Early stages of arthrospore maturation in Streptomyces.

In the sporogenesis of Streptomyces, two basic stages can be considered: sporulation septum synthesis and arthrospore maturation. Most of the information about the ultrastructural changes accompanying the sporogenesis refer to the first stage of the process, but nothing has been published about the evolution of the sporulation septum during maturation. In a previous paper, proposed three basic types of sporulation septum formation in Streptomyces. Our ultrastructural study on the evolution of the sporulation septum during the early stages of arthrospore maturation in seven species of Streptomyces indicates correlation between the sporulation septum type and its evolution during the arthrospore maturation. In types I and II the material of the annuli was incorporated into the lateral walls of the arthrospore, whereas in types II and III the deposits were lysed during the maturation. Only in type III was the arthrospore wall synthesized de novo. In type I there was total integration and in type II there was partial integration of the septum into the arthrospore wall.     

Sporogenesis in Streptomyces melanochromogenes

The mode of spore differentiation in a strain of Streptomyces melanochromogenes was followed by analysis of ultrathin sections of sporulating aerial hyphae at various stages of sporogenesis. A special accent was laid on the formation of the sporulation septum and its alterations in the course of spore delimitation and separation. Distinct differences in formation and substructure have been observed between the cross walls of vegetative hyphae and the sporulation septa.Cross walls of vegetative hyphae are formed in a way typical for Gram-positive bacteria by a centripetal annular ingrowth of cytoplasmic membrane, on which wall material immediately is deposited. The development of the sporulation septa is characterized by the accumulation of amorphous material in addition to the newly synthesized wall layer inside the invaginating cytoplasmic membrane. This amorphous septal material will later be decomposed presumably by two lytic systems which cause the separation of the spores. The central region of the finished sporulation septum is perforated by microplasmodesmata. Spores are released by a break down of the surface sheath. The complete spores are enveloped by a twolayered cell wall and the spiny surface sheath.     

Chemical and Morphological Studies of Bacterial Spore Formation : I. The Formation of Spores in Bacillus cereus

Experimental conditions were developed whereby a culture of Bacillus cereus formed spores with reasonable synchrony following a growth cycle of some 8 hours. The cytology of this metamorphosis was studied by dark phase contrast, bright-field microscopy and electron microscopy of thin sections. Particular attention has been paid to the changes in chromatin patterns and these have been correlated with quantitative chemical estimations of the nucleic acids. The cell commencing sporulation contains two compact chromatin bodies and twice the spore amount of deoxyribonucleic acid. Following fusion of the two chromatin bodies, one-half of this chromatin becomes located at a cell end. A transverse septum growing inwards from, and remaining attached to, the inner surface of the cell wall separates this end-piece of chromatin and some associated cytoplasm from the rest of the cell to form the primordial spore. Although the synthesis of deoxyribonucleic acid ceases during the segregation process, it recommences in this organism and continues at a linear rate as the spore develops. Tracer studies with radioactive phosphorus indicated that this further synthesis is confined to the non-spore portion of the sporangium. Although the net synthesis of ribonucleic acid ceased prior to the onset of sporogenesis, some evidence of a turnover of this fraction during the sporulation process was found.     

The Bacillus SpoIIGA protein is targeted to sites of spore septum formation in a SpoIIE-independent manner

The process of bacterial cell division involves the assembly of a complex of proteins at the site of septation that probably provides both the structural and the cytokinetic functions required for elaboration and closure of the septal annulus. During sporulation in Bacillus subtilis , this complex of proteins is modified by the inclusion of a sporulation-specific protein, SpoIIE, which plays a direct role in gene regulation and also has a genetically separable role in determining the gross structural properties of the specialized sporulation septum. We demonstrate by both green fluorescent protein (GFP) fusions and indirect immunofluorescence microscopy that SpoIIGA, a protein required for proteolytic cleavage of pro-σ^E, is also targeted to the sporulation septum. Septal localization of SpoIIGA–GFP occurred even in the structurally abnormal septum formed by a SpoIIE null mutant. We also report the isolation of a spoIIGA homologue from Bacillus megaterium , a species in which the cells are significantly larger than those of B . subtilis . We have exploited the physical dimensions of the B . megaterium sporangium, in conjunction with wide-field deconvolution microscopy, to construct three-dimensional projections of sporulating cells. These projections indicate that SpoIIGA–GFP is initially localized in an annulus at the septal periphery and is only later localized uniformly throughout the septa. Localization was also detected in a B . subtilis spo0H null strain that fails to construct a spore septum. We propose that SpoIIGA is sequestered in the septum by an interaction with components of the septation machinery and that this interaction begins before the construction of the asymmetric septum.     

