesis. Nalidixic acid particularly inhibits the synthesis of stable RNA, probably that of ribosomal RNA. We suggest that DNA gyrase-catalyzed alterations in DNA structure are involved in the regulation of the gene expressional program of outgrowing B. subtilis spores.     

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     

Coordinating DNA replication with cell division: lessons from outgrowing spores

Progress in solving the long-standing puzzle of how a cell coordinates chromosome replication with cell division is significantly aided by the use of synchronous cell populations. Currently three systems are employed for obtaining such populations: the Escherichia coli 'baby machine', the developmentally-controlled cell cycle of Caulobacter crescentus, and Bacillus subtilis germinated and outgrowing spores. This review examines our current understanding of the relationship between replication and division and how the use of B. subtilis outgrowing spores and, more recently its combination with immunofluorescence microscopy, has contributed significantly to this important area of biology. About 20 years ago, and also more recently, this system was used to show convincingly that termination of DNA replication is not essential for a central septum to form, raising the possibility that the early stages of division occur well before termination. It has also been demonstrated that there is no major synthesis of the division initiation proteins, FtsZ and DivIB, linked to initiation, progression or completion of the first round of chromosome replication accompanying spore outgrowth. This has led to the suggestion that the primary link between chromosome replication and cell division at midcell is not likely to occur through a control over the levels of these proteins. Very recent work has employed a combination of the use of B. subtilis outgrowing spores with immunofluorescence microscopy to investigate the relationship between midcell Z ring assembly and the round of chromosome replication linked to it. The results of this work suggest a role for initiation and progression into the round of replication in blocking midcell Z ring formation until the round is complete or almost complete, thereby ensuring that cell division occurs between two equally-partitioned chromosomes.     

Mechanism of the hydrolysis of 4-methylumbelliferyl-beta-D-glucoside by germinating and outgrowing spores of Bacillus species

AIMS: To determine the mechanism of the hydrolysis of 4-methylumbelliferyl-beta-D-glucopyranoside (beta-MUG) by germinating and outgrowing spores of Bacillus species. METHODS AND RESULTS: Spores of B. atrophaeus (formerly B. subtilis var. niger, Fritze and Pukall 2001) are used as biological indicators of the efficacy of ethylene oxide sterilization by measurement of beta-MUG hydrolysis during spore germination and outgrowth. It was previously shown that beta-MUG is hydrolysed to 4-methylumbelliferone (MU) during the germination and outgrowth of B. atrophaeus spores (Chandrapati and Woodson 2003), and this was also the case with spores of B. subtilis 168. Germination of spores of either B. atrophaeus or B. subtilis with chloramphenicol reduced beta-MUG hydrolysis by almost 99%, indicating that proteins needed for rapid beta-MUG hydrolysis are synthesized during spore outgrowth. However, the residual beta-MUG hydrolysis during spore germination with chloramphenicol indicated that dormant spores contain low levels of proteins needed for beta-MUG uptake and hydrolysis. With B. subtilis 168 spores that lacked several general proteins of the phosphotransferase system (PTS) for sugar uptake, beta-MUG hydrolysis during spore germination and outgrowth was decreased >99.9%. This indicated that beta-MUG is taken up by the PTS, resulting in the intracellular accumulation of the phosphorylated form of beta-MUG, beta-MUG-6-phosphate (beta-MUG-P). This was further demonstrated by the lack of detectable glucosidase activity on beta-MUG in dormant, germinated and outgrowing spore extracts, while phosphoglucosidase active on beta-MUG-P was readily detected. Dormant B. subtilis 168 spores had low levels of at least four phosphoglucosidases active on beta-MUG-P: BglA, BglH, BglC (originally called YckE) and BglD (originally called YdhP). These enzymes were also detected in spores germinating and outgrowing with beta-MUG, but levels of BglH were the highest, as this enzyme's synthesis was induced ca 100-fold during spore outgrowth in the presence of beta-MUG. Deletion of the genes coding for BglA, BglH, BglC and BglD reduced beta-MUG hydrolysis by germinating and outgrowing spores of B. subtilis 168 at least 99.7%. Assay of glucosidases active on beta-MUG or beta-MUG-P in extracts of dormant and outgrowing spores of B. atrophaeus revealed no enzyme active on beta-MUG and one enzyme that comprised > or =90% of the phosphoglucosidase active on beta-MUG-P. Partial purification and amino-terminal sequence analysis of this phosphoglucosidase identified this enzyme as BglH. CONCLUSIONS: Generation of MU from beta-MUG by germinating and outgrowing spores of B. atrophaeus and B. subtilis is mediated by the PTS-driven uptake and phosphorylation of beta-MUG, followed by phosphoglucosidase action on the intracellular beta-MUG-P. The major phosphoglucosidase catalyzing MU generation from beta-MUG-P in spores of both species is probably BglH. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new insight into the mechanism of uptake and hydrolysis of beta-MUG by germinating and outgrowing spores of Bacillus species, in particular B. atrophaeus. The research reported here provides a biological basis for a Rapid Readout Biological Indicator that is used to monitor the efficacy of ethylene oxide sterilization.     

