Cortex content of asporogenous mutants of Bacillus subtilis.

A method for the measurement of muramic lactam, which is specifically located in the cortical peptidoglycan of bacterial spores, was developed as a quantitative assay method for spore cortex content. During sporulation of Bacillus subtilis 168, muramic lactam (i.e., spore cortex) began to appear at state IV of sporulation and continued to increase over most of the late stages of sporulation. Spore cortex contents of various spo mutants of B. subitils were surveyed. Cortex was not detected in mutants in which sporulation was blocked earlier than stage II sporulation. Spores of spo IV mutant had about 40% of the cortex content of the wild-type spores. One spo III mutant had a low amount of cortex, but four others had none.     

Mutations in the gerP locus of Bacillus subtilis and Bacillus cereus affect access of germinants to their targets in spores

The gerP1 transposon insertion mutation of Bacillus cereus is responsible for a defect in the germination response of spores to both L-alanine and inosine. The mutant is blocked at an early stage, before loss of heat resistance or release of dipicolinate, and the efficiency of colony formation on nutrient agar from spores is reduced fivefold. The protein profiles of alkaline-extracted spore coats and the spore cortex composition are unchanged in the mutant. Permeabilization of gerP mutant spores by coat extraction procedures removes the block in early stages of germination, although a consequence of the permeabilization procedure in both wild type and mutant is that late germination events are not complete. The complete hexacistronic operon that includes the site of insertion has been cloned and sequenced. Four small proteins encoded by the operon (GerPA, GerPD, GerPB, and GerPF) are related in sequence. A homologous operon (yisH-yisC) can be found in the Bacillus subtilis genome sequence; null mutations in yisD and yisF, constructed by integrational inactivation, result in a mutant phenotype similar to that seen in B. cereus, though somewhat less extreme and equally repairable by spore permeabilization. Normal rates of germination, as estimated by loss of heat resistance, are also restored to a gerP mutant by the introduction of a cotE mutation, which renders the spore coats permeable to lysozyme. The B. subtilis operon is expressed solely during sporulation, and is sigma K-inducible. We hypothesize that the GerP proteins are important as morphogenetic or structural components of the Bacillus spore, with a role in the establishment of normal spore coat structure and/or permeability, and that failure to synthesize these proteins during spore formation limits the opportunity for small hydrophilic organic molecules, like alanine or inosine, to gain access to their normal target, the germination receptor, in the spore.     

Bichemical genetics of bacterial sporulation

Summary Pleitropic interactions among genes controlling the formation of bacterial spores and of sporulation-associated products are studied. In order to obtain sporulation mutants, spores have been germinated in the presence of chloramphenicol and then treated with nitrosoguanidine. In the most favorable conditions 25% of sporulation mutants have been found among the 40% surviving bacteria. This number is at least four times higher than the number of auxotrophic mutants, therefore a rough estimate of the number of genes involved in sporulation is 800.Rapid plate-tests have been developed for the oxidation of terrazolium salts, the formation of various proteolytic enzymes and the production of antibiotics. Although the exact biochemical nature of the products is not yet known, the results suggest that distinct factors, probably various enzymes (including several proteases) are detected by these tests. All of them are associated with spore formation and absent from a large number of sporulation mutants. Using these tests, the phenotypes of 500 randomly selected sporulation mutants were determined. No important differences were found between asporogenous and oligosporogenous mutants. The number of mutants deficient for several sporulation-associated characters is large, pleiotropic interactions following a defined pattern are observed. Statistical analysis indicates the existence of a unidirectional pleiotropic system. All the results agree with the hypothesis of sequential gene activation. Consequently, the sporulation-associated characters can be ordered into a linear sequence, presumably reflecting the consecutive steps in spore formation. The order obtained is the following: gelatinase, proteases acting on casein and on denatured albumin, oxidation of tetrazolium No 7, digestion of protamine, production of antibiotics (against a Staphylococcus and a Bacillus), hydrolysis of hemoglobin, oxidation of tetrazolium No 2, digestion of native albumin, synthesis of elastase. Another category of mutants, blocked in a late step of sporulation and apparently derepressed for the formation of elastase, is also described.In conclusion, arguments are put forward in favor of sequential gene activation. Sporulation genes, related by unidirectional pleiotropic interactions, form a sporulon. Generalization of this concept to other differentiating systems (a differon), its predictions and possible experimental confirmation are considered.The author was a Gosney Research Fellow in 1966/67, on leave of absence from the Centre National de la Recherche Scientifique, Paris. Present adress: see end of paper.     