On the nature of sporogenesis in some aerobic bacteria

Washed vegetative cells of various species of aerobic spore-forming bacteria sporulate abundantly when shaken in distilled water in air. The spores thus formed possess the same heat resistance as spores formed in a complete growth medium. Various factors influencing sporogenesis in water are described. Glucose in low concentration completely suppresses sporogenesis under these conditions and the suppression is relieved by the presence of ammonia as an exogenous source of nitrogen. Various amino acid and purine antimetabolite analogues inhibit sporogenesis and their inhibitory effects are completely reversed by much smaller amounts of the corresponding metabolites. Sporogenesis is thus regarded as a de novo synthesis of spore proteins from preexisting endogenous (enzyme) proteins. Cells low in protein fail to sporulate and the capacity of the cell to adaptively attack maltose and trehalose is strongly interfered with after the cell is irreversibly committed to sporulation, but not before that. Evidence is advanced supporting the hypothesis that sporogenesis is an endogenous process which commences when the supply of exogenous energy and carbon is depleted. It utilizes low molecular weight nitrogenous substances liberated by the degradation of preexisting enzyme proteins of the vegetative cell. Sporogenesis and adaptive enzyme formation are regarded as competitive synthetic processes, both utilizing endogenous enzyme proteins. The events of sporogenesis suggest that this process may be an adaptive protein synthesis, analogous to adaptive enzyme synthesis.     

Visualization of Penicillin-Binding Proteins during Sporulation of Streptomyces griseus

We used fluorescein-tagged β-lactam antibiotics to visualize penicillin-binding proteins (PBPs) in sporulating cultures of Streptomyces griseus. Six PBPs were identified in membranes prepared from growing and sporulating cultures. The binding activity of an 85-kDa PBP increased fourfold by 10 to 12 h of sporulation, at which time the sporulation septa were formed. Cefoxitin inhibited the interaction of the fluorescein-tagged antibiotics with the 85-kDa PBP and also prevented septum formation during sporulation but not during vegetative growth. The 85-kDa PBP, which was the predominant PBP in membranes of cells that were undergoing septation, preferentially bound fluorescein-6-aminopenicillanic acid (Flu-APA). Fluorescence microscopy showed that the sporulation septa were specifically labeled by Flu-APA; this interaction was blocked by prior exposure of the cells to cefoxitin at a concentration that interfered with septation. We hypothesize that the 85-kDa PBP is involved in septum formation during sporulation of S. griseus.     

Microcycle sporogenesis in some streptomycetes without shift down treatment

Conditions for microcycle sporogenesis in two streptomycete strains without shifting the culture were found. The sporulation in Streptomyces granaticolor took place after 24 h of cultivation. The dry mass was increasing till 32 h probably due to production of a hydrophobic substance resembling fibrous sheath of aerial hyphae and spores. Ultrathin section of microcycle spores are presented.     

BIOCHEMICAL CHANGES IN YEAST DURING SPORULATION. I. FATE OF NUCLEIC ACIDS AND RELATED COMPOUNDS

Changes in nuclear figures and in activities of nucleic acid and protein syntheses were observed mainly on Saccharomyces cerevisiae G2-2 during sporogenesis. Patterns of DNA synthesis and of meiosis show that the sporogenic process in yeast was divided into an induction phase (I-phase), a DNA-synthesizing phase (S-phase) and a maturation phase (M-phase). Meiotic figures appeared most frequently at the end of the S-phase at approximately 12 hr in sporulation culture. In M-phase visible spores formed. The amount of protein increased in the initial 7 hr culture of 1-phase, then decreased in the S- and M-phases. But in sporulation culture of the asporogenic diploid strain 3c × a, protein did not decrease. RNA increased within 3 hr of the I-phase then stopped increasing. DNA synthesis occurred critically during S-phase, i.e. between 7 and 12 hr. and was somewhat resumed during the later part of M-phase. Oligodeoxyri-bonucleotide content decreased in the I- and M-phases and increased temporarily. Deoxyribosides decreased linearly during the sporogenic processes. Based on these results and results of experiments estimating the incorporation of ¹⁴C-uracil into nucleic acid and ¹⁴C-amino acid mixture into protein fractions, the roles of nucleic acid synthesis activities in meiosis and in sporulation are discussed.     

The relation between sporulation and the induction of antibiotic synthesis and of amino acid uptake in Bacillus brevis

The induction and localization of tyrocidine-synthesizing enzymes is shown to be parallel, during growth of Bacillus brevis (ATCC 8185, American Type Culture Collection, Rockville, Md.), with the induction of uptake of constitutive amino acids and of components of pantetheine, a coenzyme of tyrocidine synthesis. Antibiotic synthesis appears at the end of logarithmic growth when the first soluble enzymes may be obtained from homogenates. During this period, binding proteins for metabolite uptake were isolated by intensive sonication which, when studied by chromatography, were identified by the appearance of low molecular weight fractions binding the radioactively marked metabolites; their induction was prevented by addition of rifampicin. The major purpose of this study was a comparison of antibiotic production and sporulation, the progress of which was followed by electron microscopy. The onset of tyrocidine synthesis and metabolite uptake coincided with the appearance of septum formation indicating that sporulation had progressed to stage II. With the progress of spore encapsulation, the tyrocidine production migrated from the soluble fraction into the forespore, terminating with the separation of forespores from the sporangium membrane. The resulting concentration of antibiotic in the forespore may indicate its function in sporulation, the nature of which, however, was not explored.     

