Emergence and development of resistance to antimicrobial chemicals and heat in spores of Bacillus subtilis

The emergence and development of chemical and thermal resistance in spores of Bacillus subtilis was examined. The chemicals studied were of the disinfectant type: glutaraldehyde, hypochlorite, hypochlorite-methanol and povidone-iodine. Growth and sporulation were followed by electron microscopy and resistance assigned to specific stages in relation to ⁴⁵Ca and DPA accumulation. A sequential development of resistance was observed with thermal resistance appearing first at early Stage V corresponding to maturation of cortex and deposition of rudimentary spore coat material. Chemical resistance coincided with middle to late Stage V dependent on the chemical concerned. A progressive development of resistance was observed on prolonged incubation in sporulation medium and was affected by inclusion of lysozyme in the spore washing sequence.     

Emergence of resistance to glutaraldehyde in spores of Bacillus subtilis 168

Abstract The emergence of resistance to glutaraldehyde in spores of Bacillus subtilis 168 was examined. Resistance to an organic solvent (toluene), heat and lysozyme were included for comparison. A sequential development of resistance was observed, with toluene resistance occuring early on in sporulation (stages III and IV), thermal resistance at early stage V, lysozyme resistance at middle stage V and glutaraldehyde resistance arising late in stage V. Studies with sporulation mutants also indicate that glutaraldehyde resistance is acquired even later than lysozyme resistance and may therefore possibly be considered as a very late marker event for sporulation, characterizing late stages of B. subtilis 168 spore formation.     

Emergence and development of chlorhexidine resistance during sporulation of Bacillus subtilis 168

Abstract During sporulation of Bacillus subtilis strain 168 initiated by step-down conditions, resistance to chlorhexidine diacetate (CHA) developed at about t _3.5, before heat but after toluene resistance. Mutants blocked at stage IV of sporulation were sensitive to all three treatments. Stage V mutants were toluene resistant but moderately sensitive to heat and CHA. A stage VI mutant was resistant to all three treatments. Thus, chlorhexidine resistance is likely to be a result of spore coat, rather than of cortex, development.     

A two-dimensional protein gel electrophoresis study of the heat stress response of Bacillus subtilis cells during sporulation

The heat resistance of spores of Bacillus subtilis formed at 30 degrees C was enhanced by pretreatment at 48 degrees C for 30 min, 60 min into sporulation, for all four strains examined. High-resolution two-dimensional gel electrophoresis showed the generation and/or overexpression of 60 proteins, 11 of which were specific to heat shock, concurrent to this acquired thermotolerance. The greatest number of new proteins was observed between 30 and 60 min after heat shock, and the longer the time between exponential growth and heat treatment, the fewer differences were observed on corresponding protein profiles. The time at which heating produced the maximum increase in spore resistance and the most new proteins on two-dimensional gels occurred before alkaline phosphatase and dipicolinic acid production and corresponded to stage I or II of sporulation. The stress proteins formed disappeared later in sporulation, suggesting that heat shock proteins increase spore heat resistance by altering spore structure rather than by repairing heat damage during germination and outgrowth.     

Roles of DacB and spm proteins in clostridium perfringens spore resistance to moist heat, chemicals, and UV radiation

Clostridium perfringens food poisoning is caused mainly by enterotoxigenic type A isolates that typically possess high spore heat resistance. Previous studies have shown that alpha/beta-type small, acid-soluble proteins (SASP) play a major role in the resistance of Bacillus subtilis and C. perfringens spores to moist heat, UV radiation, and some chemicals. Additional major factors in B. subtilis spore resistance are the spore's core water content and cortex peptidoglycan (PG) structure, with the latter properties modulated by the spm and dacB gene products and the sporulation temperature. In the current work, we have shown that the spm and dacB genes are expressed only during C. perfringens sporulation and have examined the effects of spm and dacB mutations and sporulation temperature on spore core water content and spore resistance to moist heat, UV radiation, and a number of chemicals. The results of these analyses indicate that for C. perfringens SM101 (i) core water content and, probably, cortex PG structure have little if any role in spore resistance to UV and formaldehyde, presumably because these spores' DNA is saturated with alpha/beta-type SASP; (ii) spore resistance to moist heat and nitrous acid is determined to a large extent by core water content and, probably, cortex structure; (iii) core water content and cortex PG cross-linking play little or no role in spore resistance to hydrogen peroxide; (iv) spore core water content decreases with higher sporulation temperatures, resulting in spores that are more resistant to moist heat; and (v) factors in addition to SpmAB, DacB, and sporulation temperature play roles in determining spore core water content and thus, spore resistance to moist heat.     