Altered sporulation and respiratory patterns in mutants of Bacillus subtilis induced by acridine orange

Bott, K. F. (The University of Chicago, Chicago, Ill.), and R. Davidoff-Abelson. Altered sporulation and respiratory patterns in mutants of Bacillus subtilis induced by acridine orange. J. Bacteriol. 92:229-240. 1966.-The addition of acridine orange to vegetative cultures of Bacillus subtilis induces the formation of sporulation mutants at a frequency of 20% or greater. These mutants are grouped into seven categories which reflect their different morphological properties. They are altered in their vegetative metabolism, as indicated by abnormal growth on synthetic media. Sporulation of these mutants is impaired at several levels, all of which are stable upon repeated subculturing. The initial stages of sporulation which require no increased metabolic activity (proteolytic enzyme activity and antibiotic production) are functional in all strains, but glucose dehydrogenase activity, an enzyme associated with early synthetic functions in spore synthesis, is significantly reduced. Reduced nicotinamide adenine dinucleotide oxidase is slightly depressed. It is suggested that acridine orange interacts with a cellular constituent controlling respiration and consequently prevents an increased metabolic activity that may be associated with normal spore synthesis.     

Growth, sporulation, and enzyme defects of glucosamine mutants of Bacillus subtilis

Two glucosamine (GCA)-requiring mutants have been isolated which grow on glucose minimal or nutrient sporulation medium only in the presence of either GCA or acetyl-GCA. They lack the l-glutamine-d-fructose-6-phosphate aminotransferase (EC 2.6.1.13), which is repressible by GCA and whose activity in the standard strain decreases after cessation of growth. But the mutants can grow on GCA as sole carbon and ammonia source, because GCA induces the synthesis of 2-amino-2-deoxy-d-glucose-6-phosphate ketol-isomerase (deaminating) (EC 5.3.1.10). With respect to sporulation, the GCA-requiring mutants are in a serious dilemma, as GCA represses the onset of massive sporulation and yet a small amount of GCA-6-phosphate derivatives is necessary to allow sporulation. When GCA is continuously provided in small quantities, sporelike particles are produced which contain little or no spore cortex but a normal spore coat. Apparently, GCA derivatives are needed especially for cortex formation. Many of the sporelike particles can produce colonies after octanol, but not after heat treatment. When they are purified by treatment with lysozyme and sodium dodecylsulfate, they do not show the decrease in optical density at 600 nm typical of germination nor do they produce offspring.     

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.     

ExsY and CotY are required for the correct assembly of the exosporium and spore coat of Bacillus cereus

The exosporium-defective phenotype of a transposon insertion mutant of Bacillus cereus implicated ExsY, a homologue of B. subtilis cysteine-rich spore coat proteins CotY and CotZ, in assembly of an intact exosporium. Single and double mutants of B. cereus lacking ExsY and its paralogue, CotY, were constructed. The exsY mutant spores are not surrounded by an intact exosporium, though they often carry attached exosporium fragments. In contrast, the cotY mutant spores have an intact exosporium, although its overall shape is altered. The single mutants show altered, but different, spore coat properties. The exsY mutant spore coat is permeable to lysozyme, whereas the cotY mutant spores are less resistant to several organic solvents than is the case for the wild type. The exsY cotY double-mutant spores lack exosporium and have very thin coats that are permeable to lysozyme and are sensitive to chloroform, toluene, and phenol. These spore coat as well as exosporium defects suggest that ExsY and CotY are important to correct formation of both the exosporium and the spore coat in B. cereus. Both ExsY and CotY proteins were detected in Western blots of purified wild-type exosporium, in complexes of high molecular weight, and as monomers. Both exsY and cotY genes are expressed at late stages of sporulation.     