Inhibition of meiosis in Saccharomyces cerevisiae by ammonium ions: Interference of ammonia with protein metabolism

Meiosis and sporogenesis in yeast are completely blocked by ammonia added in low concentration (10 mM) to the sporulation medium. Premeiotic DNA synthesis is not initiated in the presence of ammonium ions. The inhibitor interferes with protein turnover by reducing both synthesis and breakdown. The in vitro activities of proteinases A and B in sporulation medium supplemented with ammonia are much lower than in the control. This may partially explain the effect of ammonium ions on protein metabolism in vivo.Abbreviations PSP presporulation medium - SPM sporulation medium     

Sporulation of Streptomyces antibioticus ETHZ 7451 in submerged culture.

Streptomyces antibioticus ETHZ 7451 formed spores in cultures grown in a liquid medium from either a spore or a mycelium inoculum. The spores formed were similar to those formed on surface-grown cultures, except for reduced heat resistance. Both types of spores were sensitive to lysozyme, which is unusual for Streptomyces spores. Glucose and other carbon sources, which promoted different growth rates, did not affect sporulation efficiency. Nitrogen sources, such as casamino acids, that allowed high growth rates suppressed the sporulation. A remarkable repression was also observed in media with some nitrogen sources that promoted noticeably lower growth rates. In permissive media, with nitrogen sources that permitted relatively high growth rates, sporulation was conditioned to the consumption of ammonium in the medium, but not to that of other nitrogen sources, such as asparagine. Phosphate did not show a repressive effect on sporulation in the assayed conditions.     

Morphological stages of Bacillus subtilis sporulation and resistance to fusidic acid.

During spore development of Bacillus subtilis both protein synthesis and sporulation become resistant to the antibiotic fusidic acid. This resistance develops at the time when asymmetric prespore septa are formed. Simultaneously ribosomes lose their ability to bind fusidic acid, as demonstrated by their affinity chromatography with the immobilized drug. Mutants resistant to fusidic acid during growth are oligosporogenous; their sporulation development is blocked before septum formation. These results indicate that normal ribosomes are needed for prespore septation sporulation; only after septation can protein synthesis be maintained, throughout the development period, by fusidate resistant ribosomes.     

Glycogen and trehalose accumulation during colony development in Streptomyces antibioticus

Streptomyces antibioticus accumulated glycogen and trehalose in a characteristic way during growth on solid medium. Glycogen storage in the substrate mycelium took place during development of the aerial mycelium. The concentration of nitrogen source in the culture medium influenced the time at which accumulation started as well as the maximum levels of polysaccharide stored. Degradation of these glycogen reserves was observed near the beginning of sporulation. The onset of sporogenesis was always accompanied by a new accumulation of glycogen in sporulating hyphae. During spore maturation the accumulated polysaccharide was degraded. No glycogen was observed in aerial non-sporulating hyphae or in mature spores. Trehalose was detected during all phases of colony development. A preferential accumulation was found in aerial hyphae and spores, where it reached levels up to 12% of the cell dry weight. The possible roles of both carbohydrates in the developmental cycle of Streptomyces are discussed.     

Inhibitory effect of penicillin on spore-specific functions inBacillus polymyxa 2459

Penicillin at concentrations non-inhibitory to the vegetative growth was found to inhibit sporulation inBacillus polymyxa 2459. The effect of penicillin was shown to be at the level of spore-specific mucopeptide synthesis. Penicillin had no effect on the early events such as DNA and protein synthesis in sporogenesis The sensitive period of inhibition was between T₀ to T₂ hours of sporulation.     

A vital stain for studying membrane dynamics in bacteria: a novel mechanism controlling septation during Bacillus subtilis sporulation

At the onset of sporulation in Bacillus subtilis, two potential division sites are assembled at each pole, one of which will be used to synthesize the asymmetrically positioned sporulation septum. Using the vital stain FM 4-64 to label the plasma membrane of living cells, we examined the fate of these potential division sites in wild-type cells and found that, immediately after the formation of the sporulation septum, a partial septum was frequently synthesized within the mother cell at the second potential division site. Using time-lapse deconvolution microscopy, we were able to watch these partial septa first appear and then disappear during sporulation. Septal dissolution was dependent on sigma E activity and was partially inhibited in mutants lacking the sigma E-controlled proteins SpoIID, SpoIIM and SpoIIP, which may play a role in mediating the degradation of septal peptidoglycan. Our results support a model in which sigma E inhibits division at the second potential division site by two distinct mechanisms: inhibition of septal biogenesis and the degradation of partial septa formed before sigma E activation.