Effects of orphan histidine kinases on clostridial sporulation progression and metabolism

Solventogenesis and sporulation of clostridia are the main responsive adaptations to the acidic environment during acetone–butanol–ethanol (ABE) fermentation. It was hypothesized that five orphan histidine kinases (HKs) including Cac3319, Cac0323, Cac0903, Cac2730, and Cac0437 determined the cell fates between sporulation and solventogenesis. In this study, the comparative genomic analysis revealed that a mutation in cac0437 appeared to contribute to the nonsporulating feature of ATCC 55025. Hence, the individual and interactive roles of five HKs in regulating cell growth, metabolism, and sporulation were investigated. The fermentation results of mutants with different HK expression levels suggested that cac3319 and cac0437 played critical roles in regulating sporulation and acids and butanol biosynthesis. Morphological analysis revealed that cac3319 knockout abolished sporulation (Stage 0) whereas cac3319 overexpression promoted spore development (Stage VII), and cac0437 knockout initiated but blocked sporulation before Stage II, indicating the progression of sporulation was altered through engineering HKs. By combinatorial HKs knockout, the interactive effects between two different HKs were investigated. This study elucidated the regulatory roles of HKs in clostridial differentiation and demonstrated that HK engineering can be effectively used to control sporulation and enhance butanol biosynthesis.     

Identification and characterization of a gene cluster involved in manganese oxidation by spores of the marine Bacillus sp. strain SG-1.

The marine Bacillus sp. strain SG-1 forms spores that oxidize manganese(II) as a result of the activities of uncharacterized components of its spore coat. Nucleotide sequence analysis of chromosomal loci previously identified through insertion mutagenesis as being involved in manganese oxidation identified seven possible genes (designated mnxA to mnxG) in what appears to be an operon. A potential recognition site for the sporulation, mother-cell-specific, RNA polymerase sigma factor, sigmaK, was located just upstream of the cluster, and correspondingly, measurement of beta-galactosidase activity from a Tn917-lacZ insertion in mnxD showed expression at mid-sporulation to late sporulation (approximately stage IV to V of sporulation). Spores of nonoxidizing mutants appeared unaffected with respect to their temperature and chemical resistance properties and germination characteristics. However, transmission electron microscopy revealed alterations in the outermost spore coat. This suggests that products of these genes may be involved in the deposition of the spore coat structure and/or are spore coat proteins themselves. Regions of the deduced protein product of mnxG showed amino acid sequence similarity to the family of multicopper oxidases, a diverse group of proteins that use multiple copper ions to oxidize a variety of substrates. Similar regions included those that are involved in binding of copper, and the addition of copper at a low concentration was found to enhance manganese oxidation by the spores. This suggests that the product of this gene may function like a copper oxidase and that it may be directly responsible for the oxidation of manganese by the spores.     

Spore Refractility in Variants of Bacillus cereus Treated with Actinomycin D

Refractility as indicated by light microscopy, electron microscopy of thin sections, and freeze fracture etching was increased and maintained in a cortexless mutant, A(-)1, of Bacillus cereus var. alesti by the addition during sporulation stage 4 of actinomycin D, which prevents the terminal lysis of spore core associated with sporulation in this organism. (45)Calcium uptake levels and dipicolinic acid (DPA) content were similarly maintained. The location of these components appears to be in the spore protoplast. In the parent A(-), treated with actinomycin D during stage 4, spore particles with similar morphology to the mutant, that is without a cortex and with the characteristics of refractility, were obtained. A major difference in sensitivity to actinomycin D between the processes of (45)Ca uptake and DPA synthesis was observed. Some heat resistance in A(-) made cortexless by actinomycin D could be observed. These studies indicate that the role of the cortex is not to produce the dehydrated refractile spore state but to maintain it.     

A method for the determination of bacterial spore DNA content based on isotopic labelling, spore germination and diphenylamine assay; ploidy of spores of several Bacillus species.