Mechanisms of killing of spores of Bacillus subtilis by dimethyldioxirane

AIMS: To determine the mechanisms of Bacillus subtilis spore resistance to and killing by a novel sporicide, dimethyldioxirane (DMDO) that was generated in situ from acetone and potassium peroxymonosulfate at neutral pH. METHODS AND RESULTS: Spores of B. subtilis were effectively killed by DMDO. Rates of killing by DMDO of spores lacking most DNA protective alpha/beta-type small, acid-soluble spore proteins (alpha- beta- spores) or the major DNA repair protein, RecA, were very similar to that of wild-type spore killing. Survivors of wild-type and alpha- beta- spores treated with DMDO also exhibited no increase in mutations. Spores lacking much coat protein due either to mutation or chemical decoating were much more sensitive to DMDO than were wild-type spores, but were more resistant than growing cells. Wild-type spores killed with this reagent retained their large pool of dipicolinic acid (DPA), and the survivors of spores treated with DMDO were sensitized to wet heat. The DMDO-killed spores germinated with nutrients, albeit more slowly than untreated spores, but germinated faster than untreated spores with dodecylamine. The killed spores were also germinated by very high pressures and by lysozyme treatment in hypertonic medium, but many of these spores lysed shortly after their germination, and none of these treatments were able to revive the DMDO-killed spores. CONCLUSIONS: DMDO is an effective reagent for killing B. subtilis spores. The spore coat is a major factor in spore resistance to DMDO, which does not kill spores by DNA damage or by inactivating some component needed for spore germination. Rather, this reagent appears to kill spores by damaging the spore's inner membrane in some fashion. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrates that DMDO is an effective decontaminant for spores of Bacillus species that can work under mild conditions, and the killed spores cannot be revived. Evidence has also been obtained on the mechanisms of spore resistance to and killing by this reagent.     

Honey-coloured,sessile Endogone spores

Summary Wall structure is described in the parent and resting spores of an Endogone sp. with honey-coloured, sessile spores. Wall thickness increases in the parent spore and subtending hypha by passage of material through the plasmalemma, or by formation of an apparently separate inner wall and degeneration of the trapped cytoplasm. Structure and development of the multi-layered wall of the mature resting spore are described. Unusual features are: 1. the incorporation of many pigment granules into the coloured outer wall, 2. the presence between the outer coloured and inner transparent walls of a tripartite membrane and adjacent layer with a regular periodicity and 3. a sectored layer with a crystalline component. The structure of the wall is discussed with reference to that of other mucoraceous fungi, to spore germination and to the mechanism of wall formation.     

Decreased particulate NADH oxidase activity in Bacillus subtilis spores after polymyxin B treatment

The activities of several enzymes of polymyxin B-treated dormant and germinated spores of Bacillus subtilis were examined. The particulate NADH oxidase of the antibiotic-treated spores showed considerably lower specific and total activities compared with those of untreated ones. The specific and total NADH oxidase activities of untreated spores increased 12- and 15-fold respectively during germination, whereas increases during germination of polymyxin B-treated spores were inhibited. The specific and total activities of particulate NADH cytochrome c reductase of dormant spores were decreased by polymyxin B treatment in almost the same proportion as those of the particulate NADH oxidase. The specific activity of NADH dehydrogenase of dormant spores remained unchanged after antibiotic treatment but the total activity fell considerably. The activities of other enzymes examined were similar for untreated dormant and germinated spores and antibiotic-treated spores. The respiration of polymyxin B-treated dormant spores was inhibited at the same time as the start of germination. Morphologically, polymyxin B-treated dormant spores lost a laminar structure of the cortex and details of the spore protoplast. The inhibitory mechanism of particulate NADH oxidase activity of polymyxin B-treated dormant spores is discussed.     