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.     

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.     

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.     

Isolation and Properties of a Temperature-Sensitive Sporulation Mutant of Bacillus subtilis

A thermosensitive sporulation mutant (t(s)-4) of Bacillus subtilis was isolated, and its morphological, physiological, and enzymatic properties were investigated. This mutant is able to grow equally well at 30 and 42 C, but is unable to sporulate at the higher temperature. Electron microscope studies have shown that the t(s)-4 mutant is blocked at stage zero of spore development. This was further confirmed by its inability to produce antibiotic when grown at the restrictive temperature and by the relatively low ribonucleic acid (RNA) and protein turnover during the stationary growth phase, characteristic for stage zero asporogenic mutants. At the permissive temperature, however, antibiotic production and RNA and protein turnover took place at the rate normally found in sporogenic strains of B. subtilis. The above properties were not altered in the parent strain when grown at either 30 or 42 C. By shifting cultures of the t(s)-4 mutant from 30 to 42 C and from 42 to 30 C at different stages of growth, we have been further able to show that the event affected at the high temperature takes place at a very early stage of spore development. As a consequence of this early block in the sporulation process, the t(s)-4 mutant grown at 42 C became defective in the late spore-specific enzymes involved in the biosynthesis of dipicolinic acid. This study suggests that the sporulation process is mediated by a regulatory protein which is altered in the thermosensitive mutant when grown at the restrictive temperature. As a result of this alteration, a pleiotropic phenotype is produced which has lost the ability to catalyze the late biochemical reactions required for spore formation.     

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.     

Bacillus subtilis diacylglycerol kinase (DgkA) enhances efficient sporulation

The sn-1,2-diacylglycerol kinase homologue gene, dgkA, is a sporulation gene indispensable for the maintenance of spore stability and viability in Bacillus subtilis. After 6 h of growth in resuspension medium, the endospore morphology of the dgkA mutant by standard phase-contrast microscopy was normal; however, after 9 h, the endospores appeared mostly dark by phase-contrast microscopy, suggesting a defect in the spores. Moreover, electron microscopic studies revealed an abnormal cortex structure in mutant endospores 6 h after the onset of sporulation, an indication of cortex degeneration. In addition, a significant decrease in the dipicolinic acid content of mutant spores was observed. We also found that dgkA is expressed mainly during the vegetative phase. It seems likely that either the DgkA produced during growth prepares the cell for an essential step in sporulation or the enzyme persists into sporulation and performs an essential function.     

Sporulation in Bacillus subtilis. The appearance of sulpholactic acid as a marker event for sporulation

1. The synthesis of sulpholactic acid in sporulating cultures of Bacillus subtilis was studied. 2. Sulpholactic acid was first detected about 4h after the initiation of sporulation and 1h before refractility. The rate of synthesis paralleled that of the other events of sporulation examined. 3. Sulpholactic acid accounted for 1.7% of the material of the spore. 4. Because the addition of chloramphenicol in the earlier stages of sporulation inhibited formation of the compound, it is likely that the enzymes concerned are synthesized de novo during sporulation. 5. In asporogenous mutants only those blocked at a late stage and showing partial refractility were able to produce sulpholactic acid. This correlation makes sulpholactic acid a useful marker event in sporulation.     

Electron microscope studies of conditional spore cortexless mutants of Bacillus sphaericus.

Spore cortex of conditional cortexless mutants of Bacillus sphaericus 9602 was not detectable by electron microscopy unless the medium was supplemented with meso-alpha,epsilon-diaminopimelic acid during sporulation. Other spore structures appeared normal. Spore shape was quite irregular in the absence of meso-alpha,epsilon-diaminopimelic acid.     

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.