A reliable method for measuring the spore DNA content, based on radioactive DNA labelling, spore germination in absence of DNA replication and diphenylamine assay, was developed. The accuracy of the method, within 10-15%, is adequate for determining the number of chromosomes per spore, provided that the genome size is known. B subtilis spores were shown to be invariably monogenomic, while those of larger bacilli Bacillus megaterium, Bacillus cereus and Bacillus thuringiensis, often, if not invariably, contain two genomes. Attempts to modify the spore DNA content of B subtilis by altering the richness of the sporulation medium, the sporulation conditions (liquid or solid medium), or by mutation, were apparently unsuccessful. An increase of spore size with medium richness, not accompanied by an increase in DNA content, was observed. The implication of the apparently species-specific spore ploidy and the influence of the sporulation conditions on spore size and shape are discussed.     

Interference Contrast and Phase Contrast Microscopy of Sporulation and Germination of Bacillus megaterium

The techniques of Nomarski interference contrast microscopy and phase-contrast microscopy were compared for their utility in monitoring sporulation and germination in Bacillus megaterium. The Nomarski technique permitted rapid and easy delineation of septation and engulfment during sporulation, whereas with phase contrast microscopy these stages were not detected at all. The later stages of sporulation were easily seen by either technique. Thus, of the seven stages of sporulation as recognized by the electron microscopy of thin sections, five can now be routinely detected quantitatively by optical microscopy: septation (stage II), engulfment (stage III), phase-dark forespore (corresponding to cortex formation, stage IV), phase-bright spore in a sporangium (corresponding to coat formation, stage V), and the free spore (stage VII). This means that now only stage I (axial filament) and stage VI (maturation of the refractile spore) require electron microscopy for routine detection. There was no advantage in using Nomarski optics for germination studies.     

Effect of radioprotective agents in sporulation medium on Bacillus subtilis spore resistance to hydrogen peroxide, wet heat and germicidal and environmentally relevant UV radiation

Aims: To determine the effects of cysteine, cystine, proline and thioproline as sporulation medium supplements on Bacillus subtilis spore resistance to hydrogen peroxide (H₂O₂), wet heat, and germicidal 254 nm and simulated environmental UV radiation. Methods and Results: Bacillus subtilis spores were prepared in a chemically defined liquid medium, with and without supplementation of cysteine, cystine, proline or thioproline. Spores produced with thioproline, cysteine or cystine were more resistant to environmentally relevant UV radiation at 280–400 and 320–400 nm, while proline supplementation had no effect. Spores prepared with cysteine, cystine or thioproline were also more resistant to H₂O₂ but not to wet heat or 254‐nm UV radiation. The increases in spore resistance attributed to the sporulation supplements were eliminated if spores were chemically decoated. Conclusions: Supplementation of sporulation medium with cysteine, cystine or thioproline increases spore resistance to solar UV radiation reaching the Earth’s surface and to H₂O₂. These effects were eliminated if the spores were decoated, indicating that alterations in coat proteins by different sporulation conditions can affect spore resistance to some agents. Significance and Impact of the Study: This study provides further evidence that the composition of the sporulation medium can have significant effects on B. subtilis spore resistance to UV radiation and H₂O₂. This knowledge provides further insight into factors influencing spore resistance and inactivation.     

Biochemical genetics of bacterial sporulation. IV. Sequential development of resistances to chemical and physical agents during sporulation of Bacillus subtilis

Summary Resistances to various chemical agents appear sequentially during the sporulation of B. subtilis, with the following order: xylene-toluene-benzene-octanol-butanol-methanol, ethanol, chloroform, acetone, dioxane-pyridine-TCA, phenol. Heat-resistance increases gradually: resistance to 80°C for 10 min appears simultaneously with that to TCA and phenol, but spore maturation, as detected by heating at 90°C for two hours, continues for another 120 minutes. Various solvents and temperatures can be used as specific markers for the later stages of sporulation. Such markers cover more than a third of the entire process. Both chemical and temperature resistance markers are useful tools in the study of late sporulation events in wild type and in sporulation mutants.     

Electron Microscopy of the Altered Spore Morphology of a Ribonucleic Acid Polymerase Mutant of Bacillus subtilis

Electron microscopy was used to analyze sporulating cells and spores of Bacillus subtilis mutants (Rif(r)) which are resistant to rifampin, an inhibitor of ribonucleic acid polymerase. The spores of Rif-18 are pleomorphic and frequently exhibit terminal knobs. These knobs first occur during late stage IV and early stage V of sporulation and are extensions of the inner and outer spore coats. Since the rifampin resistance and altered spore morphology of Rif-18 are 100% cotransformable, these data suggest that the altered spore morphology is the result of an alteration in ribonucleic acid polymerase genes. The morphology and physical dimensions are also reported for spores from Rif-11, Rif-15, and Rif-21. Significant differences in size from the wild type were observed for these mutants.     