Synthesis of poly(A)-containing RNA in outgrowing spores of Bacillus subtilis

The synthesis of poly(A)-containing RNA in outgrowing spores of Bacillus subtilis was studied. A significant amount of RNA puls-labelled with 3H-uridine is polyadenylated. With the beginning of RNA synthesis in outgrowing spores labelled poly(A)-containing RNA was detected. The amount of poly(A)-RNA during the outgrowth and first cell division remains constant. Besides poly(A)-RNA the synthesis of tRNA and rRNA occurs. These results indicate a simultaneous activation of synthesis of tRNA, rRNA as well as of poly(A)-containing RNA during outgrowth of B. subtilis spores.     

Analysis of Outgrowth of Bacillus subtilis Spores Lacking Penicillin-Binding Protein 2a

The loss of Bacillus subtilis penicillin-binding protein (PBP) 2a, encoded by pbpA, was previously shown to slow spore outgrowth and result in an increased diameter of the outgrowing spore. Further analyses to define the defect in pbpA spore outgrowth have shown that (i) outgrowing pbpA spores exhibited only a slight defect in the rate of peptidoglycan (PG) synthesis compared to wild-type spores, but PG turnover was significantly slowed during outgrowth of pbpA spores; (ii) there was no difference in the location of PG synthesis in outgrowing wild-type and pbpA spores once cell elongation had been initiated; (iii) outgrowth and elongation of pbpA spores were dramatically affected by the levels of monovalent or divalent cations in the medium; (iv) there was a partial redundancy of function between PBP2a and PBP1 or -4 during spore outgrowth; and (v) there was no difference in the structure of PG from outgrowing wild-type spores or spores lacking PBP2a or PBP2a and -4; but also (vi) PG from outgrowing spores lacking PBP1 and -2a had transiently decreased cross-linking compared to PG from outgrowing wild-type spores, possibly due to the loss of transpeptidase activity.     

Mechanisms of killing of spores of Bacillus subtilis by iodine, glutaraldehyde and nitrous acid

Treatment of wild-type spores of Bacillus subtilis with glutaraldehyde or an iodine-based disinfectant (Betadine) did not cause detectable mutagenesis, and spores (termed alpha-beta-) lacking the major DNA-protective alpha/beta-type, small, acid-soluble proteins (SASP) exhibited similar sensitivity to these agents. A recA mutation did not sensitize wild-type or alpha-beta- spores to Betadine or glutaraldehyde, nor did spore treatment with these agents result in significant expression of a recA-lacZ fusion when the treated spores germinated. Spore glutaraldehyde sensitivity was increased dramatically by removal of much spore coat protein, but this treatment had no effect on Betadine sensitivity. In contrast, nitrous acid treatment of wild-type and alpha-beta- spores caused significant mutagenesis, with alpha-beta- spores being much more sensitive to this agent. A recA mutation further sensitized both wild-type and alpha-beta- spores to nitrous acid, and there was significant expression of a recA-lacZ fusion when nitrous acid-treated spores germinated. These results indicate that: (a) nitrous acid kills B. subtilis spores at least in part by DNA damage, and alpha/beta-type SASP protect against this DNA damage; (b) killing of spores by glutaraldehyde or Betadine is not due to DNA damage; and (c) the spore coat protects spores against killing by glutaraldehyde but not Betadine. Further analysis also demonstrated that spores treated with nitrous acid still germinated normally, while those treated with glutaraldehyde or Betadine did not.     

Mechanisms of killing of Bacillus subtilis spores by hypochlorite and chlorine dioxide

