Physiology of Sporulation of Clostridia

RONALD G. LABBE and N-J. REMI SHIH*

Food Microbiology Laboratory, Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA and, *Department of Chemical Engineering and Material Science, University of California Davis, Davis, CA 95616-5294, USA

I. INTRODUCTION

Exposure of Clostridia to conditions of nutrition depletion activates alternate metabolic pathways. Sporulation is the ultimate example of this adaptation response, which begins at the onset of stationary phase. Unlike most adaptive responses, bacterial sporulation involves several metabolic pathways associated with morphological, physiological and biochemical changes and can affect end products associated with energy metabolism. Several changes involve metabolites associated with stationary phase and sporulation. However, not all are sporulation-related or specific. Studies of the sporulation process at the molecular level have just begun in the genus Clostridium but are extensive in the case of aerobic sporeformers, Bacillus subtilis in particular.1'2 This chapter presents some aspects of the current knowledge about how sporulation affects the metabolism of the Clostridia and focuses on the methods that promote in vitro sporulation and the regulation of sporulation-associated products. Not surprisingly, most studies of sporulation of Clostridia have focused on species of medical or economic importance.

The Clostridia: Molecular Biology and Pathogenesis ISBN 0-12-595020-9

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II. MORPHOLOGICAL CHANGES DURING SPORULATION

The morphological events during sporulation of Clostridium species are similar to those occurring during sporulation of Bacillus species. That is, they are divided into seven stages (0-VII) based on electron microscopic studies and associated physical and biochemical properties.3"7 Stage VII is associated with the autolysis of the sporangium and release of the mature spore. The sporulation process is thought to be induced by nutrient deprivation which causes a drop in the GTP pool.8

Vegetative and sporulating cells of the same Clostridium species often exhibit distinct shapes. For example, in a glucose-rich medium, even in stationary phase, Clostridium perfringens cells are relatively short, whereas in a starch-based sporulation medium, they are longer. Although this is not the case in all Clostridia, the change of morphology provides a useful tool in laboratory observations. Sporulation is also fundamental in the classification of the Clostridia. In addition, in the case of one species, C. perfringens, an enterotoxin is produced during the sporulation process. From a practical viewpoint the production of bacterial spores also minimizes storage and handling requirements.

III. IN VITRO SPORULATION

Although sporulation generally is initiated by a lack of nutrients, Clostridia need an energy source for macromolecular synthesis during sporulation and the cells may die if one or more nutrients are completely absent. However, not all Clostridium species can sporulate well in laboratory media because conditions for their sporulation can be highly specific. For example, Clostridium cellulo-lyticum sporulates following cellobiose starvation. A level of 30% sporulation occurs if cells are starved during the exponential phase of growth, otherwise no spores or very few spores are observed.9

High sporulation levels of certain Clostridium species such as C. perfringens, are notoriously difficult to achieve, which has resulted in numerous modifications of defined and complex media. Several reagents such as caffeine, theophylline, isobutylmethylxanthine, and papaverine in the methylxanthine family have been reported to enhance sporulation in the Clostridia although their effects are somewhat strain-specific and concentration-dependent.10-13 The effect of sporulation enhancement by these compounds is characterized by reduced growth due to their ability to inhibit macromolecular synthesis.14 Because the compounds in the methylxanthine family are purine analogs, Setlow and Sacks15 concluded that their mechanisms of action are similar to that of purine deprivation in B. subtilis, namely, a drop in the GTP pool. Therefore, it is not surprising that guanosine ( 1 him) can suppress sporulation and increase vegetative growth of C. perfringens.^ Human bile salts also enhance the sporulation of some Clostridium species, although an excess amount of human bile salts may result in poor growth and result in little sporulation.17

Rapidly metabolizable carbohydrates such as glucose are generally avoided in sporulation media because they are vigorously fermented by certain saccharo-lytic species of Clostridium, resulting in considerable acid production.18'19 In fact, improved sporulation of C. perfringens has been reported by adjusting the initial pH value of the sporulation medium to 7.8 with sodium carbonate,20 which contributes to the buffering capacity of the medium.21

The optimal temperature for sporulation of C. perfringens is between 35 and 40°C. Although a few strains of this species can sporulate at high temperatures (>40°C),22 they require the exogenous addition of a-amylase to sporulate in starch-based media23 since amylase production by these strains is minimal at higher temperatures.24'25 Addition of exogenous C. perfringens enterotoxin has also been reported to hasten the sporulation process of this organism26 although the mechanism is unknown.