Sporulation in Bacillus subtilis. Morphological changes

1. When Bacillus subtilis was grown in a medium in which sporulation occurred well-defined morphological changes were seen in thin sections of the cells. 2. Over a period of 7.5hr. beginning 2hr. after the initiation of sporulation the following major stages were observed: axial nuclear-filament formation, spore-septum formation, release of the fore-spore within the cell, development of the cortex around the fore-spore, the laying down of the spore coat and the completion of the corrugated spore coat before release of the spore from the mother cell. 3. The appearance of refractile bodies and 2,6-dipicolinic acid and the development of heat-resistance began between 5 and 6.5hr. after initiation of sporulation. 4. The appearance of 2,6-dipicolinic acid and the onset of refractility appeared to coincide with a diminution of electron density in the spore core and cortex. 5. Heat-resistance was associated with the terminal stage, the completion of the spore coat. 6. The spore coat was composed of an inner and an outer layer, each of which consisted of three or four electron-dense laminae. 7. Serial sections through cells at an early stage of sporulation showed that the membranes of each spore septum were always continuous with the membranes of a mesosome, which was itself in close contact with the bacterial or spore nucleoid. 8. These changes were correlated with biochemical events occurring during sporulation.     

The effect of sporulation temperature on sporal characteristics ofBacillus subtilis A

Spores ofBacillus subtilis A were produced at different temperatures (23°–49°C) and examined for a number of sporal characteristics. Spore heat resistance increased with sporulation temperature to 45°C, with spores grown at 49°C showing a dramatic reduction in resistance. Spore crops showed biphasic thermal death curves whether enumerated on germination medium with or without calcium dipicolinate. This strain produces both rough and smooth variants. Of the spores produced at 23°C, 99% were rough, had a density of 1.305, and an average core/core + cortex volume ratio of 0.1838. At 49°C, 99% were smooth, had a density of 1.275, and an average volume ratio of 0.3098. Between these temperatures both spore types were produced. There appeared to be no direct correlation with sporulation temperature, heat resistance, and dipicolinate content. There was an increase in both the magnesium and calcium contents to 45°C with a dramatic reduction at 49°C. The 1.305 density spores had higher calcium and dipicolinate contents than the 1.275 spores, although both spore types showed biphasic thermal death curves. The mechanisms involved in determining which spore type (rough/smooth) is produced at a specific growth temperature is unknown.Florida Agricultural Experiment Station Journal Series Number R-00312.     

Inhibition of the development of the spore septum and membranes in Bacillus cereus by beta-phenethyl alcohol

Remsen, C. C. (Syracuse University, Syracuse, N.Y.), D. G. Lundgren, and R. A. Slepecky. Inhibition of the development of the spore septum and membranes in Bacillus cereus by beta-phenethyl alcohol. J. Bacteriol. 91:324-331. 1966.-The effect of phenethyl alcohol (PEA) upon the initial stages of sporulation in Bacillus cereus was studied with an electron microscope. PEA (0.35%) completely inhibited the development of the spore septum and forespore membranes. Some of the treated cells did form the axial filament of chromatin material regarded as the first stage in sporulation, but this was delayed by 4 to 5 hr compared with untreated cells. The definite effect upon these membrane systems lends support to the belief that the primary site of PEA inhibition may be upon the bacterial membrane. The fine-structure details observed during the initial stages of sporulation in untreated cells were in agreement with the structure published for other Bacillus species.     

Sporulation in Bacillus subtilis. Correlation of biochemical events with morphological changes in asporogeneous mutants

A comparison was made of morphological changes and successive, mainly biochemical, marker events for sporulation in 14 asporogenous mutants. The morphological and biochemical sequences are linked so that arrested development in one is accompanied by corresponding effects in the other. Thus mutants that fail to produce both protease and antibiotic do not progress beyond stage 0, formation of alkaline phosphatase appears to be associated with the transition from stage II to stage III and glucose dehydrogenase with that from stage III to stage IV. Stage II mutants may produce ;pygmy' cells or other bizarre cell-division forms. The biochemical sequence is dependent in the sense that if the occurrence of any one event is blocked that of all the succeeding events is also blocked. This has implications for biochemical models that have been proposed to explain the temporal sequence observed in spore development.