AIMS: To determine the mechanisms of Bacillus subtilis spore killing by hypochlorite and chlorine dioxide, and its resistance against them. METHODS AND RESULTS: Spores of B. subtilis treated with hypochlorite or chlorine dioxide did not accumulate damage to their DNA, as spores with or without the two major DNA protective alpha/beta-type small, acid soluble spore proteins exhibited similar sensitivity to these chemicals; these agents also did not cause spore mutagenesis and their efficacy in spore killing was not increased by the absence of a major DNA repair pathway. Spore killing by these two chemicals was greatly increased if spores were first chemically decoated or if spores carried a mutation in a gene encoding a protein essential for assembly of many spore coat proteins. Spores prepared at a higher temperature were also much more resistant to these agents. Neither hypochlorite nor chlorine dioxide treatment caused release of the spore core's large depot of dipicolinic acid (DPA), but hypochlorite- and chlorine dioxide-treated spores much more readily released DPA upon a subsequent normally sub-lethal heat treatment than did untreated spores. Hypochlorite-killed spores could not initiate the germination process with either nutrients or a 1 : 1 chelate of Ca2+-DPA, and these spores could not be recovered by lysozyme treatment. Chlorine dioxide-treated spores also did not germinate with Ca2+-DPA and could not be recovered by lysozyme treatment, but did germinate with nutrients. However, while germinated chlorine dioxide-killed spores released DPA and degraded their peptidoglycan cortex, they did not initiate metabolism and many of these germinated spores were dead as determined by a viability stain that discriminates live cells from dead ones on the basis of their permeability properties. CONCLUSIONS: Hypochlorite and chlorine dioxide do not kill B. subtilis spores by DNA damage, and a major factor in spore resistance to these agents appears to be the spore coat. Spore killing by hypochlorite appears to render spores defective in germination, possibly because of severe damage to the spore's inner membrane. While chlorine dioxide-killed spores can undergo the initial steps in spore germination, these germinated spores can go no further in this process probably because of some type of membrane damage. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide information on the mechanisms of the killing of bacterial spores by hypochlorite and chlorine dioxide.     

Characteristics of developing mitochondrial genetic and respiratory functions in germinating fungal spores.

Spores of the fungus Botryodiplodia theobromae began a cyanide-sensitive oxygen consumption immediately upon exposure to a liquid medium, and spore germination and respiration were not affected by ethidium bromide, D-threochloramphenicol, and acriflavin until later during germ tube emergence. These inhibitors of the mitochondrial genetic system all inhibited total cell protein synthesis to the same intermediate degree from the outset of incubation. When spores were incubated in water under non-germinating conditions, protein synthesis and oxygen uptake proceeded at initial rates almost identical to those seen in spores germinating in the presence of the three mitochondrial system inhibitors. Although the spores respired at rapid rates from the onset of incubation, no cytochrome absorption peaks could be observed in mitochondrial fractions prepared from ungerminated spores; they were readily observed in germinated spores, however. When the spores were germinated in the presence of inhibitors of the mitochondrial system, an excess of cytochrome c was observed in the near absence of cytochromes a and b. The results indicate that the ungerminated spores of this organism contain a preserved, potentially functional aerobic respiratory system which requires cycloheximide-sensitive ribosome activity to become functional when the spores are inoculated into a liquid medium.     

Honey-coloured,sessile Endogone spores: II. Changes in fine structure during spore development

Summary The fine structure of honey-coloured, sessile Endogone spores is described from initiation of the mother spore to dormancy of the resting spore. Three unusual organelles occur viz. pigment granules, large crystals and selfduplicating bacteria-like organisms. The first two are very numerous, and are specifically associated with spore formation. The pigment granules are involved in the deposition of the honey-coloured wall, and change into myelin-like figures when cytoplasm moves from the mother into the resting spore. The crystals, whose function is not known, are most conspicuous just before the resting spore reaches dormancy. The bacteria-like organisms, which may be actinomycete spores living symbiotically in the fungus, multiphy greatly as the spore enters dormancy. The dormant spore contains very little cytoplasm compressed into a fine network between very large polygonal oil globules and large round bodies thought to contain a storage polysaccharide.     