The specific carbohydrate composition of the sporulation media also affects the ability to sporulate. For example, sporulation of C. perfringens is inhibited by high concentrations (>15 ihm) of glucose, maltose, mannose, lactose and sucrose but is unaffected by the presence of high amounts (>30 mM) of ribose, galactose and fructose.27 In the case of Clostridium botulinum a level of 270 mM glucose decreases sporulation from 80% to 30%.28 However, some strains of C. botulinum require glucose for growth.29 A high concentration of glucose also inhibits sporulation of Clostridium thermosaccharolyticum30 but stimulates sporulation in Clostridium acetobutylicum.3] Ababouch and Busta32 have reported that an increase in glucose concentration (from 0.2% to 1%) delays the onset of sporulation in Clostridium sporogenes and results in a tenfold increase in the number of spores (per millilitre).

Starch and dextrin are used as carbohydrate sources in sporulation media of C. perfringens,33'34 This organism can sporulate in the presence of 8 mM glucose when soluble starch and dextrin are included in a defined medium. The type of starch is also of importance, with soluble starch resulting in higher spore levels than potato, corn or arrowroot starch.35 Substitution of starch with raffinose can also enhance sporulation in some strains of C. perfringens.36

Supplementation of complex sporulation medium with 1 mM Mn2+ and Zn2+ enhances sporulation of C. botulinum and results in a one-log increase in spores. However, Fe2+ is less effective at the same concentration37 and less sporulation of C. botulinum is observed if Cu2+ (0.5-1.0 mM) is present in the medium together with Zn2+.38

IV. MOLECULAR REGULATION OF SPORULATION

It has long been suggested that the sporulation processes in Bacillus spp. and Clostridium spp. share similar molecular mechanisms. Nevertheless, little attention has been paid to the genetic analysis of the latter, despite its considerable biotechnical potential.39'40

In Bacillus spp. phosphorylation of the regulatory protein SpoOA controls the initiation of sporulation and also changes in gene expression that occur during transition into stationary phase.41'42 Homologues of the B. subtilus spoOA and spoIVB genes have been identified in six Clostridium species with three highly conserved regions within the effector domain.43 Such homologs were absent in several nonsporulating bacteria closely related to B. subtilus. Disruption of the spoOA homolog in C. acetobutylicum by integrative recombination produces a Spo~ phenotype and abolishes the solvent biosynthesis properties of the parent strain.

Small cytoplasmic RNA (scRNA) is a stable and abundant RNA of approximately 270 nucleotides from B. subtilus.44 It is a member of the signal-recognition particle (SRP)RNA family and consists of four domains based on secondary structure with domains I and II necessary for formation of heat-resistant spores.45 Recent evidence indicates that scRNA of C. perfringens is of a size similar to that of B. subtilus scRNA with about 70% primary sequence homology.46 Functional analysis showed that C. perfringens scRNA allows the formation of heat-resistant spores in a scRNA-depleted B. subtilus strain. This suggests that conservation of domains I and II, needed for efficient sporulation, has conferred a selective advantage throughout eubacterial evolution.

Finally, there is evidence that some of the more complex regulatory features of sporulation in B. subtilus are conserved in C. acetobutylicum. Transposon insertions can occur at 12 or more chromosomal sites which normally express the developmental signals which induce solvent production and sporulation 41 The variety of solvent classes observed in such mutants suggest several levels of control of solventogenesis in C. acetobutylicum. In B. subtilus four sporulation-specific sigma factors, oE, oF, oG, and oK, are involved in the sporulation process. Homologous genes for these sigma factors are present in C. acetobutylicum, and have been cloned and sequenced.48'49 Hybridization analysis indicates the presence of oH, oD and spoIID homologs, and again indicates the evolutionary conservation of molecular mechanisms of sporulation in members of the extremely diverse Bacillus and Clostridium genera.48'49

Physiology of sporulation 25 V. METABOLITES ASSOCIATED WITH THE SPORULATION PROCESS