Germination of spores of Bacillus subtilis with dodecylamine

AIMS: To determine the properties of Bacillus subtilis spores germinated with the alkylamine dodecylamine, and the mechanism of dodecylamine-induced spore germination. METHODS AND RESULTS: Spores of B. subtilis prepared in liquid medium were germinated efficiently by dodecylamine, while spores prepared on solid medium germinated more poorly with this agent. Dodecylamine germination of spores was accompanied by release of almost all spore dipicolinic acid (DPA), degradation of the spore's peptidoglycan cortex, release of the spore's pool of free adenine nucleotides and the killing of the spores. The dodecylamine-germinated spores did not initiate metabolism, did not degrade their pool of small, acid-soluble spore proteins efficiently and had a significantly lower level of core water than did spores germinated by nutrients. As measured by DPA release, dodecylamine readily induced germination of B. subtilis spores that: (a) were decoated, (b) lacked all the receptors for nutrient germinants, (c) lacked both the lytic enzymes either of which is essential for cortex degradation, or (d) had a cortex that could not be attacked by the spore's cortex-lytic enzymes. The DNA in dodecylamine-germinated wild-type spores was readily stained, while the DNA in dodecylamine-germinated spores of strains that were incapable of spore cortex degradation was not. These latter germinated spores also did not release their pool of free adenine nucleotides. CONCLUSIONS: These results indicate that: (a) the spore preparation method is very important in determining the rate of spore germination with dodecylamine, (b) wild-type spores germinated by dodecylamine progress only part way through the germination process, (c) dodecylamine may trigger spore germination by a novel mechanism involving the activation of neither the spore's nutrient germinant receptors nor the cortex-lytic enzymes, and (d) dodecylamine may trigger spore germination by directly or indirectly activating release of DPA from the spore core, through the opening of channels for DPA in the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide new insight into the mechanism of spore germination with the cationic surfactant dodecylamine, and also into the mechanism of spore germination in general. New knowledge of mechanisms to stimulate spore germination may have applied utility, as germinated spores are much more sensitive to processing treatments than are dormant spores.     

GIBBERELLIC ACID-INDUCED GERMINATION OF SPORES OF ANEMIA PHYLLITIDIS: NUCLEIC ACID AND PROTEIN SYNTHESIS DURING GERMINATION

The distribution and synthesis of nucleic acids and proteins during gibberellic acid-induced germination of spores of Anemia phyllitidis were studied in order to relate biochemical activity with morphogenetic aspects of germination. Germination is accompanied by the hydrolysis of storage protein granules and the localized appearance of cytoplasmic RNA, protein, and insoluble carbohydrates in a small area adjoining the spore wall and surrounding the nucleus. The protoplast of the spore enlarges in this region, the spore wall breaks and a protonemal cell is formed which contains many chloroplasts. A second division in the spore at right angles to the first yields a rhizoid cell. Autoradiography of ³H-thymidine incorporation has shown that DNA is synthesized both in the nucleus and in the immediately surrounding cytoplasm of the germinating spore until some time after the first division, although a strictly nuclear DNA synthesis is observed later. Synthesis of RNA and proteins is limited to the presumptive regions of the germinating spore which become the protonema and rhizoid, shifting to specific sites in these cells as germination proceeds. The nucleus of the spore continues to be biosynthetically active long after it ceases to divide.     

Synchytrium solstitiale: reclassification based on the function and role of resting spores

Studies were made about resting spores of Synchytrium solstitiale, a chytrid that causes false rust disease of yellow starthistle (YST). During evaluation of this fungus for biological control of YST, a protocol for resting spore germination was developed. Details of resting spore germination and study of long-term survival of the fungus were documented. Resting spores from dried leaves germinated after incubating them on water agar at least 7 d at 10-15 C. Resting spores were viable after storage in air-dried leaves more than 2 y at room temperature, suggesting they have a role in off-season and long-term survival of the fungus. Each resting spore produced a single sorus that contained a single sporangium, which on germination released zoospores through a pore. YST inoculated with germinated resting spores developed symptoms typical of false rust disease. All spore forms of S. solstitiale have been found to be functional, and the life cycle of S. solstitiale has been completed under controlled laboratory and greenhouse conditions. Resting spore galls differ from sporangial galls both morphologically and biologically, and in comparison, each sporangial gall cleaves into several sori and each sorus produces 5-25 sporangia that rupture during release of zoospores. For this reason S. solstitiale should be reclassified as diheterogallic sensu Karling (Am J Bot 42:540-545). Because resting spores function as prosori and produce an external sorus, S. solstitiale is best placed in into the subgenus Exosynchytrium.     

Germination of spores of Bacillus brevis and inhibition by gramicidin S: a stratagem for survival

Germinating spores of Bacillus brevis are sensitive to inhibition by gramicidin S prior to emergence whereas once emergence is underway inhibition is lost and newly emerged vegetative cells are not affected by the antibiotic. Under conditions of overcrowding the concentration of antibiotic released is sufficient to render the germinated spores non-viable although dormant spores still retain their viability. Considering these points and the manner in which spore populations germinate we outline a strategy of germination for survival of Bacillus brevis .