A. Regulation of enzymes during sporulation

Clostridia synthesize certain compounds and enzymes when they sporulate, but not all are sporulation-specific or related to sporulation. Some sporulation media contain complex carbohydrates such as starch or dextrin. Amylolytic action during sporulation in such media contributes to cell growth and sporulation by providing metabolizable, short-chain carbon sources. a-Amylase synthesis may be regulated by sporulation.27 Cells of C. perfringens at stages II—III of sporulation are committed to complete sporulation and fail to synthesize a-amylase when transferred to a medium optimized for a-amylase synthesis. Conversely, a-amylase synthesis decreases when vegetative cells are induced to sporulate.50 In C. perfringens, eight amylolytic enzymes, which can be divided into two distinct groups according to the immunological and biochemical properties of the a-amylase they produce, are excreted during vegetative cell growth. Sporulation affects the amount of these eight amylolytic enzymes in the culture supernatant fluid (Figure 2.1). The intensification of amylase bands 1, 7, and 8, and the disappearance of bands 5 and 6 is observed when C. perfringens sporulates.27 Synthesis of the highest levels of a a-amylase require the presence of a small amount (6-10 min) of a simple sugar while higher levels inhibit sporulation.50 It has been proposed that in Gram-positive bacteria the regulation of amylolytic enzymes involves a global regulatory mechanism. A irans-acting factor, CcpA, encoded by the ccpA gene, and closely related to the Lac and Gal repressors, can bind to a responsive element in many genes encoding proteins that are regulated by mechanisms similar to that of a-amylase.51 Similar to ccpA, a regulatory gene (regA) has been identified in C. acetobutylicum. This gene encodes a RegA protein that contains a DNA-binding domain homologous to CcpA in B. subtilis.52 A mutation in the ccpA gene allows sporulation and amylase production of B. subtilis in the presence of excess glucose.5" 54

A chymotrypsin-like intracellular protease has been isolated from sporulating C. perfringens cells and may function in the processing of enterotoxin but is less important in spore coat protein synthesis.55 The relationship between protease levels and sporulation has also been studied using Spo~ mutants of C. perfringens. Protease activity of Spo~ mutants is reduced by 87-88% compared with wild type. The wild type also has drastically reduced protease levels when grown under conditions that repress sporulation. A serine-type protease rather than metallo-type is associated with sporulation.56 Like C. perfringens, C. cellulo-lyticum strains lacking proteolytic activity also fail to induce sporulation.9

The nitrogenase activity of C. butyricum is reportedly low in vegetative cells, increases at Stage III of sporulation (forespore formation) and then decreases

Figure 2.1 Distribution of extracellular amylolytic activities in the Clostridium perfringens NCTC 8679 culture fluid during vegetative growth (Veg) (mid-log phase) and sporulation (Spo). Amylases were precipitated by ammonium sulfate, dialysed, concentrated by ultrafiltration and separated by Polyacrylamide gel electrophoresis under native conditions. Zymograms of amylase activity were prepared by immersing gels in soluble starch followed by a brief exposure to iodine. Minor amylase activity was enhanced by white dotted lines: reproduced from Shih.25

Figure 2.1 Distribution of extracellular amylolytic activities in the Clostridium perfringens NCTC 8679 culture fluid during vegetative growth (Veg) (mid-log phase) and sporulation (Spo). Amylases were precipitated by ammonium sulfate, dialysed, concentrated by ultrafiltration and separated by Polyacrylamide gel electrophoresis under native conditions. Zymograms of amylase activity were prepared by immersing gels in soluble starch followed by a brief exposure to iodine. Minor amylase activity was enhanced by white dotted lines: reproduced from Shih.25

when sporulation continues.57 Similar observations have been reported in Clostridium pasteurianum,58

B. Toxins and proteins

Some C. perfringens type A strains produce an enterotoxin (CPE) which causes food poisoning59 and which is discussed at length in Chapter 19. The amount of enterotoxin increases dramatically during sporulation.18,60-62 Although the enterotoxin gene, cpe, has been cloned in Escherichia coli, the amount of enterotoxin produced by E. coli is far less than that produced by C. perfringens during sporulation.63 The enterotoxin was proposed to be a component of the spore coat.64 However, recent studies indicate that spore coat proteins are not immunologically identical to CPE.65 The presence of trace amounts of entero-toxin-like protein in vegetative cells of C. perfringens has been reported.62'63

Clostridium Sporulation Pathway

Figure 2.2 Electron micrographs of Clostridium perfringens inclusion bodies. A, Sporulation-associated inclusion body (NCTC 8679): reproduced from Loffler and Labbe,68 with permission. B, Inclusion body from vegetative cells (NCTC 8238): reproduced from Garcia-Alvarado et al.,70 with permission.

Thus, CPE is unlikely to be a component of C. perfringens spores. By ligating the cpe promoter to the E. coli gusA gene, which encodes (3-glucuronidase, it has been shown that the induction of cpe gene expression is an early stationary phase event.66 The enterotoxin is induced between Stage 0 and Stage III of sporulation; mutants blocked at later stages still produce the toxin.67 Because virtually all enterotoxin-negative strains also sporulate it appears more likely that the cpe gene plays no role in sporulation but is activated by transcriptional factors which also control sporulation genes. Results of on-going studies of cpe regulation at the transcriptional level may provide explanations for the high levels of enterotoxin produced during sporulation of this organism (see Chapter 27).

An inclusion body associated with the sporulation of enterotoxin-positive strains of C. perfringens has been reported (Figure 2.2a). It possesses biological activity and is composed of C. perfringens enterotoxin.68'69 Another type of inclusion body is present in vegetative cells of entertoxin-positive and entero-toxin-negative strains of C. perfringens, especially when they are incubated above 40°C (Figure 2.2b), and, as viewed by phase-contrast microscopy, is remarkably similar in appearance to a small spore.70 It differs from the inclusion

Figure 2.2 Electron micrographs of Clostridium perfringens inclusion bodies. A, Sporulation-associated inclusion body (NCTC 8679): reproduced from Loffler and Labbe,68 with permission. B, Inclusion body from vegetative cells (NCTC 8238): reproduced from Garcia-Alvarado et al.,70 with permission.

body associated with sporulation in that it is refractile when observed by phase-contrast microscopy, is not surrounded by vacuoles, and is composed of none-nterotoxin-related protein.

Unlike C. perfringens enterotoxin, the production of Clostridium difficile toxin A (also an enterotoxin) is not sporulation-specific.71 However, the protein toxins of Clostridium bifermentans, a novel class of insecticidal toxins, are synthesized during sporulation.72 C. botulinum type C2 neurotoxin is reportedly associated with sporulation, whereas type A and E toxins are produced by vegetative cultures.8 Like Bacillus species, Clostridia synthesize several small, acid-soluble spore proteins (SASPs) which account for about 20% of the spore protein during sporulation and are degraded during germination.73,74 The amino acid sequences of two major SASPs (SASP-a and SASP-(3) of C. bifermentans are almost identical and are very similar to those of C. perfringens,74

C. Acids, solvents and other byproducts

Many Clostridia produce solvents such as ethanol, acetone or butanol by converting the organic acids produced from carbohydrate sources in the medium. Clostridium acetobutylicum excretes acetic and butyric acids which cause the pH of the medium to fall below 5.0 at the onset of the stationary phase of growth, although the intracellular pH of C. acetobutylicum is unaffected.75 The acids are converted to acetone, butanol and ethanol after prolonged incubation as a detoxification process in response to the accumulation of the acid end-products.76 Although sporulation also is observed at the time of organic acid accumulation, it is not a prerequisite for solvent production.77'78 The overall yield of solvents depends on the total amount of acidic products converted from the carbon source in the growth medium rather than the degree of sporulation of C. acetobutylicum. At a low initial glucose concentration, C. acetobutylicum is unable to produce acetone and butanol because of the low levels of acetic and butyric acids excreted.80 In contrast, high-sporulating strains of Clostridium felsineum have been reported which produce less butyric and propionic acid but twice as much butanol than low-sporulating strains.81

It has been recently noted that sterile, concentrated supernatants of C. perfringens can promote sporulation of certain homologous and heterologous strains of this organism.82 The effect of this sporulation factor is concentration-dependent. The substance responsible for this reaction is still unidentified but it is heat- and acid-stable, and possesses a molecular weight of less than 5000. Although its physiological role is unclear it is produced by both vegetative and sporulating cultures of C. perfringens and also by enterotoxin-positive and enterotoxin-negative strains.

In summary, the conditions of nutrition deprivation that induce sporulation also induce other metabolic responses. It is not surprising that regulatory factors expressed during stationary phase can affect metabolic pathways. However, details of the mechanisms of sporulation in the genus Clostridium and their sporulation-associated metabolites have yet to be clarified. The multiple regulatory events controlling vegetative cell growth, sporulation and enzyme and solvent production and excretion in Clostridium species indicate the diversity of this genus.

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B: Genome Organization and Molecular Genetics

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